1. health and fitness
2. disease

Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Science and Technology 1303
Comparative Cell Biology in
Diplomonads
ELIN EINARSSON
ACTA
UNIVERSITATIS
UPSALIENSIS
UPPSALA
2015
ISSN 1651-6214
ISBN 978-91-554-9374-5
urn:nbn:se:uu:diva-264541
Dissertation presented at Uppsala University to be publicly examined in A1:111a, BMC,
Husargatan 3, Uppsala, Friday, 4 December 2015 at 09:15 for the degree of Doctor of
Philosophy. The examination will be conducted in English. Faculty examiner: Professor Scott
Dawson (UC Davies, USA).
Abstract
Einarsson, E. 2015. Comparative Cell Biology in Diplomonads. Digital Comprehensive
Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1303. 84 pp.
Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9374-5.
The diplomonads are a diverse group of eukaryotic flagellates found in microaerophilic
and anaerobic environments. The most studied diplomonad is the intestinal parasite Giardia
intestinalis, which infects a variety of mammals and cause diarrheal disease. Less is known
about Spironucleus salmonicida, a parasite of salmonid fish, known to cause systemic infections
with high mortality.
We created a transfection system for S. salmonicida to study cellular functions and virulence
in detail (Paper I). The system was applied to explore the mitochondrion-related organelle
(MRO) in S. salmonicida. We showed that S. salmonicida possesses a hydrogenosome (Paper
II) with a higher metabolic capacity than the corresponding MRO of Giardia, the mitosome.
Evolutionary analysis of key hydrogenosomal proteins showed ancient origin, indicating
their presence in the ancestral diplomonad and subsequent loss in Giardia. Annexins are of
evolutionary interest since these proteins are found across all kingdoms. Annexin-like proteins
are intriguingly expanded into multigene families in Giardia and Spironucleus. The annexins
of S. salmonicida were characterized (Paper III) with distinct localizations to various cellular
structures, including a putative adhesion structure anterior in the cell.
The disease-causing Giardia trophozoites differentiate into infectious cysts, a process
essential for transmission and virulence of the parasite. Cysts are often spread via contaminated
water and exposed to environmental stressors, such as UV irradiation. We studied the survival
and transcriptional response to this stress factor (Paper IV) and results showed the importance
of active DNA replication machinery for parasite survival after DNA damage. In addition, we
studied transcriptional changes along the trajectory of encystation (Paper V), which revealed a
coordinated cascade of gene regulation. This was observed for the entire transcriptome as well as
putative regulators. Large transcriptional changes appeared late in the process with the majority
of differentially regulated genes encoding hypothetical proteins. We studied the localizations of
several of these to gain information of their possible function.
To conclude, the diplomonads are complex eukaryotic microbes with cellular processes
adjusted to match their life styles. The work in this thesis has provided insight of their
adaptations, differences and similarities, but also new interesting leads for future studies of
diplomonad biology and virulence.
Keywords: Giardia intestinalis, Spironucleus salmonicida, intestinal parasite, hydrogenosome,
encystation, gene regulation, transfection, diplomonad, antigenic variation, annexin
Elin Einarsson, Department of Cell and Molecular Biology, Box 596, Uppsala University,
SE-75124 Uppsala, Sweden.
© Elin Einarsson 2015
ISSN 1651-6214
ISBN 978-91-554-9374-5
urn:nbn:se:uu:diva-264541 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-264541)
In nature nothing exists alone
-
Rachel Carson
Till min farmor Signe
Cover photos by Stan Erlandsen and Andrew Hemphill
List of Papers
This thesis is based on the following papers, which are referred to in the text
by their Roman numerals.
I
Jerlström-Hultqvist J, Einarsson E, Svärd SG. (2012) Stable
transfection of the diplomonad parasite Spironucleus salmonicida. Eukaryotic Cell. 11(11):1353–61
II
Jerlström-Hultqvist J, Einarsson E, Xu F, Hjort K, Ek B, Steinhauf D, Bergqvist J, Andersson JO, Svärd SG. (2013) Hydrogenosomes in the diplomonad Spironucleus salmonicida.
Nature Communications. 4:2493
III
Einarsson E, Ástvaldsson Á, Hultenby K, Andersson JO, Svärd
SG, Jerlström-Hultqvist J. Comparative Cell Biology and Evolution of Annexins in Diplomonads. Submitted manuscript.
IV
Einarsson E, Svärd SG, Troell K. (2015) UV irradiation responses in Giardia intestinalis. Experimental parasitology.
154:25-32
V
Einarsson E, Troell K, Höppner, M, Grabherr M, Ribacke U,
Svärd SG. Coordinated Changes in Gene Expression Throughout Encystation of Giardia intestinalis. Submitted manuscript.
Reprints were made with permission from the respective publishers.
Publications not included in the thesis.
I
Franzén O, Jerlström-Hultqvist J, Einarsson E, Ankarklev J,
Ferella M, Andersson B, Svärd SG. (2013) Transcriptome profiling of Giardia intestinalis using strand-specific RNA seq.
PLoS Computational Biology. 9(3):e1003000
II
Xu F, Jerlström-Hultqvist J, Einarsson E, Ástvaldsson Á,
Svärd SG, Andersson JO. (2014). The genome of Spironucleus
salmonicida highlights a fish pathogen adapted to fluctuating
environments. PLoS Genetics. 10(2):e1004053.
III
Einarsson E, Svärd SG. (2015). Encystation of Giardia intestinalis- a journey from the duodenum to the colon. Current
Tropical Medicine Reports. 2(3):101-109. Review article.
Contents
Introduction ................................................................................................... 11 Diplomonads ............................................................................................ 11 The Giardia cell ....................................................................................... 14 Giardia classification ............................................................................... 15 The life cycle of Giardia .......................................................................... 16 The trophozoite .................................................................................... 17 Energy metabolism ................................................................................... 23 Differentiation .......................................................................................... 24 Giardia pathogenesis................................................................................ 31 Antigenic variation ................................................................................... 34 Genomics and transcriptomics ................................................................. 38 Spironucleus ............................................................................................. 40 Infection and transmission ....................................................................... 41 Genomics and transcriptomics of S. salmonicida..................................... 42 Metabolism ............................................................................................... 43 Cell biology .............................................................................................. 44 Present Investigation ..................................................................................... 46 Aim of the thesis .................................................................................. 46 Establishment of S. salmonicida as a diplomonad model system
(Paper I) ............................................................................................... 46 S. salmonicida possess hydrogenosomes (Paper II) ............................ 48 Annexin diversity revealed in S. salmonicida (Paper III) .................... 50 UV stress in G. intestinalis (Paper IV) ................................................ 53 The differentiation of G. intestinalis is a coordinated cascade of
gene regulation (Paper V) .................................................................... 54 Conclusions and future perspectives ............................................................. 58 S. salmonicida can be used as a biological model system ................... 58 Can S. salmonicida form cysts? ........................................................... 58 Hydrogenosomes: one organelle, several functions ............................ 59 S. salmonicida have an attachment organelle ...................................... 59 Giardia cysts- masters of survival ....................................................... 60 What dictates differentiation in Giardia intestinalis?.......................... 60 Pieces missing in the differentiation puzzle ........................................ 60 VSP switch event during encystation .................................................. 61 What controls the antigenic switch? .................................................... 61 Is antigenic variation a common trait of diplomonad parasites? ......... 62 Sammanfattning på svenska (Summary in Swedish) .................................... 63 Acknowledgements ....................................................................................... 66 References ..................................................................................................... 70 Abbreviations
ADI
AP
ASH
CLO
COP
Cpn60
CRMP
CRP
CWP
ECV
ER
EST
ESV
FPKM
GAG
GalNAc
HAT
HCMP
HCP
HDAC
HMT
MRO
Nek
NO
OCT
PFOR
PV
RdRP
ROS
SHMT
snoRNA
TEM
UTR
VSP
Arginine deiminase
Adaptor protein
Allelic sequence heterozygosity
Carpediemonas-like organism
Coat protein
Chaperonin 60
Cysteine-rich membrane protein
Cysteine-rich protein
Cyst wall protein
Encystation
carbohydrate-positive
vesicle
Endoplasmic reticulum
Expressed sequenced tag
Encystation specific vesicle
Fragments/kilo base/million reads
Glycosaminoglycan
N-acetyl galactosamine
Histone acetyltransferase
High cysteine membrane protein
High cysteine protein
Histone deacetylase
Histone methyltransferase
Mitochondrion-related organelle
Never in mitosis related kinase
Nitric oxide
Ornithine carbamoyltransferase
Pyruvate:ferredoxin oxioreductase
Peripheral vesicles
RNA-dependent RNA polymerase
Reactive oxygen species
Serine hydroxyl methyltransferase
Small nucleolar RNA
Transmission electron microscopy
Untranslated region
Variant-specific surface protein
Introduction
Eukaryotes display great variation and diversity, ranging from microscopic
unicellular life forms to multicellular plants and animals. The origin of eukaryotes has been under investigation for decades but it is still not clear how
eukaryotic cells evolved. The current view is that all eukaryotic cells share a
common ancestor that was already a relatively complex cell. A hallmark
feature of eukaryotes is the presence of a nucleus, where the genetic material
is stored, copied and protected. Other key features are an elaborate endomembrane system creating a possibility for sub-compartments and an actintubulin-based cytoskeleton that enable movements. All studied eukaryotes
also have mitochondria or mitochondrion-related organelles (MROs), which
often play central roles in energy transformation of the cell. The origin of the
mitochondrium is most likely a symbiotic fusion of an alphaproteobacterium with an early eukaryotic cell. It has even been suggested
that this cellular fusion was the start of the eukaryotes.
Historically single cell eukaryotes were lumped together into their own
group, the Protista. They are further divided into slime molds, unicellular
algae and protozoa. These were regarded as “simple” organisms from which
more complex species evolved. Amongst the protozoa many have a parasitic
lifestyle. As all parasites depend on their host for survival, there is a constant
communication and evolution between these two interacting organisms.
This thesis will further describe the unicellular eukaryotic parasites Giardia intestinalis and Spironucleus salmonicida. In my PhD studies I have
been focusing on different biological processes in these two organisms. By
doing so, I aimed to deepen the understanding of cellular features such as the
MROs, the cytoskeleton and the response to different stress factors in these
fascinating diplomonads.
Diplomonads
Several supergroups (or major branches) make up the eukaryotic tree of life.
Excavata consists of many groups of unicellular eukaryotes and they include
parasites of global importance such as Trypanosoma sp., Leishmania sp. and
Giardia sp. (Adl et al., 2005). The excavates all share a general flagellar
organization i.e. they are grouped together based on cell ultrastructural ob11
servations (Simpson, 2003). Most, but not all, also share the feature of a
suspension feeding groove utilized to collect food particles (Simpson, 2003).
The diplomonads are found within the taxon Fornicata together with Carpediemonas-like organisms (CLOs) and retortamonads (Figure 1). These
flagellated unicellular organisms can be found in anaerobic or microaerophilic environments e.g. intestines of animals or aquatic sediments (Simpson, 2003).
Figure 1. Phylogenetic reconstruction of Fornicata and the parabasalid Trichomonas
vaginalis. The origin of the alternative code is indicated. Figure adapted from
(Kolisko et al., 2008).
The members of Fornicata lack classical aerobic mitochondria and therefore these organisms were considered to be “early branching eukaryotes”,
suggesting that they evolved before the mitochondrial symbiosis occurred.
This theory is referred to as the archezoan scenario. Archezoans are hypothetical ancestors of eukaryotes and they lacked mitochondria but possessed
other signature features of a eukaryotic cell. However, over the last decades
MROs have been found among these “ancient” eukaryotes in the form of
mitosomes and hydrogenosomes (Hjort et al., 2010). Moreover, with information from genome sequencing and improved phylogenetic methods available, there are now indications that the deep root placement of these organisms were due to long-branch attraction artifacts (Brinkmann et al., 2005).
This is probably due to the rapid evolution of these organisms. In fact, these
“early branching” organisms may have evolved from a more complex ancestor through reductive evolution, driven by rapid evolution of their relatively
small genomes and strong selective pressure of a parasitic lifestyle. Reduc12
tive evolution is the process in which genes, organelles or functions are lost
and is a common process in parasites when the respective functionalities are
taken over by the host (Koonin, 2010).
The so far studied diplomonad MROs appear to have undergone reductive evolution (Takishita et al., 2012). Classical mitochondria contain DNA
that codes for mitochondrial genes, but the MROs in diplomonads lack
DNA. Actually, little evidence exists that the MROs found in diplomonads
would produce energy (Takishita et al., 2012). Giardia harbors small MROs
(mitosomes) that have no role in energy production, but instead are involved
in Fe-S cluster synthesis, another important function of mitochondria (Tovar
et al., 2003). In contrast, for the CLO examined to date, much larger organelles can be found (Takishita et al., 2012) but their metabolic capacity remains unknown. Their larger size might implicate that these organelles may
be involved in more metabolic pathways than the giardial mitosomes (Takishita et al., 2012). Almost nothing is known about the organelles in retortamonads.
The parabasalids, a sister-clade to diplomonads, are known to harbor hydrogenosomes. Most information of the hydrogenosome comes from studies
in the parasite Trichomonas vaginalis (Makiuchi and Nozaki, 2014; Rada et
al., 2011; Schneider et al., 2011). The hydrogenosome can generate ATP by
converting pyruvate or malate to CO2, acetate and hydrogen (Müller, 1993).
Fe-S cluster synthesis and amino acid metabolism are processes executed by
the hydrogenosome in this organism (Schneider et al., 2011). Similar to most
other defined MROs, the T. vaginalis organelle do not contain a genome. A
protein transport machinery is therefore present to import the hydrogenosomal proteins with a N-terminal targeting signal, which then functions for
recognition (Rada et al., 2011).
Diplomonads are further divided into two subdivisions based on molecular
phylogeny; Giardiinae (Giardia and Octomitus) and Hexamitinae (Spironucleus, Trepomonas and Hexamita). These groups contain parasites, commensals and free-living organisms. All studied members of Hexamitinae employ
an alternative genetic code (Figure 1) where UAG and UAA encode glutamine leaving UGA as the only stop codon (Keeling and Doolittle, 1997).
Most diplomonads have a highly unusual cellular organization with double
karyomastigonts. In other words, each cell possesses two (identical or similar) nuclei and two flagellar apparatuses (Simpson, 2003). Interestingly,
monokaryotic organisms can be found in CLO, retortamonads and enteromonads as well. These contain only one karyomastigont resembling half of
a typical diplomonad cell. Enteromonads were previously considered to be
closely related to Giardiinae based on the observed morphological similarities (Simpson, 2003). However recent phylogenetic analysis placed enteromonads within the Hexamitinae (Kolisko et al., 2008). The paraphyletic
13
state of enteromonads raises the question what was the state of the ancestral
diplomonad. It has been suggested that either the double karyomastigont
morphology arose several times independently or that monokaryotic cells
evolved several times by secondary reduction from dikaryotic ancestral cells
(Kolisko et al., 2008). More data is needed to be able to discriminate between these evolutionary complex scenarios.
I will further introduce the characteristics of the parasites Giardia and
Spironucleus. The most studied diplomonad to date is G. intestinalis and we
are just beginning to gain insight of the characteristics of S. salmonicida.
The Giardia cell
The discovery of Giardia occurred more than 300 years ago. The first reported observation was made by the great Dutch microscopist Antonie van
Leeuwenhoek who reported his finding in a letter dated to November 4 1681
(Dobell, 1920). The organisms reported were observed in his own diarrheal
stools and they were described as follows:
“All these described particles lay in a clear transparent medium, in which I
have at times seen very prettily moving animalcules, some rather large, others
somewhat smaller than a blood corpuscle, and all of one and the same structure. Their bodies were somewhat longer, than broad, and their belly which
was flattened, provided with several feet, with which they made such a
movement through the clear medium and the globules that we might fancy
we saw a pissabed running up against the wall. But although they made a rapid movement with their feet, yet they made but slow progress” (Dobell,
1920).
This observation together with the disease symptoms that he reported fits
very well with the description of the intestinal parasite Giardia. It took another 178 years until the parasite was re-discovered and described in greater
detail by Lambl, who assigned the organism the name Cercomonas intestinalis. A few years later (1889) it was discovered in Sweden for the first time
when the intestinal content of an executed murderer was studied (Muller,
1889). The current genus name Giardia was first used by Kunstler in 1882
and the name Giardia lamblia was proposed in 1915 (Adam, 2001). Many
Giardia species was discovered in following years and assigned names
based on their host specificity or morphology. In 1952, Filice proposed the
species names G. duodenalis (infecting mammals), G. muris (infecting rodents) and G. agilis (infecting amphibians) based on the morphology of the
median body (Adam, 2001). Today, Giardia causing human infections have
three names used as synonyms namely G. lamblia, G. duodenalis and G.
intestinalis. For simplicity I will use the name G. intestinalis throughout this
thesis and Giardia refers to G. intestinalis unless otherwise indicated.
14
Giardia classification
Today it is known that Giardia infects a wide variety of different animals
(Cacciò and Ryan, 2008). Apart from the study by Filice, three additional
Giardia species have been described based on their morphology and differences in DNA sequence. These were given the names G. microti (infecting
voles), G. psittaci (infecting psittacine birds) and G. ardae (infecting herons), see Table 1. Differences were observed in the ventral disc, lateroventral flange and in cysts (Adam, 2001).
Molecular epidemiology tools (i.e. PCR and related techniques) have
been important to elucidate the host specificity and understanding the pathogenicity of Giardia isolates obtained from a variety of sources (Adam,
2001). It was shown that G. intestinalis consists of eight assemblages (or
genotypes). Only assemblages A and B infect humans, but these are also
found in a wide range of other mammals, whereas the remaining (C-H) appear more host-specific (Cacciò and Ryan, 2008). It has been suggested that
G. intestinalis should be divided into different species, since the evidence of
subgroupings within this species exists (Monis et al., 2009), see Table 1.
Genetic variation has been observed within the assemblages and consequently this created sub-assemblages. The isolates within sub-assemblages are
closely related but not identical. Assemblage A and B can be divided into
four distinct sub-assemblages (Monis et al., 2003; Ryan and Cacciò, 2013).
Table 1. Recognized Giardia species, assigned assemblages, host associations and
the proposed species. Table adapted from Monis, 2009.
Species
Assemblage
G. intestinalis
A
B
C-D
E
F
G
H
G. agilis
G. muris
G. psittaci
G. ardeae
G. microti
Suggested species
name
G. duodenalis
G. enterica
G. canis
G. bovis
G. cati
G. simondi
Host(s)
Humans and other mammals
Humans and other mammals
Dogs
Hoofed animals
Cats
Rodents
Marine mammals
Amphibians
Rodents
Birds
Birds
Rodents
Human infections of assemblage B (~58% of the cases) are more common
worldwide compared to assemblage A (~37%). Interestingly, this proportion
is not altered when comparing data from developing and developed countries
but the prevalence of mixed infections is higher (~5%) in the developing
countries (Ryan and Cacciò, 2013).
15
A link between different genotypes and phenotypes has been under investigation since the early 1990s. The pathogenicity was significantly higher in
experimental infections with assemblage B (isolate GS/M) than assemblage
A (Isr isolate) in human volunteers (Nash et al., 1987). Other studies showed
a significant link between symptomatic disease and assemblage B (Gelanew
et al., 2007) and an association between development of persistent diarrhea
and assemblage B (Homan and Mank, 2001). In contrast, another study associated assemblage A to have the highest probability to develop symptoms,
even though assemblage B was more prevalent and exhibited higher parasite
burden (Haque et al., 2005). This indicates that sub-assemblages might behave differently in hosts and therefore further studies are needed to connect
genotypes and disease phenotypes. Moreover, the development of giardiasis
is complex and also different host factors might influence how disease progression develops (see p. 31). The possibility to establish axenic in vitro
cultures also differs between assemblages (Adam, 2001). Assemblages B
cultures grow typically slower in vitro compared to assemblage A isolates
(Karanis and Ey, 1998).
Assemblages A and B are regarded to have zoonotic potential, but many
of the assemblages found in animals are not genetically identical to those of
human origin. More sensitive multi-locus typing strategies would be needed
to further investigate this matter (Ryan and Cacciò, 2013).
The life cycle of Giardia
The ability to spread and establish infection of the host is a key characteristic
of a successful parasite. To do this, parasites need to survive outside of their
hosts and to be able to switch between different life forms (differentiate).
Many parasites (and also free living organisms) have the ability to exit the
proliferative cell cycle and transform into dormant stages.
Giardia has a simple life cycle with two main stages; the proliferating trophozoite and the infectious cyst. There are also two intermediate stages, the
encyzoite and the excyzoite, that only exist during the differentiation steps,
encystation and excystation. Giardia infection is initiated by ingestion of
infectious cysts, which are stimulated to excyst by the acidic milieu in the
stomach and presence of bile and trypsin in the duodenum (Ankarklev et al.,
2010). The emerging excyzoites quickly transform into trophozoites that
attach to the intestinal epithelial cells where they proliferate and cause disease. Encystation starts as the trophozoite sense a change in the environment
as the cell is transported further down in the small intestine. The cell responds by forming a cyst wall that enables the parasite to survive outside the
host for several weeks in cold water (Ankarklev et al., 2010). The encystation process is essential for transmission and survival of the parasite.
16
I will give further details of the different life stages and the differentiation
process in the sections that follow.
The trophozoite
The photogenic trophozoite of G. intestinalis has the shape of a flattened
teardrop and is approximately 12-15 µm long and 5-9 µm wide and 5 µm
thick. Trophozoites have two nuclei positioned anteriorly and they are equal
in size. The cytoskeleton involves a median body and four pairs of flagella
that behave differently during motility. The ventral disc is the perhaps most
remarkable feature of these cells and this organelle mediates attachment to
different surfaces (Figure 2).
The cytoskeleton
The cytoskeletons of many parasites (e.g. apicomplexans and trypanosomes)
are highly specialized to facilitate their complex life cycles and vital for retaining the cell shape and integrity during infection (Frénal and SoldatiFavre, 2009; Gull, 1999). The cytoskeleton of Giardia is mainly built of
complex microtubule structures. The microtubule cytoskeleton consists of
structures commonly found in flagellated protists (eight flagella and two
mitotic spindles). It also possesses unique structures such as the ventral disc,
median body, funis and axoneme-associated elements (Dawson, 2010).
The ventral disc is used for attachment to the intestinal microvilli (or inert
surfaces) thereby resisting the peristaltic flow of the intestine and allowing
colonization. The ventral disc is considered a virulence factor due its importance for infection. The disc consists of a highly ordered and complex
spiral microtubule array with microribbons that extend along the spiral and
crossbridge structures linking the microribbons together (Dawson, 2010).
Surrounding the disc is another highly ordered structure known as the lateral
crest. It is a “bare area” region located in the center of the disc structure,
lacking microtubules and containing membrane bound vacuoles (Figure 2B)
(Dawson, 2010).
Attachment and detachment are rapid processes that occur within seconds
in vitro. The mechanism of attachment by Giardia has been under investigation since the 1970s. An early theory suggested that the beating of the ventral flagella pair generate a hydrodynamic force that result in a suction-based
attachment of the disc (Holberton, 1974). The attachment was later studied
in detail and is a stepwise process (House et al., 2011). The trophozoite
skims the surface and first creates contact with the surface using the ventrolateral flange (Figure 2B). Thereafter the lateral crest forms a continuous
seal with the surface. The lateral shield then forms an increased connection
and lastly the bare area makes contact to the surface. Only the lateral crest
and the bare area make contact to the attached surface whilst the disc re17
mains dome-shaped (House et al., 2011). Morpholino-based knockdown of
the median body protein resulted in a flattened disc structure and weakened
the attachment ability of Giardia in vitro highlighting the importance of the
dome-shape for proper attachment (Woessner and Dawson, 2012). Several
additional disc-associated proteins have been identified using a proteomics
approach (Hagen et al., 2011).
Giardia uses flagellar motility to find suitable sites for attachment and flagella are hence important for parasite survival. The cell division and cytokinesis also require flagellar beating for completion (Dawson and House,
2010). The trophozoite has eight flagella (9+2 microtubule arrangement)
organized into four bilaterally symmetrical flagellar pairs; the anterior, the
caudal, the posterolateral and the ventral pair (Figure 2B). The beating of
these four pairs produces complex movements that are used for positioning
and orientation prior to attachment (Dawson, 2010). However, the flagellar
beating itself is not necessary for maintenance of attachment of the parasite
(Hagen et al., 2011). The flagella originate from eight basal bodies positioned in close proximity to the nuclei (Dawson and House, 2010). During
mitosis the basal bodies appear to be centers for signaling (Aurora kinase)
(Davids et al., 2008). The flagella are reorganized during cell division in a
highly complex manner and the newly divided trophozoite inherits four parental and four newly synthesized flagella (Nohynková et al., 2006).
The median body is another microtubule structure of this parasite and is responsible for the characteristic “crooked smile” in Giardia images. The
structure has unknown function but is hypothesized to have a role in ventral
disc biogenesis (Sagolla et al., 2006).
Another structure with unknown function is the funis, but it has a suggested role in maintaining the cell shape and a special movement (the dorsal
tail flextion). This movement can facilitate detachment by lifting the dorsal
part of the cell (Benchimol et al., 2004).
18
Figure 2. Schematic overview of the Giardia trophozoite characteristics are described in (A). The two nuclei have a ploidy of 2N, are equal in size and both transcriptionally active. The basal bodies are placed between the nuclei as well as the
central mitosomes (black). Mitosomes are also scattered in the cytoplasm. The peripheral vesicles (PVs) are situated close to the plasma membrane for transport and
uptake of material. The ventral surface of the cell is shown in (B) with the characteristic ventral disc positioned in the anterior part of the cell. The attachment of the
trophozoite is aided by ventrolateral flange, lateral crest, lateral shield and the bare
area. The four flagellar pairs are used for motility.
The genome of Giardia contains only a single very divergent actin gene and
lacks the core set of actin-binding proteins (Morrison et al., 2007). However,
the actin forms filaments and the actin cytoskeleton are required for membrane trafficking, cytokinesis and cellular morphology (Paredez et al., 2011).
Annexins are found across eukaryotes and they provide a link between lipid
membranes and the cytoskeleton as they bind phospholipids in a Ca2+ dependent manner (Hofmann et al., 2010). Giardia possesses an expanded
Annexin-like gene family (the α-giardins), which are associated with the
cytoskeleton. The giardins is a Giardia-specific cytoskeleton family and they
are divided into α-giardins, striated fiber (SF) assemblins (β- and δ-giardin)
and γ-giardin. The β- and δ-giardin are associated to the ventral disc (Palm et
al., 2005). γ-giardin has no other known homologs and is a novel Giardia
protein (Nohria et al., 1992).
The α-giardins are diverged annexin homologs and 21 members can be
found in the genome of Giardia intestinalis. They are numbered α-1-19 with
α-7 existing as three variants: α-7.1, α-7.2 and α-7.3 (Weiland et al., 2005).
19
Some are clustered on the same chromosome and likely results of gene duplication events and subsequent divergence (Weiland et al., 2005). Several of
the α-giardins have been found to be immunodominant and recognized by
human serum from giardiasis patients; among them the plasma membrane
localized α-1-giardin (Weiland et al., 2003; Wenman et al., 1993). Alpha-1
giardin binds to glycosaminoglycans (GAGs), implicating that this surfaceassociated protein has the potential to interact with membranes of the host
cells (Weiland et al., 2003). In addition, α-1 giardin has been found on the
surface of excyzoites and hence was tested as a vaccine antigen candidate
successfully in murine models (Jenikova et al., 2011; Weiland et al., 2003).
Characterization studies have shown the localizations of other α-giardins
to different cytoskeletal structures such as the flagella, lateral crest and the
plasma membrane (Kim et al., 2013; Saric et al., 2009; Vahrmann et al.,
2008; Weiland et al., 2005). Recently the protein structures of α-11, α-14
and α-1 giardin were solved (Pathuri et al., 2007, 2009; Weeratunga et al.,
2012). Overall the three proteins have the typical Annexin fold but they differ in the calcium coordination scheme both among themselves as well as to
annexins of other eukaryotes (Weeratunga et al., 2012). Interestingly, the
binding capacity of α-1 giardin to phospholipids is regulated by calcium. At
high calcium levels the interaction is disrupted. It has been hypothesized that
α-1-giardin binds to host cells, awaiting the assembly of the ventral disc.
Changes of calcium levels in the duodenum would allow detachment caused
by structural changes of α-1 giardin and progression of trophozoite formation (Weeratunga et al., 2012). However, this theory needs experimental
validation.
The Annexin-like gene family of S. salmonicida is characterized in Paper
III.
Protein transport systems
As mentioned, many processes and classical eukaryotic features are absent
or minimalistic in Giardia. Cellular features such as peroxisomes, a Golgi
apparatus and a classical endo-lysosomal system are missing. However,
three giardial membrane systems are recognized; the endoplasmic reticulum
(ER), peripheral vesicles (PVs) and mitosomes (see p. 22) (Faso and Hehl,
2011).
The ER is continuous with the nuclear envelope as in other eukaryotes
and extends bilaterally throughout the cell body (Soltys et al., 1996). Current
information suggests that the ER, together with ESVs during differentiation
(see p. 28), is solely responsible for the secretory system.
Giardia possesses members of the core machinery of membrane transport
such as three coat complexes (COPII, clathrin and COPI) and two adaptor
proteins (AP) complexes. In addition, few Rab GTPases and SNAREs (soluble N-ethyl-maleimide-sensitive factor attachment protein receptors) exist
20
indicating limited interaction of membranes or organelles (Marti et al.,
2003a; Morrison et al., 2007).
Glycosylation is a common post-translational modification of eukaryotic
proteins and it involves the ER and Golgi compartments. In Giardia only
addition of N-acetyl glucosamine(s) in the ER has been described (Robbins
and Samuelson, 2005).
There are no lysosomes or peroxisomes in Giardia. The PVs have dual
roles of both performing endo- and exocytosis. The vesicles are underlying
the plasma membrane on the dorsal side and in the bare area (Figure 2A).
The PVs appear to have lysosomal properties and contain hydrolases and
proteases (McCaffery and Gillin, 1994; Thirion et al., 2003). The uptake of
nutrients from the environment is believed to be an important additional role
of the PVs. The mechanisms behind the uptake and transport back to the ER
remain to be explored (Wampfler et al., 2014). Since the Golgi apparatus is
absent, the secreted proteins are directly transported from the ER via secretory vesicles (the PVs) to their final destination (Marti et al., 2003b). Secreted
proteins are targeted by N-terminal signal peptides, but in the case of variant-specific surface proteins (VSPs) they are combined with signals from a
semi-conserved C-terminal region (Faso and Hehl, 2011). There are probably alternative secretion signals since trophozoites are known to secrete metabolic enzymes upon interaction with host cells, but the pathway behind the
export of these proteins is currently unknown (Ringqvist et al., 2008).
The nuclei
A peculiarity of Giardia and other diplomonads is that they are binuclear.
The nuclei both have the same size and are transcriptionally active (Kabnick
and Peattie, 1990). However, the amount and clustering of nuclear pores are
different for each nucleus within the same cell. This indicates that the nuclei
are not functionally equal (Benchimol, 2004).
The genome content of each nucleus cycles between 2N and 4N in the
proliferating trophozoite stage (Bernander et al., 2001) i.e. the cell contains
four to eight copies of the genome. The nuclei replicate almost synchronously (Wiesehahn et al., 1984) and with semi-open mitosis leading to each
daughter cell inheriting one copy of each parental nucleus (Sagolla et al.,
2006; Yu et al., 2002). Thus the nuclei remain independent during mitosis in
trophozoites, in contrast to division during differentiation (see p. 30). G.
intestinalis (strain WB) possesses five chromosomes and each nucleus contains at least one of each (Morrison et al., 2007; Yu et al., 2002). It has been
shown that the number and size of chromosomes can differ among G. intestinalis isolates. The number of chromosomes could differ from nine to eleven per nucleus (Tůmová et al., 2007). Taken together it is now clear that the
two nuclei are not exactly identical and could differ in behavior.
Genetic manipulations of Giardia has been challenging due to its tetraploid status. When introducing circular DNA as plasmids to parasites of the
21
isolate WB, they are maintained as episomes and linearized DNA is inserted
in the chromosomes by homologous recombination. Isolate GS (assemblage
B) fails to maintain episomes and integrate introduced DNA into the chromosome (Singer et al., 1998). Moreover, episomes are only present in one of
the nuclei (Poxleitner et al., 2008).
The mitosome
Mitosomes are highly reduced forms of mitochondria that do not contain any
genome and have lost the capacity to generate energy (Makiuchi and Nozaki,
2014). They have, however, retained some features known to mitochondria
such as presence of a double-membrane, synthesis of iron-sulphur (FeS)
clusters and requirement of translocation signals for import of mitosomal
proteins (Dolezal et al., 2005; Tovar et al., 2003). In addition to Giardia,
mitosomes can be found in several parasitic protists living in oxygen-poor
environments e.g. Cryptosporidium parvum (Riordan et al., 1999), Entamoeba histolytica (Mai et al., 1999; Tovar et al., 1999) and the Microsporidia
(Williams et al., 2002).
The discovery of a Cpn60 in the genome was the first evidence for a
MRO in Giardia (Roger et al., 1998). The discovery of the actual organelle,
the mitosome, revealed that they are tiny (~100 nm) and ranging in number
from 25-100 per cell (Tovar et al., 2003). They appear to be in two forms;
peripheral mitosomes are scattered in the cytosol and several tightly packed
mitosomes lined in between the nuclei (Figure 1). The central mitosomes
migrate with the basal bodies of the caudal flagella during mitosis (Regoes et
al., 2005).
The genome of the protomitochondria, most likely a α-proteobacteria, has
partly been transferred into the host’s nucleus during evolution and hence
protein import pathways were created. The size of the proteome of the tiny
mitosome is difficult to estimate but 20 proteins have been verified as mitosomal in the first proteomics study (Jedelský et al., 2011). A targeting signal,
present at the N-terminal or internally, ensure that nuclear encoded proteins
are delivered to the organelle (Dolezal et al., 2006). Only few mitosomal
proteins appear to have N-terminal pre-sequences, but have been found and
verified experimentally for IscU, IscA and ferredoxin. The targeting peptides
are very short (10-18 amino acids) and are cleaved by the giardial mitosomal
processing peptidase (MPP) upon arrival to the organelle (Dolezal et al.,
2005; Regoes et al., 2005). Thus the giardial MPP is likely only needed for
processing few mitosomal proteins (Smíd et al., 2008). Additional mitosomal proteins appear to have internal localization signals such as IscS, Cpn60
and mtHsp70 (Dolezal et al., 2005; Regoes et al., 2005).
Import of proteins across double-membraned organelles is executed by
protein complexes. The translocation requires stored energy in the form of
membrane potential (Dolezal et al., 2006). The import machinery found in
giardial mitosomes is very reduced but known components are porin Tom40
22
of the outer membrane (Dagley et al., 2009) and inner membrane translocases Pam18 and Pam16 (Jedelský et al., 2011). Recently, the translocase of the
inner membrane, a highly divergent Tim44, was elegantly discovered utilizing a compartment-specific biotinylation strategy (Martincová et al., 2015).
Applying the same technique, a mitosomal outer membrane protein 35
(MOM35) and 13 additional mitosomal proteins were discovered with unknown function (Martinkova, 2015).
The formation of Fe-S clusters is mediated by the Fe-S cluster assembly
complex that facilitates the maturation of Fe-S proteins. The mitosomes contain all key components of the assembly complex, namely IscS, IscU, IscA,
ferredoxin, glutarredoxin and Nfu (Jedelský et al., 2011). More direct studies
of the complete proteome of this organelle will reveal additional pathways
and functions.
The MROs of S. salmonicida is described and characterized in Paper II.
Energy metabolism
Giardia has a minimalistic metabolic capacity as many other microaerophilic
parasites. The parasite lacks pathways for de novo biosynthesis of pyrimidines and purines and depends on the host for nucleotide salvage (Morrison
et al., 2007). Most metabolic enzymes are soluble and present in the cytosol,
thus they are not sub-compartmentalized. Trophozoites mainly use glycolysis and arginine dihydrolase pathways for energy production.
The preferred sugar glucose is converted into pyruvate and end products
of glucose catabolism are acetate, ethanol, alanine and CO2. However, small
changes in oxygen concentration can affect the metabolism of trophozoites
and influence the end product formation (Adam, 2001). Despite anaerobic
metabolism, oxygen radicals are generated and a detoxification mechanism
is necessary. Moreover, the intestinal environment fluctuates in oxygen levels and reactive oxygen species (ROS) are generated by the host (Adam,
2001). The oxygen-sensitive parasite lack conventional ROS scavenging
pathways such as catalase and superoxide dismutase systems. Instead Giardia depends on thioredoxin-like proteins, NADH oxidase, flavodiiron protein, flavohemoglobin and superoxide reductase for detoxification.
Arginine is used as an energy source for the parasite and is imported into
the cell via an arginine-ornithine transporter. The arginine dihydrolase pathway consists of three enzymes; arginine deiminase (ADI), ornithine carbamoyltransferase (OCT) and carbamate kinase (CK) (Brown et al., 1998). It
has been estimated that generation of ATP is faster utilizing arginine compared to glucose. The arginine hydrolase pathway is rarely found in eukaryotes, but has been reported for T. vaginalis (Brown et al., 1998). Giardia
scavenges most amino acids and especially cysteine have been noted to be
23
needed for in vitro growth (Adam, 2001). Cysteine is abundant in VSPs and
also provides additional protection from oxygen (Adam, 2001).
De novo synthesis of lipids and fatty acids were believed to be absent in
the parasite. Recent findings based on genomic data, support that some remodeling or synthesis of some lipids are possible (Yichoy et al., 2011). Lipid
metabolism changes during encystation and the sphingolipid pathway is
differentially expressed (Sonda et al., 2008).
Differentiation
Cells must constantly monitor their surroundings and respond to changes
accordingly to survive. Giardia must go through two differentiation steps
during its life cycle; the trophozoite formation (excystation) and cyst formation (encystation) (Figure 3).
Proliferating trophozoites attached to enterocytes are covered by a mucus
blanket and surrounded in a nutrient-rich environment with near neutral pH
and low bile concentration. Detached trophozoites are subjected to the intestinal lumen were the environment varies depending on location and host
nutrition. However, the pH is slightly alkaline, oxygen levels lower and the
bile concentration is higher.
It is possible to study the entire life cycle of Giardia in vitro and several
protocols have been developed for this purpose. The initial work towards an
in vitro protocol was performed using infected mice to determine where in
the gut cyst forms were highly abundant and thereby predicting important
biological signals (Gillin et al., 1987). Cysts were found in the mid to lower
part of the jejunum early in infections and in the large intestine and cecum in
later stages (Gillin et al., 1987). By increasing the amount of primary bile
salts in the growth media, encystation was induced in vitro. This method was
later revised (Boucher and Gillin, 1990) and is now the most used encystation protocol throughout the last years. It is commonly known as the 2-step
protocol; trophozoites are starved from bile in a pre-encystation media (pH
7) followed by incubation in encystation media containing high bile concentration and lactic acid with pH 7.8 (Boucher and Gillin, 1990). Several other
protocols have been developed, since but all share the features of lipid starvation and an elevated pH, which are likely environmental changes the parasite encounters on the journey through the intestine (Kane et al., 1991; Luján
et al., 1996; Sun et al., 2003). The lipid starvation is accomplished using
different strategies, either by increasing the bile concentration (Kane et al.,
1991) or using delipidated serum (Luján et al., 1996). The efficiency of in
vitro encystation varies between methods and between laboratories, reflecting the complexity of this process.
Most prominent in the transformation from trophozoite to cyst is the formation of the 300 nm thick cyst wall. This protective layer consists of pro24
teins and the carbohydrate β(1-3)-N-Acetyl-D-galactosamine (GalNAc). The
cyst wall proteins are transported in encystation-specific vesicles (ESVs) and
differentiation is often divided in an early and late phase based on the progression of cyst wall synthesis (Figure 3). Cysts secreted in fecal material
can survive for several weeks in cold water (Olson et al., 2004).
Figure 3. The life cycle of Giardia is divided into two differentiation steps; encystation and excystation. The water resistant infectious cyst is ingested by the host and
excystation is triggered in the stomach. The excyzoite is released from the cyst and
quickly undergoes cytokinesis and assemble the adhesive disc. The trophozoite is
replicating in the small intestine and cause disease. Environmental changes trigger
the trophozoite to start encysting. There is a “point of no return” in the process after
which the cell cannot revert back to proliferation. This differentiation process is
divided into early and late phases. Cyst wall material is transported in ESVs (green)
and ECVs (purple). The ventral disc and flagella are internalized as the cell round up
and enter dormancy. An intermediate pre-cyst stage with single flagella is commonly seen in vitro. DNA replication occurs late in encystation, giving the mature tetranucleated cyst a final ploidy of 16N.
Induction of encystation
It is still unknown what stimuli are required for initiation of encystation in
the host. However, to induce encystation in vitro it is not sufficient to only
increase the pH or bile concentration of the growth media (Gillin et al.,
25
1987; Morf et al., 2010). Hence, both these two signals are necessary, but
little is known how the intracellular signaling is activated and mediated.
Intracellular signaling in eukaryotes are often controlled and mediated via
MAP (mitogen activated protein) kinases (English et al., 1999). Giardia
possesses two homologs of ERK1 and ERK2 (extracellular mitogen regulated kinases 1 and 2), which localize to different structures. ERK2 localizes to
nuclei and caudal flagella in trophozoites and appear cytoplasmic in encysting cells, whereas ERK1 seems associated to the median body, basal bodies
and lateral crest of the disc (Ellis et al., 2003). However, no transcription
factors have been shown to be regulated by these ERK proteins in Giardia
and it remains to be investigated if the putative MEK and MEKK are involved in activating the ERK1/2 proteins (Ellis et al., 2003). Several studies
on individual signaling proteins have been performed (Bazán-Tejeda et al.,
2007; Ellis et al., 2003; Gibson et al., 2006; Kim et al., 2005; Lauwaet et al.,
2007), but much remains to be explored to elucidate the complete signaling
pathway for this parasite.
The commitment to differentiate into a cyst appears to reach a “point of
no return”. When the induction signal has been present for 3-6 hours, the
trophozoites cannot revert back to proliferation (Sulemana et al., 2014).
Slightly later (5-8 hours) encystation specific vesicles (ESVs) formation is
prominent among trophozoites in the population (Faso et al., 2013a; Hehl et
al., 2000; Morf et al., 2010), proposing that when cyst wall production has
reached a certain stage, the cells will complete the encystation.
The encystation response is heterogeneous in the population in vitro;
some cells spontaneously form cysts even in absence of encystation media,
whereas some will never induce the process. Experiments with gerbils infected with Giardia of the isolate WB, showed that trophozoites and encysting cells were distributed evenly in the small intestine (Erlandsen et al.,
1996). This suggests that there are a variety of cell stages in vivo as well.
Moreover, changes of encystation efficiency have been observed among
different clones of the isolate WB (Erlandsen et al., 1996; Kane et al., 1991).
Adding to the complexity, parasites from assemblage A have greater success
in growth and differentiation in vitro compared to parasites from other assemblages (Ankarklev et al., 2010). However, these isolates can grow and
differentiate better in vivo. All differentiation studies completed so far have
used the isolate WB (assemblage A). Much more could be learned regarding
initiation factors and signaling from studying parasites from different assemblages both under in vivo and in vitro settings.
Transcriptional response during encystation
The promoter regions in Giardia are short and A/T rich without any detectable TATA box that initiates transcription (Adam, 2001). Only four of the
twelve general transcription factors have been found (Morrison et al., 2007).
The first giardial transcription factor studied in detail was a Myb-like protein
26
(Giardia Myb). It regulates key genes such as the cwp1-3 and g6pi-B, an
important enzyme in the synthesis of GalNAc sugars in the cyst wall, as well
as myb itself (Sun et al., 2002).
Other transcription factors have been identified and primarily reported to
regulate the expression of CWPs (Chuang et al., 2012; Lin et al., 2013; Su et
al., 2011; Sun et al., 2006; Wang et al., 2007; Worgall et al., 2004). Further
studies are needed to investigate how these interact to modulate the massive
induction of the mentioned genes.
To date two studies have been performed to investigate the transcriptional
response during encystation (Birkeland et al., 2010; Morf et al., 2010). The
first study used SAGE technology to study the entire life cycle (Birkeland et
al., 2010). Only 42 genes were found upregulated during encystation; the
three cyst wall proteins (CWP1-3), genes for UDP-GalNAc synthesis, the
excyzoite surface protein HCNCp and 13 hypothetical proteins (Birkeland et
al., 2010). In the other study, only the first seven hours of the process were
investigated using microarrays. Two encystation protocols (2-step and
delipidated serum) were used in the study to reduce any off-target effects.
The commonly used two-step protocol induced 29 genes and the cholesterolfree serum method 37 genes, generating a core set of only 18 genes induced
early in encystation (Morf et al., 2010). At least one binding site for the
GiMyb was found among the regulated core genes, suggesting this to be a
signature motif of encystation genes (Morf et al., 2010).
The transcriptional response of the entire encystation process and potential regulators are described in Paper V.
The regulation of gene expression is likely to occur at many different levels
during differentiation, since so few transcription factors have been identified.
The details of this regulation have largely remained incomplete up to date.
Epigenetic changes contribute to the differential gene expression as histone acetylation decrease during encystation (Sonda et al., 2010). Modifying
the histone acetylation levels by a histone deacetylase (HDAC) inhibitor
results in repression of encystation-specific genes and blocks cyst formation
(Sonda et al., 2010). This highlights the importance of chromatin structure
for regulation of the transcriptional response during differentiation. Further
studies could reveal more details in the interplay of epigenetic markers and
gene regulation.
RNA helicases can interact with HDACs in higher eukaryotes and Giardia possesses several SF2 RNA helicases. The expression levels of two of
these increase during encystation and could potentially participate in posttranscriptional silencing through interaction of the RNAi pathway (Gargantini et al., 2012). RNAi and/or microRNAs appear to regulate the antigenic
variation and recently the regulatory role of small RNAs (sRNAs) in encystation was investigated (Liao et al., 2014; Prucca et al., 2008; Saraiya et al.,
2011). Deep sequencing showed increasing levels of endogenous small inter27
fering (siRNA) originating from telomeric retrotransposons and from clusters in the genome during encystation (Liao et al., 2014). None of the targets
predicted for the siRNAs have been experimentally validated. In addition,
tRNAs derived siRNAs increased during late encystation but their function
in the process requires further studies.
The proteome during encystation
The encystation process has been subjected to proteomic analyses as an important complement to the available transcriptional data. The first 14 hours
of encystation have been investigated during which the ESVs develop and
mature (Faso et al., 2013a). The proteome appears to be overall robust, since
many proteins were overlapping between the selected time points. The largest changes were reported for the early time points as several proteins implicated in metabolic pathways were differentially expressed (e.g. protein folding, cytoskeleton regulatory components and Nek kinases). Indications of
that the VSP diversity is affected during encystation were found. The poorly
defined high cysteine membrane proteins (HCMPs) were another group of
secreted proteins that changed during encystation (Faso, 2013). Possibly
these cysteine rich surface proteins share a regulatory mechanism during this
process. Only modest changes were reported for the later time points (8-12
h) and proteins in carbohydrate biogenesis pathways were enriched (Faso et
al., 2013a).
To date no proteomic study exits that includes the complete transformation from trophozoite to mature cyst. This, together with analyses of posttranslation modifications and a completer picture of encystation-induced
gene expression changes should be generated.
The building of the cyst wall
Massive amounts of cyst wall material is secreted from the ER and transported to the plasma membrane during encystation. To facilitate the transportation, encystation-specific vesicles (ESVs) are formed and this process
has been studied extensively (Faso and Hehl, 2011; Lujan, 2011) since its
discovery (Gillin et al., 1987). The water-resistant cyst wall consists of ~40
% protein and ~60 % of the carbohydrate GalNAc (Gerwig et al., 2002).
There are three main cyst wall proteins (CWP1-3) and they share several
features (Luján et al., 1995; Sun et al., 2003). They all have a central leucine-rich repeat (LRR) regions important for sorting into the ESVs and the
N-terminal signal peptides to direct them to the secretory pathway (Lujan,
2011). The C-terminal region contains a cysteine-rich domain and CWP2
differs from the others by carrying a basic extension in this region (Luján et
al., 1995). A peak in expression on the mRNA level of the CWPs occurs at
approximately 7 hours post-induction, but the newly synthesized proteins are
accumulated in the ER after about two hours post-induction. It appears likely
28
that the cargo of ESVs is sorted in the ER prior to export, since within ESVs
exclusively cyst wall material is found (Konrad et al., 2010).
Giardia lacks a constitutively expressed Golgi apparatus or any other stable delay compartment for secretory cargo. However, the features of ESVs
indicate that these organelles function as stage induced cis Golgi. The neogenesis of ESVs depends on the small GTPase Sar1 and COPII coat formation (Stefanic et al., 2009). Expression of non-functional Sar1-GTPase
variants lead to impaired ESV biogenesis caused by ER exit site (ERES)
collapse. It was further shown that nascent ESVs co-localize with ERES and
that CWP1 depends on these sorting stations for accurate trafficking (Faso et
al., 2013b). This strongly suggests that these vesicles are Golgi-like.
When the ESVs are formed, they are delayed for several hours before secretion on the cell surface. This is presumably to allow post-translational
modifications to the CWPs such as formation of disulfide bonds, isopeptidelinkages and addition of phospho-groups (Davids et al., 2004; Reiner et al.,
2001; Slavin et al., 2002). The CWP2 basic extension is modified by proteolytic processing as part of the maturation process. The processed CWP2
forms a condensed core together with CWP3, while the N-terminal of CWP2
and CWP1 remain in fluid state until secretion. The dense core is secreted
over several hours and the fluid part is secreted rapidly (Konrad et al., 2010)
indicating the complexity of the cyst wall assembly.
Apart from CWPs, further proteins have been localized to the ESVs. The
high-cysteine non-variant cyst protein (HCNCp) is one of them and localizes
to the surface of the excyzoite in the cyst (Davids et al., 2006). The tenascinlike proteins are also cysteine-rich and appear to localize to the cyst wall and
the excyzoite surface (Chiu et al., 2010).
Much less is known about the transport and incorporation of the UDPGalNAc sugar into the cyst wall. The Giardia-unique carbohydrate is synthesized via an inducible pathway consisting of five enzymes (Lopez et al.,
2003) using glucose as the starting substrate. The final step in the synthesis
is polymerization of the UDP-GalNAc and responsible is the “cyst wall synthase” (Karr and Jarroll, 2004). The gene product responsible for this activity
has not been identified yet, but activity measurements exits that verify its
existence.
The structure of the homopolymer of GalNAc appears as curled fibrils
compressed by the CWPs. Both CWP1 and 2 can bind the fibrils and the
LRRs of CWP1 have been reported to have lectin binding properties (Chatterjee et al., 2010). The lectin binding properties of CWP1 were exploited to
visualize the GalNAc homopolymer in small vesicles of encysting parasites.
These vesicles did not co-localize with ESVs suggesting another transportation pathway for the carbohydrate portion of the cyst wall (Chatterjee et al.,
2010). A recent study reported the presence of encystation carbohydratepositive vesicles (ECVs) (Midlej et al., 2013). To clarify how these vesicle
29
types interact upon cyst wall assembly and the mechanism behind it, additional studies are needed.
The nuclei fuse during late encystation
The trophozoite alternates between a tetraploid (2x2N) and octaploid (4x2N)
genome content during proliferative growth. The G2 phase is the longest
during the cell cycle and contains the restriction point to start differentiating
into the cyst form (Bernander et al., 2001; Reiner et al., 2008). During encystation, division of the two nuclei without cytokinesis occurs, forming a
pre-cyst with ploidy of 4x2N. Another round of replication give rise to the
final ploidy of 16N (4x4N) in the mature cyst. The excyzoite goes through
cellular division twice without DNA replication during the excystation process, giving rise to four trophozoites (Bernander et al., 2001).
Giardia is considered to be an asexual organism as no direct evidence of
cellular fusion has been observed. However, several homologs to genes involved in meiosis exist in the genome (Ramesh et al., 2005). In addition, the
allelic sequence heterozygosity (ASH) in the genome of WB is low, which is
surprising for an asexual organism that lacks control of differences between
the nuclei (Jerlström-Hultqvist et al., 2010; Morrison et al., 2007). The nuclei do not fuse during mitosis in trophozoites, hence the cyst nuclei were
put into focus. Indeed, the nuclei were shown to fuse in the cyst stage and
genetic material in form of episomal plasmids are transferred between them
(Poxleitner et al., 2008). This process is known as diplomixis and three meiotic genes were reported to be expressed late in encystation to facilitate homologous recombination (Poxleitner et al., 2008). Later it was shown that
also chromosomally integrated markers were exchanged between cyst nuclei
(Carpenter et al., 2012). Integrated markers were followed throughout differentiation by FISH from a clonal starting population where 76 % carried the
marker in one nucleus. After encystation, excystation and growth to confluency, the resulting trophozoites carried different amount of the marker with
one spot (43%), no spot (31%) and two spots (20%). The genetic exchange
of chromosomal material appears to be high between the nuclei in
cysts/excyzoites (Carpenter et al., 2012). Further it was shown that the
daughter trophozoites inherit one copy each of the parental nucleus after
excystation. Thus, the tethered chromosomes can recombine with those from
a neighboring nucleus and thereby diplomixis could be responsible for reducing the ASH between nuclei (Carpenter et al., 2012).
An additional study (Jiráková et al., 2012) showed that nuclear division in
the cysts occurs by semi-open mitosis and results in four daughter nuclei
from two non-sister pairs. The nucleus pairs are connected via their nuclear
envelope that remains in excyzoites. After excystation and final division,
each trophozoite inherits one pair of non-sister nuclei, consistent with (Carpenter et al., 2012)
30
Excystation
Cysts are excreted in the feces and spread through the environment to a new
host. Upon ingestion the acidic milieu in the stomach of the newly infected
host triggers excystation. Cysts are shortly translocated to the duodenum
were gastric acid is rapidly neutralized by bicarbonate (Boucher and Gillin,
1990). The exact site of excystation is not known, but presumed to be in the
upper small intestine. The excyzoite must quickly emerge from the cyst; reassemble the adhesive disc and flagella to avoid being swept away from the
site of infection (Boucher and Gillin, 1990). Emerging trophozoites also
undergo cytokinesis and become metabolically active (Bernander et al.,
2001). Hence, the process needs to be precisely timed but the mechanisms
behind signal transduction are not fully discovered. However, calcium signaling has been shown to influence the process as inhibitors of calmodulin
and protein kinase A block excystation (Reiner et al., 2003).
The in vitro protocol for excystation is carried out in two steps; cysts are
induced in an acidic solution (pH 4) followed by a transfer to a solution containing bicarbonate, trypsin and a basic pH of 8 (Boucher and Gillin, 1990).
Few details of this very rapid differentiation process are known. The cyst
wall is disrupted by release of a cysteine protease (Ward et al., 1997) and
acidic phosphatases (Slavin et al., 2002). The flagella of excyzoites emerge
from one of the poles of the cyst followed by release of the entire cell.
Excystation specific genes have been found using SAGE technology (Birkeland et al., 2010) but has not been studied further.
Giardia pathogenesis
Diarrheal disease is the leading cause of death and illness for children under
five years of age in developing countries (Kotloff et al., 2013). G. intestinalis is distributed worldwide and estimated to cause 280 million symptomatic infections per year (Lane and Lloyd, 2002). Giardiasis is part of the
WHO’s Neglected disease initiative since 2004 (Savioli et al., 2006). Transmission of the parasite is via the fecal-oral route, most often by contaminated
water and food. The parasite is commonly found in many developing countries which are considered to be endemic regions. Contaminated water is the
most common infection route in developed countries and local outbreaks
occur (Baldursson and Karanis, 2011).
The two assemblages A and B are responsible for human infections (see p.
15) causing acute to persistent symptoms. However, there are also cases of
asymptomatic patients and patients that develop chronic disease. Very little
is known about the mechanisms accountable for this spectrum of symptoms.
The pathogenesis of Giardia is multifactorial reflecting the complex interplay between the host and parasite as discussed below.
31
The disease giardiasis
The infection starts as the host ingests cysts and trophozoites attach to the
intestinal epithelial cells in a non-invasive manner. Symptoms of disease
typically manifests after approximately 6-15 days (Ankarklev et al., 2010).
The most common clinical signs of infection are diarrhea (with or without
malabsorption syndrome), nausea and weight loss (Robertson et al., 2010).
Some patients experience mild illness that resolves spontaneously. Others
suffer from long-lasting severe disease that does not respond to the normal
treatment. Most patients are usually found between these extremes (Robertson et al., 2010). Chronic infections are known to occur and could lead to
complications such as irritable bowel syndrome (IBS) or chronic fatigue
syndrome. Patients infected during a Giardia outbreak in a non-endemic area
were reported to have long-term symptoms after the parasite was cleared
(Hanevik et al., 2014).
As diarrheal disease is a major cause of death in young children in developing countries, many studies have been conducted to elucidate the role of
Giardia in this regard. Reports of both acute or persistent disease as well as
protection against diarrhea exist (Robertson et al., 2010). An extensive metaanalysis has been performed to try and resolve these conflicting findings
(Muhsen and Levine, 2012). There is no statistical evidence of association of
acute Giardia infections in children older than 5years in developing countries but an association was found in children up to 1 year old. Instead a
strong association was seen to persistent diarrhea (≤14 days) among children
in developing countries. Support was found that citizens of industrialized
countries that encounter the parasite in a waterborne outbreak or during travels to endemic areas are prone to develop acute diarrheal disease (Muhsen
and Levine, 2012). Explanations such as nutrition status, age, immune status
and differences in microflora of the small intestine (Muhsen and Levine,
2012; Robertson et al., 2010) could contribute to the differences in disease
outcome observed among individuals from developing versus developed
countries.
The influence of the host’s microflora during Giardia infections is still vastly unknown. A study in mice showed that adult mice with differences in
their microflora varied in their susceptibility to Giardia infections (Singer
and Nash, 2000). Co-infections with Helicobacter pylori and Giardia have
been observed in asymptomatic children (age 1>5) in Uganda (Ankarklev et
al., 2012). C. elegans was recently used as a model system to study effects of
Giardia on the microbiota. Commensal bacteria from humans were exposed
to Giardia and thereafter fed to C. elegans. An increased worm killing was
observed using bacteria exposed to Giardia. This suggests that the parasite
has the ability to affect the host’s microbiota and alter the interactions between them (Gerbaba et al., 2015). Lactobacillus sp. can be used as a probi32
otic and administration to mice before or during Giardia infections reduced
severity and duration of the infection (Goyal et al., 2013; Humen et al.,
2005).
Observed differences in infection outcome could of course also be due the
parasite itself. Few studies exists that links symptoms and different assemblages together in a consistent manner. Recently a correlation between flatulence and infections of assemblage B was found in children (Lebbad et al.,
2011). Giardia uses antigenic variation by switching surface proteins (see p.
34). Possibly different VSPs give rise to different response and symptoms in
the host. More studies are required to further investigate how pathogenicity
differs between assemblages.
Host-parasite interactions
The interactions of intestinal epithelial cells (enterocytes) and Giardia have
mainly been studied in vitro using axenic parasite isolates and intestinal cell
lines. Mice, and to some extent gerbils, are used as in vivo models of the
human infections. Studies in the mouse model sometimes use the rodent
parasite Giardia muris.
How Giardia respond to host cells have only been studied in vitro so far.
Transcriptomes of parasites during interaction are available and revealed
regulation of hundreds of genes including surface proteins, cysteine proteases, oxygen defense proteins and attachment associated proteins (Ma’ayeh
and Brook-Carter, 2012; Ringqvist et al., 2011). The metabolic enzymes
ADI, enolase and OCT are rapidly secreted upon interaction (Ringqvist et
al., 2008). ADI and OCT are part of the arginine dihydrolase pathway, which
is the primary source of energy for Giardia (Schofield et al., 1992). Scavenging of arginine reduces the enterocytes possibility to produce nitric oxide
(NO), which is cytotoxic to the parasite (Eckmann et al., 2000; Ringqvist et
al., 2008; Stadelmann et al., 2012). Elongation factor 1α is secreted and an
immunoreactive protein but its role during interaction is unclear (Skarin et
al., 2011). Cysteine proteases are also secreted upon interaction (Cotton et
al., 2014; Ma’ayeh and Brook-Carter, 2012) and further studies of the secretome will reveal additional released proteins.
As the trophozoites colonize the host induction of apoptosis in enterocytes have been detected (Cotton et al., 2011). Additional evidence from
human biopsies from chronically infected patients exists (Troeger et al.,
2007). Transcriptome studies of host cells during infection under in vitro
settings highlighted induction of various chemokines and apoptosis. In addition, decreased expression of genes responsible for proliferation was found
(Roxström-Lindquist et al., 2005). The cell cycle progression can be inhibited by arginine starvation caused by parasite’s consumption of arginine and
the release of ADI. The decreased rate of cell number turnover might help
the parasite to prolong its colonization (Stadelmann et al., 2012).
33
Tight junctions between the enterocytes create an important selective barrier between the intestinal lumen and underlying tissue. Giardia increases
the permeability by altering the proteins important for maintaining tight
junctions (e.g. F-actin and zonula occludens-1 (ZO-1)) (Cotton et al., 2011).
Also during chronic human infections an increase of intestinal permeability
was observed and these patients had decreased surface mucosal area
(Troeger et al., 2007). CD8+ T cells have been reported to be responsible for
diffuse microvilli shortening (Scott et al., 2004). Reduction of the brush border villi decrease the absorptive area of the intestine, resulting in malabsorption of water, disaccharides, Na+ and hypersecretion of Cl- (Scott et al., 2004;
Troeger et al., 2007). Immune responses involve both the innate and adaptive systems. Nitric oxide, reactive oxygen species and antimicrobial peptides may give a protective effect. IgA and mast cells are known to contribute to control of the infection (Solaymani-Mohammadi and Singer, 2010).
Treatment
There are several anti-giardial drugs, but the most used are 5-nitroimidazole
compounds such as metronidazole. Metronidazole is a prodrug that needs to
be reduced to become toxic, upon which DNA damage is induced in microaerophilic and anaerobic organisms (Edwards, 1993). An additional target is
the redox enzyme, thioredoxin, whose inactivation causes severe oxidative
stress (Leitsch et al., 2012). There are known cases of treatment failure and
drug resistance and the search for additional drug targets highlighted
(Leitsch, 2015).
Antigenic variation
Infectious microorganisms (bacteria, fungi and protozoans) must colonize
the host at their preferred site of infection, avoid the immune system and
once recognized, be able to avoid being destructed. Many organisms have
developed antigenic variation and change surface markers as a way to avoid
clearance from the host (Deitsch et al., 2009). Plasmodium sp. are protozoan
parasites that invades red blood cells and causes malaria. Plasmodium falciparum expresses highly variable erythrocyte membrane proteins (PfEMP1)
on the surface of the red blood cell to avoid immune clearance. PfEMP1 are
encoded by ~60 var genes, but only one is expressed at the time and this
appears to be mainly epigenetically regulated (Guizetti and Scherf, 2013).
Another protozoan exploiting antigenic variation is Trypanosoma brucei
with a massive vsg repertoire of ~1000 genes. Telomeric position, chromatin
status and gene rearrangements into an active site are features of vsg regulation (Glover et al., 2013).
34
Giardia intestinalis employs antigenic variation for immune evasion using a
large repertoire (150-300 proteins) of surface proteins. The entire surface of
trophozoites, including flagella, is covered by a single variant-specific surface protein (VSP) (Pimenta et al., 1991; Svärd et al., 1998) (Figure 3B).
This dense protein coat is exchanged over time allowing the trophozoite to
avoid antibody recognition and enables re-infection of hosts. Direct exposure
to VSP-generated monoclonal antibodies is cytotoxic to the parasite in vitro
(Nash and Aggarwal, 1986).
The immune response and the antigenic variation interplay have been
studied to some extent during mouse infections. However, a single study
exits in human volunteers that employed the isolate GS (assemblage B).
After ~14 days post-infection the expressed VSPs of the parasites recovered
from the gut had started to diversify. At the same time specific antibodies
were present. After day 21 post-infection, the initial VSPs were completely
replaced (Nash et al., 1990). Similarities to this outcome have been observed
in a mouse model using the same isolate (Bienz et al., 2001; Singer et al.,
2001; Stäger et al., 1998). Interestingly, indications of a cross protection
between assemblage A and B VSP epitopes exist. A purified mixture of
epitopes of assemblage A VSPs was used for immunization of gerbils. The
gerbils challenged with assemblage B parasites displayed some degree of
protection (Rivero et al., 2010). Moreover, VSP switching was investigated
using SCID mice lacking adaptive an immune response. Several clones of
isolate GS were used for infection and both positive and negative selection
of VSPs was observed (Singer et al., 2001). Since no antibodies are produced in these mice, other signals for switching must exist (Singer et al.,
2001). It is known that switching of VSPs occurs during in vitro growth
conditions and different isolates display different switching rates. Two VSPs
can be detected on the surface simultaneously during the switch event (Nash
et al., 2001). These observations are based on the use of very few available
VSP specific antibodies and thus could be underestimated. This indicates
that biological selection of VSPs might be triggered due to conditions apart
from pressure of the immune system.
The VSPs are a large gene family consisting of approximately 200 members.
Together they make up around 4 % of the genome suggesting that diversity
is important for the parasite. Comparative genomics revealed that vsps are
rarely placed in synteny between genomes. In addition the repertoires between isolates are divergent which suggest that recombination events occur
(Jerlström-Hultqvist et al., 2010). The majority of vsps can be found clustered together in linear arrays, but many are placed independently on the
chromosomes with no other vsp in close proximity (Adam et al., 2010). Most
of genomic information about VSPs is based on the most complete genome
of the isolate WB. Utilizing next generation sequencing techniques, more
35
Giardia genomes of high resolution would give further insight to how organization and repertoires of species differ.
Figure 4. The general structures of VSPs are shown in a schematic drawing in (A).
The N-terminal domain varies in size (20-200 kDa) and can be divided into a variable and semi-conserved domain. The N-terminal is rich in cysteine with numerous
CXXC motifs and some contain a GGCY motif. The single transmembrane domain
terminates in the cytosolic pentapeptide tail sequence CRGKA. Immunofluorescence
assay on WB trophozoites labeled with monoclonal antibody against VSP TSA417
(GL50803_113797) (green) are shown in (B). Nuclei are stained with DAPI (blue).
Scale bar 5 µm.
The VSPs are high-cysteine proteins (~12% cysteine) with frequent
CXXC motifs present (Figure 3A). The N-terminus carries a signal peptide
of 14-17 amino acids believed to direct the proteins to the surface and is
thereafter cleaved off from the remainder sequence (Adam et al., 2010). The
C-terminus carries a nearly invariant CRGKA tail that most likely remains in
the cytoplasm. Adjacent to the penta-peptide is a hydrophobic domain that
binds the plasma membrane and anchors the protein to the surface. Zinc
finger and GGCY are additional motifs found among many VSPs and were
all experimentally verified. The more conserved C-terminal is ~38 amino
acids, whereas the N-terminal domain can be highly variable in size (20-200
kDa) thus exposing different antigens to the host (Adam et al., 2010).
The VSPs are transported via PVs and secreted out into the media during
growth in vitro by an unknown mechanism (Papanastasiou et al., 1996;
36
Svärd et al., 1998). The CRGKA tail has been shown to be important for
proper transport and delivery to the plasma membrane in one study (Marti et
al., 2003a) but could not be confirmed by another (Touz et al., 2005). Recently, it was claimed that neither the N-terminal signal peptide nor the
CRGKA tail is required for proper transport (Li et al., 2013), but these results would need additional verifications since the cloning employed in the
mentioned study was not optimal. All studies used different cloning strategies and further experiments are required to see if CRGKA is needed or not.
Post-translational modifications of the penta-peptide have been observed.
The cysteine residue is palmitoylated (Papanastasiou et al., 1997) and the
arginine is citrullinated (Touz et al., 2008). The modifications are hypothesized to be important for segregation into lipid rafts or as a switching signal
respectively. How the modification of the arginine residue would induce
switching is unknown.
So far the trigger and mechanism behind the switch event is unknown. A
switch event has been reported during late encystation/excystation (von Allmen et al., 2004; Carranza et al., 2002; Svärd et al., 1998). The earliest study
(using WB) showed how the dominating VSP (TSA 417/A5) was internalized in PVs late in encystation simultaneously as new VSP transcripts started
to appear. Trophozoites after excystation displayed other VSPs (Svärd et al.,
1998). Another VSP was preferably expressed during encystation using the
same isolate (Carranza et al., 2002). An antigenic reset was also observed in
the mouse model using cysts of the GS isolate (von Allmen et al., 2004). The
inoculated cysts were VSP H7 negative, but after excystation the resulting
trophozoite population was found to express VSP H7. The antigenic reset
mechanism could be a way of re-infecting the same host or facilitate transmission to other host species (von Allmen et al., 2004).
Both nuclei of Giardia are transcriptionally active. It is fascinating how this
binucleated cell can co-regulate transcription so that one and the same VSP
is translated into protein. Moreover, it appears that the majority of vsp genes
are transcribed in a population (Franzén et al., 2013). Several vsp genes are
highly similar and can exist in multiple copies, making it complex to study
their regulation (Adam et al., 2010). Regulation by the RNAi-like pathway
and/or miRNA leading to translational inhibition occurring in the cytoplasm
has been proposed. Giardia possesses Dicer, RNA-dependent RNA polymerase (GiRdRP) and Argonoute (GiAgo) (Prucca et al., 2008). Knockdown strains of GiAgo and GiRdRP results in a decoupling of antigenic
variation and the parasites express many (possibly all) VSPs on their surface.
The authors suggest that GiRdRP transcribe double stranded RNAs for every
vsp mRNA, except the one with the highest concentration. Transcripts are
processed into siRNA by GiDicer and GiAgo degrades all but one intact
single stranded mRNA vsp that will be expressed (Prucca et al., 2008). Addi37
tionally, another group reported a miRNA mediated translational repression
mechanism (Li et al., 2011; Saraiya and Wang, 2008; Saraiya et al., 2011).
Several miRNAs are derived from short non-coding RNAs (snoRNAs) and
six miRNAs have capacity to bind 3´UTR of vsp trancsripts (Li et al., 2011).
Recently, 99 new putative miRNAs were found by sequencing of GiAgoassociated small RNAs (Saraiya et al., 2014). The potential target sites for
these miRNAs was mapped to the entire length and the 3’region of 73 vsp
mRNA. The miRNAs have the capacity to repress expression of vsp mRNA
(Saraiya et al., 2014). These models do not explain how all vsps are transcribed, how the concentration dependent selection of mRNAs is controlled
or how a switch is mediated.
Lastly, epigenetic regulation is likely to be involved in VSP expression
since histone acetylation activates vsp genes (Kulakova et al., 2006).
Regulation of VSPs appears to be multilayered and the mechanism behind
the antigenic switch remains unknown. All studies so far are based on data
studied on populations. However, antigenic variation occurs in individual
cells and single cell experiments would give important insights to contributing mechanisms.
Genomics and transcriptomics
The Giardia genome has many features typically found in eukaryotic cells,
such as linear chromosomes flanked by telomeres with TAGGG repeats
(Adam, 2001). All four core histones (H2A, H2B, H3 and H4) can be found
but the linker histone H1 is missing (Yee et al., 2007). The genomes of six
G. intestinalis isolates, representing three different assemblages (A, B and
E), are available to date. The first genome to be available was WB-C6 (assemblage A1) which has a size of ~11.7 MB distributed over five chromosomes (Morrison et al., 2007; Perry et al., 2011). The compact genome contains few introns and promoters are short and AT rich regions situated upstream of start codon. Genes are placed on both DNA strands and sometimes
overlap. Reduction of components in metabolic pathways, DNA replication
and transcription was also reported. Several genes have bacterial origin and
are candidates of lateral gene transfer (Morrison et al., 2007).
Shortly thereafter the genome of GS (assemblage B) was sequenced and
revealed unique isolate specific genes as well as differences in gene synteny.
Moreover, GS has much higher levels of ASH than WB (0.5% versus
0.01%). ASH was distributed differently into low and high ASH regions
over contigs for GS (Franzén et al., 2009). The genomes show 78% amino
acid identity in protein coding regions and the repertoire of vsp genes were
very different. The third genome represents the first non-human isolate to be
sequenced. The P15 isolate originates from a symptomatic pig (piglet no. 15)
and belongs to assemblage E (Koudela et al., 1991). Comparative genomics
38
of the three isolates revealed a core genome of ~5000 protein coding genes
which is approximately 86% of the genome. The four large gene families
VSPs, HCMPs, NEK kinases and Protein 21.1 display divergent repertoires
between genomes and are often placed in non-syntenic regions. Thus, Giardia chromosomes harbor gene-rich regions in synteny with interspersed nonsyntenic regions containing non-core genes (Jerlström-Hultqvist et al.,
2010).
The mechanism of transcriptional regulation in Giardia is not well understood. The tightly packed genome leaves little space for promoter regions
which are A/T rich without recognizable TATA box. The TATA binding
protein is highly divergent and TFIII is completely missing (Best et al.,
2004). Only 4 of 12 general transcription factors are present. In addition,
very few transcription factor motifs are found among proteins and only 9 of
29 classes of transcriptional factor motifs can be found in the genome (Iyer
et al., 2008). The few transcription factors found in the parasite have mainly
been investigated during encystation (see p. 26). Moreover, Giardia harbors
only 21 of 28 proteins of eukaryal RNAPI, RNAPII and RNAPIII complexes
(Best et al., 2004). Components of the exosome complex are missing and
nonsense mediated decay mechanism are not fully present (Chen et al., 2008;
Williams and Elmendorf, 2011).
Abundant antisense transcription was detected when analyzing the SAGE
data generated during the life cycle. An explanation suggested is that promoter regions are bidirectional. A/T rich stretches in the genome could function as cryptic promoters and increase antisense transcription (Teodorovic et
al., 2007). Transcriptomic studies have been performed in Giardia using
different techniques such as SAGE (Birkeland et al., 2010) and microarrays
(Morf et al., 2010; Müller et al., 2008; Ringqvist et al., 2011; Spycher et al.,
2013). The drawbacks of these techniques are limitations detecting lowly
expressed genes or technical issues such as template cross-hybridization.
The use of RNA sequencing (RNA-seq) can be expected during coming
years as it is affordable and gives a highly dynamic range.
The first RNA-seq study used four isolates (WB, GS, P15 and AS175) to
analyze the transcriptomes of trophozoites (Franzén et al., 2013). RNA-seq
reads were aligned to reference genomes and used to calculate digital gene
expression levels as fragments per kilobase per million fragments mapped
(FPKM). Between 94 to 98 % of all protein coding genes were found to be
transcribed using a cutoff of FPKM >0.5. This confirms that transcription is
promiscuous in this parasite.
Surprisingly, a silent region comprised of 27 ORFs was located on ~41 kb
region on chromosome 5 for WB. Highly divergent homologs were found in
P15 and were also without expression (Franzén et al., 2013). Homologs in
GS and AS175 were found scattered on contigs and their expression status is
39
unknown. The complete lack of transcription can reflect chromatin organization leading to transcriptional blockage.
The RNA-seq data was also used to analyze 3’UTR regions, since these
might contain binding sites for miRNAs or have impact on mRNA stability.
The analysis revealed un-expectantly long 3’UTRs and the length have been
conserved among isolates indicating a possible regulatory role (Franzén et
al., 2013).
The Giardia genomes contain few introns with five cis-spliced (Morrison
et al., 2007; Nixon et al., 2002; Roy et al., 2012; Russell et al., 2005) and
two trans-spliced genes (Kamikawa et al., 2011; Nageshan et al., 2011) reported. These were all confirmed in the RNA-seq data for most isolates and
additionally one novel cis-spliced gene was verified (Franzén et al., 2013).
Another interesting observation was that the GS isolate display higher
levels of ASH in the genome and these alleles are expressed. The allelic
imbalance will contribute to the presence of protein isoforms in this isolate
(Franzén et al., 2013).
Spironucleus
Members of the Spironucleus species contain both commensals and parasites. These flagellates are found in microaerophilic or anaerobic environments, typically in the gut of various animals. The species with associated
host ranges are summarized in Table 2. Species classification has historically
been based on electron microscopy studies, but it is now coupled to molecular biology tools to resolve phylogeny (Jørgensen and Sterud, 2007; Kolisko
et al., 2008). Several of the Spironucleus species can cause infections in
freshwater and marine fish species. Spironucleus vortens could be the cause
of hole-in-the-head disease in angelfish, but was also found in the intestine
of healthy fish (Paull and Matthews, 2001). Spironucleus barkhanus existed
in two morphologically similar types with different host specificity, one of
them living as a commensal in freshwater salmonids, whereas the other were
causing severe systemic infections in farmed Atlantic salmon (Jørgensen and
Sterud, 2006). Molecular genotyping of these organisms’ revealed genetic
differences and the pathogen was renamed as Spironucleus salmonicida.
Morphological differences include presence of dense bodies scattered in the
cytoplasm and a larger cell size was reported for S. salmonicida (Jørgensen
and Sterud, 2006).
40
Table 2. Recognized Spironucleus species with their associated host and reported
lifestyle (Jorgensen and Sterud, 2007).
Species
Hosts
Lifestyle
Spironucleus muris
Spironucleus meleagridis
Spironucleus columbae
Spironucleus elegans
Spironucleus vortens
Spironucleus salmonis
Spironucleus torosa
Rodents
Turkeys and partridges
Pigeons
Amphibians
Angel fish
Rainbow trout
Cod
Parasitic
Parasitic
Parasitic
Parasitic
Parastitic/Commensal
Parastitic
Commensal
Spironucleus barkhanus
Grayling
Commensal
Spironucleus salmonicida
Salmon/Artic charr
Parasitic
Infection and transmission
The systemic infection caused by Spironucleus salmonicida is termed spironucleosis and the parasite is reported to normally be found in the upper intestine of the fish host. In several cases, the parasite could be found in blood
vessels and most internal organs (Guo and Woo, 2004; Poppe et al., 1992;
Sterud et al., 2003). The parasite is reported to cause more severe disease in
Atlantic salmon than in Artic charr (Sterud et al., 2003). Experimental infection with the parasite reports a distinct blood and tissue phase (Guo and
Woo, 2004). Parasites were present in the blood during the first three to eight
weeks of infection. Thereafter the parasites were mainly found in internal
organs, muscles and skin. It is unknown what causes this re-localization.
Major sites of infection were the liver, spleen and the eye socket (Guo and
Woo, 2004). Intracellular stages of the parasite were observed in natural
infections in Artic charr (Sterud et al., 2003). Moreover, all salmon inoculated were infected and developed disease (Guo and Woo, 2004). The mortality
was close to 100 % in naturally infected farmed Atlantic salmon (Poppe et
al., 1992; Sterud et al., 2003). S. salmonicida can cause severe problems for
aquaculture as the infection and mortality associated with the parasite is
high. Recurrent problems with spironucleosis have been reported from Norway and Canada leading to economical loss (Williams et al., 2011). Infections with other Spironucleus species are often reported to be limited to the
host intestine (Williams et al., 2011).
41
Figure 4. Scanning electron microscopy picture of S. salmonicida. Image by Prof
Andrew Hemphill, University of Bern. Scale bar 5 μm.
How transmission of Spironucleus species occurs is not well described. Spironucleus muris forms cysts that are shed with feces. The cyst wall appears
to be similar to Giardia microti but the size of S. muris cysts are smaller
(Januschka et al., 1988). Cysts of S. meleagridis have been found in intestinal smears from pheasants (Wood and Smith, 2005). No cysts have been
encountered for Spironucleus infecting piscine species. Transmission of
disease has been observed from infected fishes (Guo and Woo, 2004; Kent et
al., 1992), although the route of infection is still to be described.
A cyst stage is likely for S. salmonicida, since eight putative cyst wall
proteins are present in the genome. These proteins display similarities to the
giardial CWPs and therefore expression of a S. salmonicida cyst wall protein
was tested in Giardia. As encystation was induced and preceded, the Spironucleus CWP was transported in ESVs and incorporated into the cyst wall of
Giardia cysts. This gives support to a cyst stage of S. salmonicida, even
though encystation trials in vitro have so far failed and no cyst-like structures have been observed (Xu et al., 2014).
Genomics and transcriptomics of S. salmonicida
Apart from the G. intestinalis genomes (see p. 38) much less is known about
other diplomonad genomes. The genome of S. vortens was sequenced using
Sanger technology but the assembly is problematic as the genome appears
highly repetitive. First insight into the genome of S. salmonicida were generated from genome survey (GSS) and expressed sequence tags (EST) libraries
(Andersson et al., 2007). The data showed a compact genome with G/C content of 38 % and very short 3’untranslated regions. Thereafter both S. salm42
onicida and S. barkhanus were subjected to a comparative genomics project
using EST libraries (Roxström-Lindquist et al., 2010). The results highlighted large genetic divergence between the organisms with an average identity
of 84% on amino acid level. This is similar to the identity found comparing
Giardia isolates WB and GS (Roxström-Lindquist et al., 2010). Moreover,
high frequencies of SNPs were detected for S. barkhanus genes, whereas S.
salmonicida displayed less variation. The genome size of both genomes was
estimated using flow cytometry. The haploid genome size of S. barkhanus
was around 18 Mbp, whereas for S. salmonicida it was close to 12 Mbp
(Roxström-Lindquist et al., 2010).
The complete genome of S. salmonicida was recently published and compared to Giardia genomes (Xu et al., 2014). Optical mapping was used to
resolve the size of the genome, which was reported to be 12.6 Mbp consistent with results from flow cytometric data (Xu et al., 2014). The genome
is not very repetitive and ASH levels are low. There are approximately 3000
more protein coding genes in the genome compared to Giardia and the
shared synteny is almost non-existent. The overall GC content is 33.4 % but
higher in the coding regions. Four genes are reported to contain introns and
S. salmonicida has a reduced splicing machinery, as Giardia (Xu et al.,
2014).
Generated RNA-seq data was mapped to the assembled genome and revealed clear boundaries of transcription, in contrast to the fuzzy boundaries
of transcription found in Giardia. In addition, putative regulatory elements
were found and they include a TATA-box motif, a C-rich motif and enrichment of As that likely act as initiator elements for transcription. An expansion of proteins containing Myb-binding domains is found together with
other potential transcription factors. This indicates that S. salmonicida has a
tighter transcriptional regulation of expressed genes compared to Giardia
(Xu et al., 2014).
Metabolism
Metabolism of Spironucleus species is not well characterized, but the genome data of S. salmonicida and experimental investigations of S. vortens
have yielded some insights.
The growth of S. vortens under in vitro conditions is atypically biphasic
and can be extremely fast for a eukaryote with a doubling time of ~1.8 hour-1
(Millet et al., 2011). Alanine, ethanol, acetate, CO2 and lactate are end products during glucose metabolism. Amino acids appear to be favored as carbon
source, over glucose and ethanol. Addition of arginine did not lead to production of NO, indicating that the arginine dihydrolase pathway is not active
under in vitro growth, as in contrast to Giardia and Trichomonas vaginalis
(Millet et al., 2011).
43
Comparative genomics of Giardia and S. salmonicida reveled large differences in the metabolic gene repertoire. Extended genes involved in carbohydrate metabolism, energy production and amino acid metabolism were
found. This indicates that S. salmonicida can utilize a larger variety of metabolites, as compared to Giardia (Xu et al., 2014). S. salmonicida harbors
more putative transporters in the genome making transport of a larger variety
of metabolites possible.
Hydrogen production is rare in eukaryotes. The presence of [FeFe]hydrogenases in S. salmonicida was discovered and therefore also the possibility of hydrogen metabolism (Horner, 2001). The EST data generated for
S. salmonicida revealed presence of Cpn60 and IscS in the genome indicating presence of a MRO in this diplomonad (Andersson et al., 2007). Later, S.
vortens was experimentally shown to produce large amounts of H2 at similar
levels as the hydrogenosome harboring T. vaginalis (Millet et al., 2010).
Recently, presence of hydrogenosomes was investigated using TEM and
heterologous antibodies from known MRO markers. Presence of doublemembrane organelles was found that potentially could be hydrogenosomes
(Millet et al., 2013).
Cell biology
Much remains to be explored regarding cell biology of Spironucleus species.
Plasmids to facilitate tagging of proteins are essential tools for increasing the
knowledge of protein function in an organism. The creation of tagging vector and transfection protocols for S. vortens has been described (Dawson et
al., 2008). Stable transfectants were generated using puromycin selection
and the localization of GFP-tagged Histone H3 was studied. The episomal
plasmid resided in only one nucleus and the copy number was 10-20 copies
as observed by FISH (Dawson et al., 2008). The presence of the vector in
one nucleus indicates that the nuclei do not fuse during mitosis, in line with
observations in Giardia. The creation of a plasmid system for S. salmonicida
is presented in Paper I.
The available genome of S. salmonicida has revealed several interesting
aspects worthy of further studies. The fish parasite has more cysteine proteases than Giardia and is possibly utilized by the parasite to invade tissues.
These could potentially be secreted virulence factors and need further characterization (Xu et al., 2014). The parasite encounters highly variable environments during the invasion of the host tissues and needs to be protected
against fluctuating O2 levels during systemic infection. This is reflected in
the genome as an extended oxygen scavenging system is present. More
genes are present to protect against reactive oxygen species as compared to
Giardia (Xu et al., 2014).
44
Protection against the host’s immune system is vital during infection and
S. salmonicida has many (>350) cysteine-rich proteins and some of these
carried the conserved motif [KR][KR][GSA][KR][KR] in the C-terminal.
The number of CXXC and/or CXC motifs, KKXKK motifs and a Cterminal transmembrane domain was used to classify the cysteine-rich proteins. This resulted in three different classes; Cysteine-rich membrane protein 1 (CRMP1), CRMP2 and Cysteine-rich protein (CRP). Members of
CRMP1 all contain the KKXKK motif in the C-terminal, reminiscent of the
penta-peptide CRGKA of VSPs in Giardia. The transmembrane domain
present in CRMP1 and CRMP2 is indicative for the association to external
or internal membranes. Localization of members from CRMP1 was experimentally shown to be associated to various membrane structures in the parasite. Additional experiments are needed to decide if these proteins are truly
involved in antigenic variation. An RNAi-like mechanism has been suggested to be involved in regulation of VSPs in Giardia. However, Dicer, RdRP
and Argonaute could not be identified in the genome of S. salmonicida.
Therefore, another mechanism is likely to be responsible for regulation of
CRMPs. The CRMPs do not contain N-terminal signal peptides as the VSPs;
hence secretion to the surface is performed by unknown means (Xu et al.,
2014).
45
Present Investigation
Aim of the thesis
This thesis describes the work on the two diplomonads, Spironucleus salmonicida and Giardia intestinalis, using comparative cell biology and transcriptomics approaches. The overall aim of the projects presented was to
understand different cell biological processes in these unicellular eukaryotic
parasites. The development of a transfection system in S. salmonicida will
open the door to explore cellular features in this parasite. Focus will be directed against investigating the mitochondrion-related organelle (MRO) and
to characterize the annexin gene family in S. salmonicida. This will lead to
an increased understanding, of not only diplomonad characteristics, but also
of eukaryotes in general.
The differentiation into infectious cysts, via the process of encystation, is
crucial for transmission and survival of Giardia intestinalis. The cysts are
exposed to different stress conditions prior to ingestion into a new host. We
aimed to study one such stress, namely UV irradiation, as it is also used for
treatment of drinking water. The response will be studied using transcriptomics to reveal mechanisms behind stress management in this parasite.
To ultimately prevent transmission of this parasite, much more remains to
explore regarding the process of encystation. Therefore the transcriptional
response of the process will be further studied. This will ultimately generate
information of how the transcriptional landscape alters, of vital processes
and what genes participate in the cellular transformation.
Establishment of S. salmonicida as a diplomonad model system
(Paper I)
The knowledge about diplomonads has long been focused and restricted to
Giardia intestinalis. But the creation of a stable vector tagging system in S.
vortens for transfection opened the opportunity to study cell biology functions (Dawson et al., 2008). However, difficulties in assembly of the genome
have hampered the possibilities to fully exploit S. vortens as a model organism. To further explore the diversity among the diplomonads, the genome of
Spironucleus salmonicida was sequenced. The genome sequence infor-
46
mation was used to construct a vector tagging system enabling functional
studies of proteins.
Vector constructions and establishment of S. salmonicida transfectants
The growth effect of five different drugs was evaluated on S. salmonicida
trophozoites. The antibiotics puromycin, blasticidin S and G418 displayed
the most inhibitory effects. The constructed vectors for S. salmonicida were
based on already existing Giardia vectors (Jerlström-Hultqvist et al., 2012).
The vector system was built on a cassette format with selection markers,
inserted genes and epitope tags easily exchangeable. The resistance markers
for antibiotics puromycin (pac gene), blastidicin s (bsr gene) and G418
(nptII gene) were incorporated into the vector, allowing selection of resistant
parasites. The epitope tags 3xHA, 3xMYC or 2xOLLAS can now be used
for localization studies and SF-TAP or SBP-GST for protein purification
purposes. The epitope tags are available for C-terminal or N-terminal tagging of the proteins. Settings for optimal electroporation were investigated
based on known conditions for Giardia and S. vortens, giving rise to stably
transfected cell lines.
Localization of epitope-tagged proteins in S. salmonicida
Six different proteins were selected for localization studies using epitopetagging and full length expression of the proteins were assessed by Western
blotting. IFT46, Fibrillarin and Caltractin were cloned, transfected and localized using the 3xHA epitope present in the C-terminus of proteins. Intraflagellar transport complex B (IFT46) localized to the flagella and the C-shaped
sheets in close proximity to the nuclei. Some cells displayed enrichment of
the protein in the flagellar tips which indicates trafficking of IFT along the
microtubules. Fibrillarin partially localized to the DAPI staining of the nuclei. Caltractin was present in two pyramidal foci anterior of the nuclei. Colocalization using commercial anti-centrin antibody strongly suggests that
the structure represents the basal bodies of S. salmonicida.
The possibility to integrate selection markers on the chromosome was also investigated using a protocol developed for Giardia (Gourguechon and
Cande, 2011). BiP, Sec61α and Protein disulfide isomerase (PDI) are potentially associated to the ER. They were cloned into the 2xOLLAS C-terminal
tagging vector and a unique restriction site found within each of the genes
was used to linearize the plasmid prior to transfection. Successful integration
of BiP-2xOLLAS was confirmed using a PCR approach, but could not be
verified for Sec61α-2xOLLAS and PDI-2xOLLAS. The protein expression
detected using Western blot showed expected protein sizes. The immunolocalization of the three proteins displayed similar distribution in the cell
with association to the recurrent flagella throughout the cell body and around
the nuclei.
47
Generation of double transfectants
To be able to perform co-localization studies in the parasite, we explored the
possibility to generate double transfectants. Transfectants carrying the PACIFT46-3xHA-C episome were transfected with the linearized BSR-PDI22xOLLAS-C vector and selected with blasticidin S. Transgenic puromycin
resistant cells were less sensitive to blasticidin S selection. Increasing the
blasticidin S concentration near 5-fold abolished the interaction of the drugs,
allowing selection of double transfectants. The protein localizations of double transfectants were identical to those observed in single transfectants.
The creation of this successful transfection system was further used to facilitate the studies described in Paper II and III.
S. salmonicida possess hydrogenosomes (Paper II)
All eukaryotes most likely harbor mitochondria or a MRO. Very little is
known about MROs in diplomonads, but mitosomes are found in Giardia
and S. vortens can produce hydrogen in similar amounts to the hydrogenosome-containing T. vaginalis. We set out to explore the nature of
MROs in S. salmonicida.
The MROs present in S. salmonicida produce hydrogen
The S. salmonicida draft genome was explored to find potential MRO associated genes. Gene candidates were cloned and localized using the transfection vector system described in Paper I. Chaperonin 60 (Cpn60) is a known
marker for MROs and was further characterized by integrating a HA-tagged
Cpn60 copy into the chromosome. The localization of Cpn60 was to punctuate structures scattered in the cytoplasm posterior to the nuclei. Transmission
electron microscopy (TEM) of this strain revealed double-membrane bound
organelles that were electron dense and differed in size and shape (Figure 5).
However, most often they were round to ovoid with a size of 300-400 x 200
nm. Immunoelectron microscopy was used on epitope tagged Cpn60 and
gold labeling was strong in the electron dense organelles. These results show
presence of MROs in S. salmonicida.
48
Figure 5. Transmission electron microscopy images showing S. salmonicida hydrogenosomes. A) An S. salmonicida cell with three double-membraned organelles
of different morphology (H). Nucleus and flagella are denoted as N and F, respectively. Scale bar denotes 1 ɥm. B) Enlarged view of the organelles indicated by a
box in A). Scale bar denotes 200 nm. Images by Ass. Prof. Kjell Hultenby.
In addition, proteins involved with Fe-S cluster synthesis (IscU, IscS,
Frataxin, Nfu and two Ferredoxins), protein import and homeostasis (Pam18,
Tom40, HSP70, GrpE and Jac1) were identified in the genome. All these
proteins, except Frataxin, have been reported as present in the Giardia mitosomes. The proteins were found experimentally to co-localize with Cpn60 in
the MROs. None of the proteins carried detectable amino-terminal targeting
sequences and homologues of α- or β-subunits of mitochondrial processing
peptidase are missing from the genome. However, when HA-tagged Pam18
of S. salmonicida was expressed in Giardia, the protein was recognized and
correctly imported into the mitosomes.
Hydrogen production of S. vortens is known (Millet et al., 2010) and we
detected that also S. salmonicida and S. barkhanus could produce hydrogen
but to lesser extent. Bioinformatic searches revealed the presence of five
PFORs and seven [FeFe]-hydrogenases in the genome of S. salmonicida.
This finding demonstrates that the molecular basis for hydrogen production
is present. Only PFOR5, FeHyd5 and FeHyd6 localized to the hydrogenosomes, whereas the remaining members localized to the cytoplasm.
The hydrogenase maturases HydE, HydF and HydG were present in the genome and co-localize with Cpn60. A proximity ligation assay (PLA) was
used to show direct interactions between HydF-HydG and HydF-HydF. In-
49
teractions between HydF and FeHyd2, FeHyd5 and FeHyd6 could also be
detected.
Evolutionary origin of the hydrogenosomes
The origin of [FeFe]-hydrogenase, hydrogenase maturases and PFOR were
analyzed using a phylogenetic approach. Homolog sequences from S.
vortens and the free-living diplomonad Trepomonas were included apart
from S. salmonicida. The diplomonads were monophyletic in trees for the
maturases and the topologies highlights presence of hydrogenase maturases
in the common ancestor of diplomonads and parabasalids. The origin of
[FeFe]-hydrogenase were complex, but indicated presence of multiple paralogs in the diplomonad ancestor. PFOR were monophyletic for eukaryotes
and diplomonads were found in two groups together with sequences from
Trichomonas. Tree topology indicated that four paralogous genes were present in the ancestor of diplomonads. Two of these have been lost in Giardia.
The hydrogenosome in S. salmonicida has additional functions
Comparative genomics and proteomics were used to identify and expand the
list of proteins present in the hydrogenosome present in S. salmonicida.
Known members of the hydrogenosome in T. vaginalis were used to find
homologous proteins resulting in the identification of a potential H-protein
of the glycine decarboxylase complex and serine hydroxymethyltransferase
(SHMT). The presence of these proteins argues for amino acid metabolism
in the hydrogenosome. Proteomic analysis was performed on hydrogenosomal enriched fractions generated using subcellular fractionation techniques.
Candidate proteins found in the data sets were experimentally verified by
localization studies. A divergent homolog of Acetyl-CoA-synthetase (ADPforming) was found to display hydrogenosomal localization. Presence of this
enzyme indicates that generation of ATP is possible from pyruvate degradation to acetate.
Annexin diversity revealed in S. salmonicida (Paper III)
Annexins are found in organisms spread across the kingdoms and the name
means hold/bring together. The multifunctional proteins bind lipids in a calcium dependent manner and have a conserved Annexin-fold (Lizarbe et al.,
2013). Annexins are a multigene family of 21 members (the alpha-giardins)
in Giardia intestinalis. The majority of Alpha-giardins are associated to the
cytoskeleton and/or membrane making these the best characterized Annexins
in a unicellular organism (Weiland et al., 2005). Spironucleus was reported
to have Annexins with higher similarity other eukaryotes, compared to any
Alpha-giardin (Roxström-Lindquist et al., 2010). Annexins from plants and
animals are well characterized compared to the Annexin-homologs found in
50
protists. We therefore wanted to investigate the Annexin diversity in S.
salmonicida.
Identification and features of S. salmonicida Annexins
The search of Annexin homologs resulted in 16 full-length gene copies of
which 14 were distinct genes. Annexin 3 and 9 were present in two identical
copies. The Annexins were distributed on seven of nine chromosomes with
clusters on chromosomes 3, 4 and 6. Traces of probable gene duplication
events were detected leading to expansion of this gene family. Sequence
analysis was performed to uncover signature motifs found in Annexins/Alpha-giardins. AB-loops (type II Ca2+coordination sites) were present in S. salmonicida Annexins in at least one copy, which suggest the possibility of binding calcium ions. Very few Alpha-giardins contain AB-loops
and overall the S. salmonicida annexins appears less divergent in sequence.
Post-translational modifications of proteins are common ways to specify
their functions. One such example is lipid modifications to promote association to membranes. Annexins 3 and 12 carried putative palmitoylation and
myristoylation sites.
Annexins are defined as calcium-dependent membrane binding proteins.
The association to membranes was determined for a subset of the Annexins
by membrane fractionation of transfected cell lines. The fractionation separated proteins into a hydrophobic and hydrophilic phase used for Western
blot analysis probing against the HA-tag. Annexins 1, 3, 4, 9 and 12 were
found in both membranous and cytosolic fraction, whereas 2, 5, 7, 10 and 13
were exclusively detected in the cytosolic fraction. We investigated lipidbinding preferences for Annexin homologs, Annexin 3 with four AB-loops
and Annexin 5 with a single AB-loop. The two Annexins were expressed as
GST-fusions, purified and subjected to lipid binding assays. Alpha-14
giardin was used as positive control as the binding pattern is known from
earlier studies (Pathuri et al., 2009). The proteins displayed different binding
patterns in a calcium dependent manner. Annexin 3 could bind cardiolipin,
phosphatidylserine (PS) and less to phosphatidylglycerol and had an overall
similar binding pattern to Alpha-giardin 14. Annexin 5 displayed an overall
weaker association to lipids in the assay, but interaction to phosphatidylinositol 4-phosphate (PtdIns(4)P) and sulfatide was found.
The Annexins have distinct localizations
As many of the Alpha-giardins display strong association with the cytoskeleton, we wanted to investigate the localizations of the S. salmonicida Annexins. All Annexins were cloned to carry a C-terminal HA tag, using the
vector system described in Paper I. A great variety of localizations were
observed for the tagged proteins of which some were very specific. Annexin
3 and 12 localized to all eight flagella including the internal part of the recurrent flagella. Annexins 5 and 13 show association to an undescribed struc51
ture in the anterior part of the cell. We could also observe a smaller structure
placed at midbody position in the cell. This structure was translocated into
the anterior part of a newly divided cell.
Annexin 6 localized to eight distinct foci in the anterior part of the cell and
prior cell division these were doubled in number. Two foci are localized in
between the nuclei. These structures are likely to represent the flagellar pore
complexes and the basal bodies or centrioles. Annexin 4 was seen in the
plasma membrane, including all eight flagella. Annexin 1 was observed in
the cytoplasm and internal parts of the recurrent flagella. Annexins 2 and 9
were seen as numerous cytoplasmatic foci. Annexins 8, 11 and 14 appeared
to be mainly cytosolic. Annexin 7 could not be localized with the native
promoter, but during overexpression using promoter region of Annexin 3,
the protein localized to cytoplasm.
Association to specific structures was investigated to pinpoint potential
functions of the Annexins using the proximity labeling tag ascorbic peroxidase (APEX) as a fusion partner. DAB was used as substrate for the APEX
peroxidase, forming an oxidized complex visible as a dark precipitate after
osmium treatment in TEM. Annexin 3 was associated to the microtubule
doublets of all flagella and the striated laminae that encase the cytostomal
canals that the recurrent flagella pass through. Annexin 6 revealed signal in
the vicinity of the basal bodies with prominent staining in and around the
centrioles. Annexin 5 displayed a strong staining in the anterior part of the
cell above the nuclei and plasma membrane of the flagella. A much weaker
signal with similar localization could be observed for Annexin 13.
Phylogeny of diplomonad Annexin homologs
The evolutionary relationship of eukaryotic Annexins was investigated using
phylogenetics. Sequences from selected eukaryotes were compared to Annexin homologs from hexamitid flagellates (S. salmonicida, S. vortens, S.
barkhanus and Trepomonas PC1) and the Alpha-giardins of G. intestinalis
and G. muris. The sequences from diplomonads formed a cluster with strikingly large sequence diversity compared to other Annexins. Annexins of
hexamitid flagellates form a large cluster with the Alpha-giardins nested
within and are possible sister clades as the root of the diplomonad cluster is a
likely long-branch attraction. This indicates that the Annexin family has
expanded independently in the two groups of diplomonads. G. muris homologs branch together with G. intestinalis in most cases, suggesting a functional conservation. A similar situation was detected for close relatives S.
barkhanus and S. salmonicida. Lineage-specific expansions is likely to have
occurred in Trepomonas PC1 and S. vortens as they have their own set of
homologous genes. The phylogenetic analysis suggests that Annexins were
likely to be present in the eukaryotic ancestor. To further deepen the understanding of the different functions of Annexins in eukaryotes, additional
studies from diverse protists would be useful.
52
UV stress in G. intestinalis (Paper IV)
Giardia encounters several stress stimuli during its life stages and excreted
cysts are exposed to UV irradiation in the environment. Cysts are able to
survive several months in cold water and disinfection of drinking water using UV-C is commonly used. How the parasite responds to UV-C exposure
is important knowledge as it may have implications on treatment to ensure
safe drinking water. We used RNA-seq to understand the transcriptional and
cellular response from the parasite.
Response to irradiation highlights the importance of active DNA
replication
Trophozoites, encysting cells and mature cysts were exposed to UV-C irradiation in order to study the molecular and cellular effects of UV-C irradiation. Viability after irradiation exposure, ranging from 2-100 mJ/cm2, was
measured by growth (trophozoites) or excystation followed by growth (irradiated encysting cells and cysts). Trophozoites survived all tested irradiation
doses but a slight decrease in growth was noticed already at 2 mJ/cm2. Mature cysts were unable to establish a culture after exposure to 10 mJ/cm2 and
no viable excyzoites could be detected after irradiation dose of 20 mJ/cm2.
These results indicated that trophozoites survive UV-C exposure better than
cysts, even though they never naturally encounter this stress factor.
DNA damage induced by UV is normally repaired by nucleotide excision
repair (NER) proteins, but this system is not present in Giardia (Franzén et
al., 2009; Jerlström-Hultqvist et al., 2010; Morrison et al., 2007; Novarina et
al., 2011). Other eukaryotes without NER systems rely on active DNA replication to repair UV induced damage (Novarina et al., 2011). As the Giardia
trophozoites have active DNA replication machinery, whereas the dormant
cysts do not, this could be a possible explanation for the increased survival.
We could show that UV treatment using 2 mJ/cm2 induced a cell cycle arrest
at G1/S in trophozoites using flow cytometry. The hypothesis that active
DNA replication is important for survival was tested by irradiating encysting
trophozoites. Encysting trophozoites undergo two rounds of DNA replication
during late stages of the process (Bernander et al., 2001) and UV treated
cells at that stage survived tested doses.
UV induced double stranded DNA breaks (DSB) could result in cell cycle
arrest and repair. We tested if the UV treatment resulted in DSBs using an
antibody for phosphorylated Histone H2AX on treated cells and cysts. Both
trophozoites and cysts were positively stained showing that UV-C induce
DSBs in both cell types. Thus the trophozoites and cysts subjected to RNAseq had DNA damages and were allowed three hours of recovery time prior
RNA extraction.
53
Transcription analysis of UV exposed cells
The analysis of the overall transcriptional response to UV irradiation revealed few major changes in gene expression. The amount of genes differentially expressed were 108 and 202 genes in trophozoites and cysts respectively (fold change cut off ≥1.5). Functional annotation cluster analysis was
used on differentially regulated genes to reveal enriched changes in cellular
processes. For trophozoites several stress/heat-shock proteins, kinesins and
Nek kinases were up regulated, whereas all core histones were down regulated. The majority of regulated genes for both trophozoites and cysts encode
hypothetical proteins with unknown function. The expression of VSP genes
changed upon UV exposure for both cell types.
The differentiation of G. intestinalis is a coordinated cascade of
gene regulation (Paper V)
The differentiation into infectious cysts is essential for transmission and
survival of the parasite. The encystation process has been studied over the
last decades as the entire life cycle can be completed in vitro. The majority
of studies have focused on the early phase of the process, leaving late events
poorly defined. In order to further study the encystation, we developed a new
protocol to increase the yield of mature cysts. The transcriptional response of
the transformation from trophozoite to cyst was studied using RNA-seq.
New encystation protocol give higher yield of mature cysts
To enable studies of the late phase of encystation and mature cysts, an additional differentiation protocol was developed. The Uppsala encystation
method employs high bile concentrations (10-20 times that of the growth
media) to facilitate lipid starvation and an elevated pH of 7.8. We evaluated
the newly developed protocol to the 2-step method and cholesterol starvation
using delipidated serum. Flow cytometry was used to compare these encystation protocols with regard to yield of mature 16N cysts. The Uppsala method
generated highest yield of mature cysts making studies of the late phase and
mature cysts possible at a higher resolution.
In addition, the encystation kinetics was studied by counting encystationspecific vesicles (ESVs) using an antibody against CWP1. The amount of
ESVs per cell as well as ESV positive cells in the population was counted at
five different time points. The ESVs/cell differed greatly amongst cells indicating that the encystation process was asynchronous, as reported by others
(Morf et al., 2010; Poxleitner et al., 2008; Reiner et al., 2008). However, the
median of ESV/cell and the amount of encysting cells in the population
greatly increased at 7 hours post-induction (p.i). At 14 and 22 h p.i the majority of cells (62 and 83% respectively) in the population were encysting.
54
The Uppsala protocol was subsequently used to enhance knowledge of the
transcriptional response of the process using RNA-seq.
Major changes in the transcriptional landscape occur late
The total gene expressional changes were studied during the entire differentiation process (0 h, 1.5 h, 7 h and 22 h p.i. and mature cysts). Genes expressed with different temporalities and amplitudes during the process were
selected for verification using quantitative real-time PCR (qPCR). Four biological replicates were used to validate the RNA-seq data and high level of
agreement was observed. This suggests high reproducibility in terms of periodicity and amplitude of gene expression levels using the Uppsala encystation protocol.
Non-clustered heatmaps were used to further characterize the periodicity of
gene expression along the trajectory of encystation. Cascades of up- and
down-regulation of genes throughout the encystation argue for a coordinated
pattern of developmentally linked gene regulatory activities. The profiles of
putative and known mediators of transcriptional regulation were therefore
investigated. Interestingly, both known and putative transcription factors and
repressors together with chromatin modifiers (HDACs, HATs and HMTs)
and chromatin remodeling complex SNF displayed a similar temporality.
This suggests that the encystation process is regulated in a coordinated manner both on the level of DNA accessibility and transcriptional activation.
By comparing the transcriptome of encysting cells to trophozoites, it was
evident that majority of expressional changes occurred late. Many genes
overlap between 22 hours and cysts, indicative that cysts were already present in the culture at this time point or remains expressed in the cyst stage.
Majority of up- and down-regulated genes encode hypothetical proteins.
Functional annotation cluster analysis was performed on differentially
expressed genes in order to uncover enrichment of function-related gene
groups. It was evident that certain processes were prominent during early or
late phase of encystation.
Early phase of encystation
Genes associated with “ARF/Sar superfamily”, “Glycolysis”, “Co-factor
binding” and “Ribonucleoprotein complex” (1.5 and 7 h p.i) were enriched
during early phase (1.5 and 7 h p.i.) of encystation. Formation of ESVs is the
major event during these time points. Small GTPases Sar1, Arf1 and Rab1a
are essential for proper vesicle formation and maturation (Stefanic, 2009).
Several other ER associated proteins (e.g. Rab and Sec proteins) were also
up-regulated reflecting the enhanced need for protein secretion and transportation. The cyst wall proteins were massively up-regulated (100 fold) be55
tween 1.5 to 7 h, as were most of the ribosomal proteins highlighting the
increased demand on translation.
The enzymes of the glycolysis pathway were up-regulated which suggest
an increasing need for energy during the early phase. Many enzymes responsible for synthesizing the carbohydrate component of the cyst wall were up
regulated at 7 h and peaked in expression at 22 h of encystation.
Downregulated processes included genes associated with “cytoskeleton”,
histone core” and “integral to membrane”.
A previous transcriptional study using microarray technology employed two
encystation protocols to reveal a core set of 13 genes (Morf et al., 2010).
These genes were upregulated early in our data set as well and five genes
encoding proteins with unknown functions were further studied. A Protein
21.1 localized to the nuclei, the other four proteins showed ER and/or nonESV vesicle-like localization patterns.
Large changes occurs late in encystation
Processes found enriched at 22 hours for up-regulated genes were “Glycolysis” and “Co-factor binding”. Genes associated with glycogen breakdown
were found together with enzymes responsible for building the GalNAc sugar in the “glycolysis” cluster. Two of the genes in the “Co-factor binding”
cluster were serine palmitoyltransferase 1 and serine palmitoyltransferase 2,
which are involved with the ceramide synthesis pathway, reflecting that the
lipid metabolism undergo changes during encystation as also observed by
others (Yichoy et al., 2011). The functional annotation analysis for downregulated genes showed enrichment for processes such as “Initiation factor”,
Nucleobase catabolic activity” and “Translation”. Since the cells at this time
point prepare to enter dormancy, the gradual decrease of translationassociated processes was expected.
The majority of genes that were up-regulated during late encystation encode hypothetical proteins. Many of these genes lack GO terms and are
therefore excluded from the functional annotation cluster analysis. Therefore
ten genes were selected and epitope-tagged to study their function during
differentiation. Four genes localized to non-ESV vesicles during late phase
and the excyzoite surface in mature cysts. Three proteins showed localizations to the nuclei in the late phase and in mature cysts. The remaining three
proteins localized to unknown structures in the mature cyst.
The cyst- prepared for dormancy and excystation
All core histones, meiosis-associated genes and several kinesins were enriched clusters for up-regulated genes in cysts. Many of these genes displayed similar expression profiles in the SAGE dataset and were found to be
highly expressed during excystation (Birkeland et al., 2010). Up-regulation
of histones might reflect the condensation of chromatin in the cyst or a prep56
aration of transcripts to facilitate the rapid cell division shortly after excystation. Transcription of kinesins could also be a preparation for excystation.
Kinesins are motor proteins important for flagellar assembly, which is a vital
process during excystation as the flagella are the first to emerge from the
cyst.
Cellular processes such as “Translation” and “Protein folding” were
down-regulated in mature cysts, as they are not required during dormancy.
Calcium binding proteins, such as the alpha-giardins and adhesive disc proteins were found down-regulated, indicating the large rearrangements in the
cytoskeleton in mature cysts.
Expressional changes of high cysteine proteins
Evident from the functional annotation analysis were the transcriptional
changes of cysteine rich EGF-like proteins. Majority of these were VSPs, but
also HCMPs and Tenascins were found. VSPs are surface proteins involved
with antigenic variation and major transcriptional changes were seen late in
encystation. The highest transcribed VSP from trophozoites to 22 h were
VSP 5 (50803_113797 or TSA 417) but VSPs found expressed in cysts were
different from those expressed during differentiation. This strongly indicates
that a switch event occurs late during encystation.
HCMPs and Tenascins are much less studied gene families and their expressions were also changed during differentiation. Further studies are needed to clarify the functions of HCMPs and Tenascins during the life cycle of
the parasite.
57
Conclusions and future perspectives
S. salmonicida can be used as a biological model system
The salmonid parasite S. salmonicida is easy to grow in vitro and a highquality genome is available. These two criteria and the development of a
transfection system make research on cellular functions possible. The addition of this transfection system increases the knowledge of diplomonad biology by the possibility to do comparative cell biology. This will hopefully
promote research on both Giardia and Spironucleus.
To date, over one hundred transgenic cell lines of S. salmonicida have
been generated which indicate the robustness of the system. It is possible to
integrate markers on the chromosome using single cross-over recombination
and generation of double-transfectants is routine. The possibility of colocalizations studies is valuable since heterologous antibodies in general
work poorly in diplomonads.
Fluorescent proteins have been used to great success in many organisms,
including Giardia, to study the dynamics of cellular processes via live microscopy. The use of fluorescent proteins has not been fully evaluated in S.
salmonicida. The oxygen scavenging pathways present in the parasite might
restrict the maturation of conventional fluorescent proteins, but some alternative proteins and ligands have been developed for anaerobic conditions.
The use of reporter proteins could also be utilized for in vivo imaging of
experimentally infected salmonids to follow the progression of infection.
Can S. salmonicida form cysts?
The exploration of the S. salmonicida genome generated several interesting
aspects worthy of further functional studies. One such discovery was the
presence of cyst wall homologs and enzymes synthesizing the GalNAc component of the giardial cyst wall. This suggests that a cyst stage exists in the
life cycle of S. salmonicida. The transmission route of S. salmonicida is not
clear and cyst formation has not been detected during in vitro conditions.
Initial trails using known encystation conditions for Giardia (i.e. high bile
and elevated pH) did not generate cyst-like structures. More extensive search
for conditions using reporter-protein fusions to potential encystation proteins
could give clues of trigger signals. As the parasite is found in various tissues
58
during infection, it could be possible that tissue cysts are formed similar to
Toxoplasma gondii (Dubey et al., 1998).
Hydrogenosomes: one organelle, several functions
Mitochondria or MROs are present in all eukaryotes investigated but the
origin of the organelle remains controversial. The functions and origin of the
MRO found in S. salmonicida were explored using phylogenetics and functional studies. We could show that the MRO present in S. salmonicida is a
hydrogenosome. The evolutionary analyses of hydrogenosomal proteins
indicate their presence in the diplomonad ancestor. Hydrogen production
was detected for the three Spironucleus sp. investigated and this further
strengthens the conclusion that the organelle functions as a hydrogenosome.
Indications of additional features, besides FeS cluster assembly, were found
in the form of metabolic proteins present in the organelle and ATP might be
generated by an Acetyl-CoA synthetase.
Several interesting aspects remain to explore on this subject. Further proteomic studies of the S. salmonicida hydrogenosome would reveal additional
proteins and potential functions associated to the organelle. Genomic information of additional diplomonads, including Spironucleus vortens and Spironucleus barkhanus, will provide information about the MRO diversity and
function. Ultimately this will give insights to the origin of the hydrogenosome and why MROs were required in eukaryotes.
S. salmonicida have an attachment organelle
The S. salmonicida Annexins were studied using both phylogenetics and
experimental approaches. It is evident that the Annexin homologs have expanded in the diplomonads. The reason for the expansion could be to substitute for the lack of a complex actin cytoskeleton. Maintaining integrity of the
cell through cytoskeleton-membrane interactions is likely to be important, as
several diplomonads live in harsh environments e.g. the intestine. Many of
the S. salmonicida Annexins had very specific localizations to cellular structures. Interestingly, Annexin 5 and 13 localized to an undescribed structure
in the anterior part of the cell. Information of a potential function was gathered using proximity labeling system coupled to TEM for high resolution of
the ultrastructure of the cell. The structure labelled by these two Annexins,
appear to correspond the general area used for attachment by the parasite. S.
salmonicida does not have such an obvious attachment organelle as the ventral disc of Giardia but is clearly capable of attaching to various surfaces.
Moreover, the homologs of signature disc proteins (e.g. SALP-1, median
body protein) are absent, but there are Alpha-giardins that localize to the
disc. It is tempting to speculate how this structure is used and which proteins
might interact to facilitate attachment. Perhaps it additionally plays a role
59
during invasion, since we know that S. salmonicida can invade various tissues but nothing of how this is mediated.
Giardia cysts- masters of survival
We wanted to study the effect of UV irradiation as this is a natural stress
factor that the Giardia cysts encounter. Trophozoites, encysting cells and
mature cysts were included to compare the response mechanism of different
cell stages. Transcriptomes of irradiated trophozoites and cysts were studied.
The trophozoites and encysting cells can survive UV treatment to larger
extent than cysts. The DNA replication machinery is active in these cells
enabling repair of DSBs induced by UV-C. Dormant cysts might not be able
to repair DNA damage when exposed to UV and the resulting excyzoite fail
to proceed in cytokinesis due to DNA damage. The responses to UV stress
on the transcriptional level were minor for both trophozoites and cysts, in
line with results from other protozoan parasites (Ware et al., 2010; Weber et
al., 2009; Zhang et al., 2012). However, the UV exposure can lead to accumulation of reactive oxygen species, which may influence the protein structure. Up-regulation of heat-shock proteins and chaperonins could reflect
protection of UV by limiting misfolding of proteins.
What dictates differentiation in Giardia intestinalis?
An additional encystation protocol was developed that yielded high amounts
of mature 16N cysts. This enabled us to study the entire transcriptional response during differentiation using RNA-seq, thereby increasing the detection range from previously performed studies. Transcriptional changes appeared as highly coordinated cascades of up- and down regulation of genes,
both on the level of the entire transcriptome and putative regulators. Giardia
has surprisingly few transcription factors, hence differentiation must be mediated on different levels. Histone acetylation is known to be important for
correct progression of encystation (Sonda et al., 2010). Further characterization of epigenetic mechanisms will be vital to understand how differentiation
is orchestrated. DNA methylation regulates numerous processes in eukaryotic cells (e.g. cell differentiation), but have been reported to be absent in Giardia. However, recent technology advancement could be used to reinvestigate the methylation status in Giardia. The genome of Plasmodium falciparum was discovered to be methylated and these epigenetic marks were suggested to participate in silencing of virulence genes (Ponts et al., 2013).
Pieces missing in the differentiation puzzle
Largest changes in the transcriptional landscape were seen in the late phase
of encystation and the majority of these genes encode hypothetical proteins.
60
It is possible that these genes have a function during excystation and that the
late phase of encystation is in part a preparation for that process. Much remains to be explored about encystation and even more in the much understudied process of excystation.
Parasites of assemblage B differentiate poorly under in vitro conditions.
This indicates that vital signals are missing or are different from assemblage
A parasites. Therefore all information about encystation in Giardia so far,
builds on research of one isolate. Important clues of how the signaling to
induce differentiation could be learned using more than one assemblage.
Recently we have completed sequencing the genome and transcriptome of
Giardia muris, giving the first insights into a close relative of Giardia intestinalis (Jerlström-Hultqvist and Xu, unpublished data). The transcriptome
analysis of trophozoites isolated from the upper small intestines of mice,
matches the expressional profile of encysting cells. What similarities can be
detected of the encystation process between these species? The trophozoites
we see in vitro, do they exist in vivo?
VSP switch event during encystation
The VSP coat is disassembled during encystation as the cyst wall is assembled to protect the cyst. A switch event during differentiation has been described by previous studies and hypothesized to enable re-infection in the
same host and facilitate infections of different host species (von Allmen et
al., 2004; Carranza et al., 2002; Svärd et al., 1998). It is however, still puzzling why a switch event occur during encystation if the process is repeated
shortly after excystation.
In our transcriptional data, there is a remarkable change of expression of
the VSPs during differentiation. The highest transcribed VSP gene in the
population remains until 22 h of encystation and two different VSP transcripts dominate in mature cysts. The two VSPs activated during encystation
have an identical promoter region and are 97% identical in the coding region. The activation of the same VSPs have been detected before and appear
to be preferred during encystation (Weiland and Svärd, unpublished results).
This observation indicates a time frame and potentially which VSPs (out of
hundreds) that are activated during a switch event. This opens the possibility
for further studies of the mechanisms responsible for mediating antigenic
variation.
What controls the antigenic switch?
Little is known about the epigenetic landscape in Giardia and how chromatin remodeling is participating in the gene regulation. Histone acetylation is
the only known epigenetic modification associated to activation of vsp genes
(Kulakova et al., 2006). The RNAi-like machinery in Giardia is reported
61
degrade transcripts of all vsp genes, except one. The selection of the expressed vsp is selected in a concentration dependent manner (Prucca et al.,
2008). But how is this controlled? Are there chromatin modifications responsible for silencing/poising of vsps? Additionally it would be interesting
to investigate the subnuclear localization of vsp genes using FISH. Are there
transcriptionally active areas of euchromatin and could this differ between
the nuclei?
Moreover, single cell transcriptomics should be performed to investigate
the status of vsp gene expression. Antigenic variation is after all conducted at
the single cell level.
Is antigenic variation a common trait of diplomonad parasites?
Antigenic variation is an intriguing and difficult process to study. We know
very little about if this escape mechanism is a common trait of diplomonad
parasites. However, S. salmonicida have a large cysteine-rich gene family
and members of this family can localize to the surface of the parasite. Further studies to elucidate if these cysteine-rich proteins are used to perform
antigenic variation would be most interesting. Since no components of the
RNAi machinery have been found, the regulation must be different. We have
generated a high quality assembly of the G. muris genome (JerlströmHultqvist and Xu, unpublished data). Interestingly, the vsp genes are positioned differently compared to G. intestinalis. The vsp repertoire appears to
be smaller and vsp pseudogenes are often found in arrays close to telomeric
ends of the chromosomes. This indicates that G. muris have an alternative
way of maintaining these genes and possibly also regulating them compared
to G. intestinalis.
To conclude, the diplomonads are fascinating eukaryotic microbes with cellular processes adjusted to match their life style. The work in this thesis has
provided some insight of their adaptations, differences and similarities, but
also new interesting leads for future studies of diplomonad biology and virulence.
62
Sammanfattning på svenska (Summary in
Swedish)
Variationen av eukaryota organismer är väldigt stor, allt från mikroskopiskt
encelliga till flercelliga växter och djur. Diplomonader är exempel på en
grupp encelliga mikroorganismer som lever i syrefattiga eller syrefria miljöer, till exempel i tarmar hos djur eller i bottensediment. De flesta diplomonader har fördelat arvsmassan (DNA) i två cellkärnor, vilket skiljer dem från
övriga organismer. Diplomonader har olika livsstil, en del är parasiter medan
andra är frilevande. Parasiter är beroende av en värd för att överleva, hos
vilken den orsakar sjukdom.
Diplomonaden man vet mest om är Giardia intestinalis, en parasit som
lever i tunntarmen hos däggdjur. Där orsakar den sjukdomen giardiasis med
symptom som diarré, kräkningar och magkramper, men det finns även patienter som inte upplever några symptom alls. Varför individer reagerar så
olika och exakt hur parasiten orsakar sjukdom är inte helt känt. G. intestinalis finns i hela världen och beräknas infektera trehundra miljoner människor per år. I Sverige rapporteras det cirka 1500 fall per år och merparten av
dessa har blivit smittade utomlands.
Spironucleus salmonicida är också en diplomonad parasit som infekterar
olika organ hos laxfiskar. Infektionen orsakar hög dödlighet (”salmonicida”
betyder ”lax dödare”), vilket innebär den orsakar stora ekonomiska problem
för fiskeindustrin. Väldigt lite forskning har bedrivits på Spironucleus salmonicida och det är ännu okänt hur dessa parasiter sprids mellan fiskar.
För att öka kunskapen om virulens och den biologiska kunskapen om S.
salmonicida har vi sekvenserat genomet och skapat förutsättningar för att
göra funktionella studier i parasiten. Vi har skapat en transfektionsvektor
som gör det möjligt att studera hur olika proteiner lokaliserar i cellen och på
det viset kunna dra slutsatser dess funktion (Artikel I).
Analyserna av innehållet i genomet hos fiskparasiten gav viktig information om vilka metaboliska gener och olika genfamiljer som finns i jämförelse
med den besläktade parasiten G. intestinalis. Vi hittade även gener som i
andra organismer är associerade till mitokondrien. Alla eukaryoter har en
mitokondrie eller en mitokondrie-relaterad organell (MRO), till exempel en
mitosom. Det var känt sedan tidigare att Giardia har en mitosom, men vilken
MRO som finns i övriga diplomonader var okänt. Mitosomen är en väldigt
reducerad version av en mitokondrie som förlorat kapaciteten att generera
63
energi. Vi kunde visa att Spironucleus salmonicida har en hydrogenosom,
vilket är ytterligare en annan variant av en mitokondrie (Artikel II). Vi använde transfektionssystemet (från Artikel I) och lokaliserade möjliga hydrogenosom-proteiner och visade att dessa faktiskt finns denna organell. Vi
kunde dessutom visa att Spironucleus kan generera vätgas, vilket är en trolig
biprodukt från energiproduktionen som sker i organellen. Slutligen gjordes
en evolutionär analys som visade att anfadern till Giardia och Spironucleus
troligen hade en hydrogenosom, som var en anpassning till en syrefattig
miljö.
Annexiner är ett annat exempel på evolutionärt intressanta proteiner eftersom dessa finns i både växter, djur och bakterier. Dessa proteiner är multifunktionella och har förmågan att binda membran. Intressant nog har dessa
expanderat till stora genfamiljer i både S. salmonicida och Giardia. Dessa
proteiner är välstuderade i djur och växter, men för encelliga organismer
finns bara information om deras funktion i G. intestinalis. I Giardia-celler
lokaliserar många av dessa proteiner till en specifik del av cytoskelettet som
t.ex. till flagellerna eller till den ventrala disken. Vi karaktäriserade S. salmonicidas annexiner för att öka förståelsen hur dessa proteiner används och
hur de evolverat i dessa eukaryoter (Artikel III). Som i Giardias fall, lokaliserade många av Spironucleus Annexiner till specifika strukturer i cytoskelettet eller till membranet. Att hålla ihop cytoskelettet är förmodligen otroligt
viktigt för parasiterna eftersom de lever under svåra fysiologiska förhållanden, t.ex. i tarmen. Detta kan vara en förklaring till varför dessa organismer
har många Annexiner med så specificerade uppgifter.
Sammanfattningsvis gav dessa tre studier viktiga insikter i olika cellulära
funktioner i S. salmonicida, vilka kan ha stor betydelse vid infektioner i laxfiskar. De bidrog även till en ökad förståelse om diplomonader och evolutionen av mitokondrie-relaterade organeller och annexiner.
Giardia intestinalis finns i de två livsstadierna trofozoit och cysta. Cystorna
är det infektiösa stadiet som parasiten sprids via och oftast blir man infekterad via avföringskontaminerat dricksvatten. Infektionen börjar då trofozoiten
frigörs sig från cystan i den övre delen tunntarmen och fäster på tarmcellerna. Trofozoiten är det sjukdomsframkallande stadiet och för att öka sin chans
att överleva i värden, uttrycker trofozoiten ett ytprotein. Ytproteinet byts ut
regelbundet och på så sätt förblir parasiten oupptäckt av värdens immunförsvar. Denna strategi kallas antigenisk variation och används av många patogena organismer.
När trofozoiten förflyttas längre ner i tarmen, förändras de fysiologiska
förhållandena och signaler som ett högre pH och brist på lipider (fetter) leder
till bildande av cystor (encystering). Encysteringen innebär en gradvis förändring av trofozoitens form, metabolismen sänks och rörligheten försvinner, då cystan är ett vilostadie. Under denna process transporteras stora
mängder proteiner och kolhydrater till ytan på den encysterade cellen och
64
bygger upp en cystvägg. Det är möjligt att återskapa hela G. intestinalis
livscykel in vitro i laboratoriet, utan närvaro av ett värddjur. Detta har möjliggjort detaljstudier om t.ex hur cystväggen byggs upp, men mycket mindre
är känt angående processen excystering då trofozoiten återbildas.
Cystväggen är ett skyddande hölje, vilket gör att cystorna kan överleva
flera månader i kallt vatten. Ute i miljön är cystorna exponerade för flera
olika stressfaktorer. En sådan är UV-strålning, dels från solljuset men också
då det används vid vattenrening. Vi ville dels studera hur väl parasiten överlever UV-ljus men även vilka mekanismer som används för att reparera
DNA-skadorna som uppstår (Artikel IV). Vi använde både trofozoiter och
cystor för att kunna jämföra deras respons på strålningen. Vi kunde visa att
trofozoiter kan överleva högre strålningsdoser än cystor. Detta trots att trofozoiter aldrig exponeras för UV-ljus, eftersom de växer i tarmen. Vi kunde
visa att det sannolikt beror på att trofozoiterna har aktiva mekanismer för
DNA replikering, vilket gör att de har en chans att laga DNA-skadorna som
bildats. Eftersom cystorna blir bestrålade i ett vilostadie, utan aktiv replikation, kan de inte laga skadorna och får svårt att etablera sig som trofozoiter.
Informationen från den här studien är viktig då UV bestrålning används för
att desinfektera dricksvatten.
Encysteringen är en essentiell process för parasitens överlevnad och för
att kunna spridas. Genom att öka förståelsen för encysteringensprocessen
kan man förhoppningsvis även förhindra den och på så sätt stoppa spridningen av parasiten. Vi utvecklade en bättre encysterings-metod som genererar
större mängd cystor än tidigare metoder. För första gången användes RNAsekvensering för att kunna studera förändringar i geners uttryck under differentieringen från trofozoit till cysta (Artikel V). Analysen av de transkriptionella förändringarna visade att gener aktiveras och inaktiveras på ett högst
reglerat sätt. Den observationen gällde även för de gener som möjligen deltar
i denna gen reglering, t.ex transkriptionsfaktorer och histon-modifierare. De
största förändringarna skedde i processens slutfas, vilket de flesta tidigare
liknande studier missat. De flesta gener som ökar i uttryck kodar för protein
med okänd funktion. Därför har tio sådana proteiner karakteriserats närmre,
genom lokaliserings studier. Vi hittade proteiner som finns i cellkärnorna, i
membranet under cystväggen eller i en okänd struktur i cystan.
Under differentieringen sker förändringar i uttrycket av ytproteinerna som
ger upphov till antigenisk variation, vilket är intressant eftersom dessa proteiner kan bidra till att parasiten kan åter-infektera samma värd men också
användas för att infektera värdar av en annan art.
För att sammanfatta, är diplomonader komplexa eukaryota mikroorganismer
med cellulära processer som är anpassade till deras livsstil. Arbetet i denna
avhandling har bidragit till att öka kunskapen om dessa anpassningar, deras
skillnader och likheter, men också nya spännande upptäckter inför framtida
studier av diplomonaders biologi och virulens.
65
Acknowledgements
The past fiveish years have been both amazing and at times a struggle. I
could never have completed this work without the help and support from a
bunch of people.
First I would like to thank the Swedish funding agencies that enabled me to
work on these two cool parasites. I am grateful to FORMAS and VR for
supporting the projects I have been involved with.
I would like thank:
My supervisor Staffan, thanks for being the coolest scientist and person!! I
joined your group as a master student to work on a fish parasite that I never
even heard of (actually very few had!). Who knew parasites could be so
much fun?! I’m so happy you gave me the opportunity to stay for a PhD and
for believing in me through these years. Thank you for always taking the
time to discuss stuff, for always being enthusiastic about whatever results
and for giving me the freedom to explore my own research interests!
Karin T, you are the best and I’m so happy that you wanted to be my cosupervisor. Thank you for always being so positive, energetic and for all the
encouragement! I always enjoyed our science and ice-cream combined meetings 
My extra supporters:
Jon (the Mastermind), you are an amazing colleague, friend and scientist.
You have taught me so much and it’s been a great pleasure working with
you!! Thank you for always patiently answering countless questions (it is
quicker to ask you than to Google stuff, you know).
Britta (my sister in crime), you are an amazing person and I would not have
made it this far without you! We have had so many crazy and great times at
work and in the world outside of BMC. Thanks for being the best conference
company, for the pep-talks and for all the great food through the years!
Ulf (Mr Go-big-or-go-home), you are a crazy amazing kick-ass scientist! I
will always remember our 14 liter encystation experiments  Thank you for
66
getting involved in projects dealing with a “poo-parasite”, for your patience,
for all the help and for being the best whistler ever!
Past and present members of the Giardia/Spiro group:
Johan A (Ankarstocken), you are an inspiring person and the best spexactor/spex-movie director Micro has ever known! Thank you for all the fun
times over the years!! Emma, we were only shortly in the group together but
it’s always fun to meet you! Thanks for sharing VSP data! Karin S, my very
first office roomie, thank you for all the non-science talks! Mattias, it was
fun sharing office with me and the coffee machine, right? I’m happy to
prove you wrong, I will not be a PhD student forever  (oops hope I didn’t
jinxed it now). Karin H, it was great that you joined to work on Spiro!
Daniel S, good luck in the yeast world! Cedrique, good luck with your future plans! Marcela, thank you for being such a caring person and for bringing Maite to work! Ásgeir, it’s nice to know that the Spiro era will continue
with many amazing discoveries in the future. It was great collaborating on
the annexin paper! Thanks for introducing me to “Black Death”! Dimitra,
you always do your best and that will take you far. You have such great
things ahead of you and I look forward to work with you on the “lip project”
and H3! Eva M, thank you for keeping me sane at insane times and for all
the “fettis-luncher”. And for sharing the amazing Mango with me sometimes! Sara C, my frutinho lady, I loved having you in the group, good luck
in Paris! Johan L, thank you for always having such a sunny mood! Great to
have a lice/beer brewing/parasite loving person associated to the group!
Showgy, thank you for always being positive and for spreading good energy
in the lab! Cool bananas! Livia, thank you for always being so helpful and
sweet! Jingyi, great to have you in the group and good luck with your projects! Viktor, nice that someone took the challenge of doing knock-out experiments in possibly the most difficult organisms! Anders, good luck with
fish experiments! Karin S, good luck with your project!
Visiting Giardians: Cecilia, Ariana, Nahuel, Lubos and Brendan, it was
great to have you in the group and I hope we meet up somewhere, sometime
in the future!
Collaborators: Jan A (the tree man), thanks for fun collaborations on various Spiro projects! Feifei (the queen of bioinformatics) thanks for introducing me to the world of manual annotation and for being so sweet! Kjell H,
thank you for amazing TEM pics!
The Micro people: I met so many amazing people throughout the years,
thank you all for making Micro such a great place to work at!
Cia, great spex-movie-making memories! Sonchita, always fun to hang with
you and Adi! Bhupi, sorry for the mascara on the microscope oculars!
67
Shiying, you are the sweetest!! Disa, so happy to have you back at Micro!
Marie, horse power is the only true power, right? Klas, always great to meet
you! Anna L, my cookie-girl! Miss you and your laugh in the lab! Rachel,
my loveliest chick, we were the best teaching team ever! Erik, you are a
rock star! Johan R, the greatest dancer ever! Gerhart, thank you for always
being interested, curious about everything and a true scientist! Maaike, good
luck with everything in the future! Mirthe, my favorite red-head of all
times! Thank you for always caring, listening and for being my friend!
Lina, thank you for not going to Mexico in December and for being a great
friend! (And for having the best boyfriend that helped me with SIM, tack
Fredrik E!) Martin, Mr Bowtie Friday Magnus L, Micro’s TV-star! Fredrik S, you are a great teacher! Zhen and Jonas, good luck in the Dicty
world! Andrea, thanks for bringing worms to Micro! Benjamin, always
enjoy chatting with you! Yani, good luck with finishing up! Dirui, the fastest runner! Sanna, great to have you at Micro! Markus, the best joke teller!
Magnus A, Mr Bowtie Friday no.2, good luck with the PhD! Anirban, good
luck with your projects! Bork, my Dr Schmetterling! Good luck back home!
Cedric, will France win Eurovision song contest next year?
The ICM people: I would like to thank all the ICMers that I interacted with
these past years for many great times! Some needs extra mentioning; Petar,
Özden, David F, Petter, David B-N, Alex and the rest of the ICM Beer
club crew, thank you! Anna-Karin, lycka till i Chicago! Ewa G, tack för
alla pratstunder, plattor och media! Solan, Ulrika, Erika, Akiko, Anders,
Ylva, Åsa, Lena, Ali, Erling och Sofia, tack för all hjälp med det tekniska
och det administrativa!
The IMBIM people:
Jessica, thank you for sharing my latest Poldark obsession, for being just a
WhatsApp message away and a great friend! Tack för alla dagens kram!
Franzi, my tequila wing woman! Thank you for always being so positive
and for sharing this crazy last year experience. Looking forward to your
defense! Marlen, thanks for all the good times! Good luck in Seattle! Lisa
T, thank you for saving my thesis in the last minute and for being so sweet!
Eva, good luck with photography and science in the future! Michael, good
luck with future Westerns! Thanks to the rest of corridor D7:3 for many fun
times!
The Biology of Parasitism (BoP) course of 2013: Thank you for an amazing and crazy summer! I will never forget it  Work hard, party hard! Annette (the best beach company), Sima (those damn sporozoites), Beth (best
roomie ever), Tony (RNA lord and caffeine junkie), Blake and Jennie,
thanks for being the best course assistants! Gaelle (queen of making popcorn) and Esteban, good luck in Boston!! Angelica (Team Toxo rocked),
68
Esther, Silvia, Melanie, Lorenz, Mirko, Ricardo, Shen, Hugo and Christina (hope to see you in Switzerland!). Boris (& Andrea) and Kirk, thanks
for being great course directors!
Thanks to all my friends outside of BMC! I could never have done this without you.
Therese, tack för att du alltid finns där för mig och för att jag alltid är välkommen till Luddis. Jag hade inte varit den jag är idag utan dig! René,
thanks for all the amazing food and the good times through the years! Algot,
tack för att du är den finaste hunden. Islay, tack för att jag får vara din tant
Elin! Johanna, min favvo-smålänning! Tack för alla tisdags-joggar, utan dig
hade jag blivit knäpp(are)! Magdalena, du var min allra första lab partner, vi
var och är ett fantastiskt team ju! Maria, nu är det snart din tur att bära runt
på Monster på riktigt, ser fram emot din disputation! Utan dig och Mags
hade jag aldrig hittat min ”inre-Gunde”! Hanna, tack för att alla luncher,
fikor och för alla roliga stunder i och utanför labbet! Katarina, kommer
alltid komma ihåg vår ”modell karriär”. Otroligt kul att hålla i ett Eppendorfrör tillsammans med dig! Malin, lycka till därnere i Skåneland med kommande får-farmen! Anna Å, tack för alla härliga middagar! Lycka till på din
stora dag! Jonas L, tack för jag fick författa hemma hos er, ser fram emot
bröllopet! Emelie, bästa rese- och konsertsällskapet! Tack för alla fantastiska och galna minnen. Robin, du är den envisaste jag känner! ”Man kan
inte bromsa sig ur en uppförsbacke!”.
Tack till hela Einarsson-klanen hemma i Askersund med omnejd!!
Min finaste syster Linnéa, tack för att du berikat mitt liv med diverse djur;
Comics, Pim, Lasso, Vazzen och Ammi!
Min bästa lillebror Henke, tack för att du är så mycket mer tekniskt smartare
än mig och för att du hjälper mig!
Mamma, tack för all fantastisk mat, för att du alltid verkar veta exakt vad
jag behöver och för att du alltid finns där.
Pappa, tack för att du hjälpt mig att flytta runt här i Uppsala och för att du
alltid lagar min cykel. Hur hårt jag än jobbar, så vinner du alltid! Ska man
göra nått, så ska man göra det ordentligt.
Min bästa Farmor, tack för allt du gett mig. Du fick aldrig se mig bli en
färdig doktor, men jag hoppas du och Farfar är stolta däruppe i himmelen!
69
References
Adam, R.D. (2001). Biology of Giardia lamblia. Clin. Microbiol. Rev. 14, 447–475.
Adam, R.D., Nigam, A., Seshadri, V., Martens, C.A., Farneth, G.A., Morrison,
H.G., Nash, T.E., Porcella, S.F., and Patel, R. (2010). The Giardia lamblia vsp
gene repertoire: characteristics, genomic organization, and evolution. BMC Genomics 11, 424.
Adl, S.M., Simpson, A.G.B., Farmer, M.A., Andersen, R.A., Anderson, O.R., Barta,
J.R., Bowser, S.S., Brugerolle, G., Fensome, R.A., Fredericq, S., et al. (2005).
The new higher level classification of eukaryotes with emphasis on the taxonomy of protists. J. Eukaryot. Microbiol. 52, 399–451.
von Allmen, N., Bienz, M., Hemphill, A., and Müller, N. (2004). Experimental infections of neonatal mice with cysts of Giardia lamblia clone GS/M-83-H7 are
associated with an antigenic reset of the parasite. Infect. Immun. 72, 4763–4771.
Andersson, J.O., Sjögren, A.M., Horner, D.S., Murphy, C.A., Dyal, P.L., Svärd,
S.G., Logsdon, J.M., Ragan, M.A., Hirt, R.P., and Roger, A.J. (2007). A genomic survey of the fish parasite Spironucleus salmonicida indicates genomic
plasticity among diplomonads and significant lateral gene transfer in eukaryote
genome evolution. BMC Genomics 8, 51.
Ankarklev, J., Jerlström-Hultqvist, J., Ringqvist, E., Troell, K., and Svärd, S.G.
(2010). Behind the smile: cell biology and disease mechanisms of Giardia species. Nat. Rev. Microbiol. 8, 413–422.
Ankarklev, J., Hestvik, E., Lebbad, M., Lindh, J., Kaddu-Mulindwa, D.H., Andersson, J.O., Tylleskär, T., Tumwine, J.K., and Svärd, S.G. (2012). Common coinfections of Giardia intestinalis and Helicobacter pylori in non-symptomatic
Ugandan children. PLoS Negl. Trop. Dis. 6, e1780.
Baldursson, S., and Karanis, P. (2011). Waterborne transmission of protozoan parasites: review of worldwide outbreaks - an update 2004-2010. Water Res. 45,
6603–6614.
Bazán-Tejeda, M.L., Argüello-García, R., Bermúdez-Cruz, R.M., Robles-Flores, M.,
and Ortega-Pierres, G. (2007). Protein kinase C isoforms from Giardia duodenalis: identification and functional characterization of a beta-like molecule during encystment. Arch. Microbiol. 187, 55–66.
Benchimol, M. (2004). Giardia lamblia: behavior of the nuclear envelope. Parasitol.
Res. 94, 254–264.
Benchimol, M., Piva, B., Campanati, L., and de Souza, W. (2004). Visualization of
the funis of Giardia lamblia by high-resolution field emission scanning electron
microscopy--new insights. J. Struct. Biol. 147, 102–115.
Bernander, R., Palm, J.E., and Svärd, S.G. (2001). Genome ploidy in different stages
of the Giardia lamblia life cycle. Cell. Microbiol. 3, 55–62.
Best, A.A., Morrison, H.G., McArthur, A.G., Sogin, M.L., and Olsen, G.J. (2004).
Evolution of eukaryotic transcription: insights from the genome of Giardia
lamblia. Genome Res. 14, 1537–1547.
70
Bienz, M., Siles-Lucas, M., Wittwer, P., and Müller, N. (2001). vsp gene expression
by Giardia lamblia clone GS/M-83-H7 during antigenic variation in vivo and in
vitro. Infect. Immun. 69, 5278–5285.
Birkeland, S.R., Preheim, S.P., Davids, B.J., Cipriano, M.J., Palm, D., Reiner, D.S.,
Svärd, S.G., Gillin, F.D., and McArthur, A.G. (2010). Transcriptome analyses
of the Giardia lamblia life cycle. Mol. Biochem. Parasitol. 174, 62–65.
Boucher, S.E., and Gillin, F.D. (1990). Excystation of in vitro-derived Giardia
lamblia cysts. Infect. Immun. 58, 3516–3522.
Brinkmann, H., van der Giezen, M., Zhou, Y., Poncelin de Raucourt, G., and
Philippe, H. (2005). An empirical assessment of long-branch attraction artefacts
in deep eukaryotic phylogenomics. Syst. Biol. 54, 743–757.
Brown, D.M., Upcroft, J.A., Edwards, M.R., and Upcroft, P. (1998). Anaerobic
bacterial metabolism in the ancient eukaryote Giardia duodenalis. Int. J. Parasitol. 28, 149–164.
Cacciò, S.M., and Ryan, U. (2008). Molecular epidemiology of giardiasis. Mol.
Biochem. Parasitol. 160, 75–80.
Carpenter, M.L., Assaf, Z.J., Gourguechon, S., and Cande, W.Z. (2012). Nuclear
inheritance and genetic exchange without meiosis in the binucleate parasite Giardia intestinalis. J. Cell Sci. 125, 2523–2532.
Carranza, P.G., Feltes, G., Ropolo, A., Quintana, S.M.C., Touz, M.C., and Luján,
H.D. (2002). Simultaneous expression of different variant-specific surface proteins in single Giardia lamblia trophozoites during encystation. Infect. Immun.
70, 5265–5268.
Chatterjee, A., Carpentieri, A., Ratner, D.M., Bullitt, E., Costello, C.E., Robbins,
P.W., and Samuelson, J. (2010). Giardia cyst wall protein 1 is a lectin that binds
to curled fibrils of the GalNAc homopolymer. PLoS Pathog. 6, e1001059.
Chen, Y.-H., Su, L.-H., and Sun, C.-H. (2008). Incomplete nonsense-mediated
mRNA decay in Giardia lamblia. Int. J. Parasitol. 38, 1305–1317.
Chiu, P.-W., Huang, Y.-C., Pan, Y.-J., Wang, C.-H., and Sun, C.-H. (2010). A novel
family of cyst proteins with epidermal growth factor repeats in Giardia lamblia.
PLoS Negl. Trop. Dis. 4, e677.
Chuang, S.-F., Su, L.-H., Cho, C.-C., Pan, Y.-J., and Sun, C.-H. (2012). Functional
redundancy of two Pax-like proteins in transcriptional activation of cyst wall
protein genes in Giardia lamblia. PloS One 7, e30614.
Cotton, J.A., Beatty, J.K., and Buret, A.G. (2011). Host parasite interactions and
pathophysiology in Giardia infections. Int. J. Parasitol. 41, 925–933.
Cotton, J.A., Bhargava, A., Ferraz, J.G., Yates, R.M., Beck, P.L., and Buret, A.G.
(2014). Giardia duodenalis cathepsin B proteases degrade intestinal epithelial
interleukin-8 and attenuate interleukin-8-induced neutrophil chemotaxis. Infect.
Immun. 82, 2772–2787.
Dagley, M.J., Dolezal, P., Likic, V.A., Smid, O., Purcell, A.W., Buchanan, S.K.,
Tachezy, J., and Lithgow, T. (2009). The protein import channel in the outer mitosomal membrane of Giardia intestinalis. Mol. Biol. Evol. 26, 1941–1947.
Davids, B.J., Mehta, K., Fesus, L., McCaffery, J.M., and Gillin, F.D. (2004). Dependence of Giardia lamblia encystation on novel transglutaminase activity.
Mol. Biochem. Parasitol. 136, 173–180.
Davids, B.J., Reiner, D.S., Birkeland, S.R., Preheim, S.P., Cipriano, M.J., McArthur,
A.G., and Gillin, F.D. (2006). A new family of giardial cysteine-rich non-VSP
protein genes and a novel cyst protein. PloS One 1, e44.
Davids, B.J., Williams, S., Lauwaet, T., Palanca, T., and Gillin, F.D. (2008). Giardia
lamblia aurora kinase: a regulator of mitosis in a binucleate parasite. Int. J. Parasitol. 38, 353–369.
71
Dawson, S.C. (2010). An insider’s guide to the microtubule cytoskeleton of Giardia.
Cell. Microbiol. 12, 588–598.
Dawson, S.C., and House, S.A. (2010). Life with eight flagella: flagellar assembly
and division in Giardia. Curr. Opin. Microbiol. 13, 480–490.
Dawson, S.C., Pham, J.K., House, S.A., Slawson, E.E., Cronembold, D., and Cande,
W.Z. (2008). Stable transformation of an episomal protein-tagging shuttle vector in the piscine diplomonad Spironucleus vortens. BMC Microbiol. 8, 71.
Deitsch, K.W., Lukehart, S.A., and Stringer, J.R. (2009). Common strategies for
antigenic variation by bacterial, fungal and protozoan pathogens. Nat. Rev. Microbiol. 7, 493–503.
Dobell, C. (1920). The Discovery of the Intestinal Protozoa of Man. Proc. R. Soc.
Med. 13, 1–15.
Dolezal, P., Smíd, O., Rada, P., Zubácová, Z., Bursać, D., Suták, R., Nebesárová, J.,
Lithgow, T., and Tachezy, J. (2005). Giardia mitosomes and trichomonad hydrogenosomes share a common mode of protein targeting. Proc. Natl. Acad. Sci.
U. S. A. 102, 10924–10929.
Dolezal, P., Likic, V., Tachezy, J., and Lithgow, T. (2006). Evolution of the molecular machines for protein import into mitochondria. Science 313, 314–318.
Dubey, J.P., Lindsay, D.S., and Speer, C.A. (1998). Structures of Toxoplasma
gondii tachyzoites, bradyzoites, and sporozoites and biology and development
of tissue cysts. Clin. Microbiol. Rev. 11, 267–299.
Eckmann, L., Laurent, F., Langford, T.D., Hetsko, M.L., Smith, J.R., Kagnoff, M.F.,
and Gillin, F.D. (2000). Nitric oxide production by human intestinal epithelial
cells and competition for arginine as potential determinants of host defense
against the lumen-dwelling pathogen Giardia lamblia. J. Immunol. Baltim. Md
1950 164, 1478–1487.
Edwards, D.I. (1993). Nitroimidazole drugs--action and resistance mechanisms. I.
Mechanisms of action. J. Antimicrob. Chemother. 31, 9–20.
Ellis, J.G., Davila, M., and Chakrabarti, R. (2003). Potential involvement of extracellular signal-regulated kinase 1 and 2 in encystation of a primitive eukaryote,
Giardia lamblia. Stage-specific activation and intracellular localization. J. Biol.
Chem. 278, 1936–1945.
English, J., Pearson, G., Wilsbacher, J., Swantek, J., Karandikar, M., Xu, S., and
Cobb, M.H. (1999). New insights into the control of MAP kinase pathways.
Exp. Cell Res. 253, 255–270.
Erlandsen, S.L., Macechko, P.T., van Keulen, H., and Jarroll, E.L. (1996). Formation of the Giardia cyst wall: studies on extracellular assembly using immunogold labeling and high resolution field emission SEM. J. Eukaryot. Microbiol.
43, 416–429.
Faso, C., and Hehl, A.B. (2011). Membrane trafficking and organelle biogenesis in
Giardia lamblia: use it or lose it. Int. J. Parasitol. 41, 471–480.
Faso, C., Bischof, S., and Hehl, A.B. (2013a). The proteome landscape of Giardia
lamblia encystation. PloS One 8, e83207.
Faso, C., Konrad, C., Schraner, E.M., and Hehl, A.B. (2013b). Export of cyst wall
material and Golgi organelle neogenesis in Giardia lamblia depend on endoplasmic reticulum exit sites. Cell. Microbiol. 15, 537–553.
Franzén, O., Jerlström-Hultqvist, J., Castro, E., Sherwood, E., Ankarklev, J., Reiner,
D.S., Palm, D., Andersson, J.O., Andersson, B., and Svärd, S.G. (2009). Draft
genome sequencing of giardia intestinalis assemblage B isolate GS: is human
giardiasis caused by two different species? PLoS Pathog. 5, e1000560.
72
Franzén, O., Jerlström-Hultqvist, J., Einarsson, E., Ankarklev, J., Ferella, M., Andersson, B., and Svärd, S.G. (2013). Transcriptome profiling of Giardia intestinalis using strand-specific RNA-seq. PLoS Comput. Biol. 9, e1003000.
Frénal, K., and Soldati-Favre, D. (2009). Role of the parasite and host cytoskeleton
in apicomplexa parasitism. Cell Host Microbe 5, 602–611.
Gargantini, P.R., Serradell, M.C., Torri, A., and Lujan, H.D. (2012). Putative SF2
helicases of the early-branching eukaryote Giardia lamblia are involved in antigenic variation and parasite differentiation into cysts. BMC Microbiol. 12, 284.
Gelanew, T., Lalle, M., Hailu, A., Pozio, E., and Cacciò, S.M. (2007). Molecular
characterization of human isolates of Giardia duodenalis from Ethiopia. Acta
Trop. 102, 92–99.
Gerbaba, T.K., Gupta, P., Rioux, K., Hansen, D., and Buret, A.G. (2015). Giardia
duodenalis-induced alterations of commensal bacteria kill Caenorhabditis elegans: a new model to study microbial-microbial interactions in the gut. Am. J.
Physiol. Gastrointest. Liver Physiol. 308, G550–G561.
Gerwig, G.J., van Kuik, J.A., Leeflang, B.R., Kamerling, J.P., Vliegenthart, J.F.G.,
Karr, C.D., and Jarroll, E.L. (2002). The Giardia intestinalis filamentous cyst
wall contains a novel beta(1-3)-N-acetyl-D-galactosamine polymer: a structural
and conformational study. Glycobiology 12, 499–505.
Gibson, C., Schanen, B., Chakrabarti, D., and Chakrabarti, R. (2006). Functional
characterisation of the regulatory subunit of cyclic AMP-dependent protein kinase A homologue of Giardia lamblia: Differential expression of the regulatory
and catalytic subunits during encystation. Int. J. Parasitol. 36, 791–799.
Gillin, F.D., Reiner, D.S., Gault, M.J., Douglas, H., Das, S., Wunderlich, A., and
Sauch, J.F. (1987). Encystation and expression of cyst antigens by Giardia
lamblia in vitro. Science 235, 1040–1043.
Glover, L., Hutchinson, S., Alsford, S., McCulloch, R., Field, M.C., and Horn, D.
(2013). Antigenic variation in African trypanosomes: the importance of chromosomal and nuclear context in VSG expression control. Cell. Microbiol. 15,
1984–1993.
Gourguechon, S., and Cande, W.Z. (2011). Rapid tagging and integration of genes
in Giardia intestinalis. Eukaryot. Cell 10, 142–145.
Goyal, N., Rishi, P., and Shukla, G. (2013). Lactobacillus rhamnosus GG antagonizes Giardia intestinalis induced oxidative stress and intestinal disaccharidases: an
experimental study. World J. Microbiol. Biotechnol. 29, 1049–1057.
Guizetti, J., and Scherf, A. (2013). Silence, activate, poise and switch! Mechanisms
of antigenic variation in Plasmodium falciparum. Cell. Microbiol. 15, 718–726.
Gull, K. (1999). The cytoskeleton of trypanosomatid parasites. Annu. Rev. Microbiol. 53, 629–655.
Guo, F.C., and Woo, P.T.K. (2004). Experimental infections of Atlantic salmon
Salmo salar with Spironucleus barkhanus. Dis. Aquat. Organ. 61, 59–66.
Hagen, K.D., Hirakawa, M.P., House, S.A., Schwartz, C.L., Pham, J.K., Cipriano,
M.J., De La Torre, M.J., Sek, A.C., Du, G., Forsythe, B.M., et al. (2011). Novel
structural components of the ventral disc and lateral crest in Giardia intestinalis.
PLoS Negl. Trop. Dis. 5, e1442.
Hanevik, K., Wensaas, K.-A., Rortveit, G., Eide, G.E., Mørch, K., and Langeland,
N. (2014). Irritable bowel syndrome and chronic fatigue 6 years after giardia infection: a controlled prospective cohort study. Clin. Infect. Dis. Off. Publ. Infect. Dis. Soc. Am. 59, 1394–1400.
Haque, R., Roy, S., Kabir, M., Stroup, S.E., Mondal, D., and Houpt, E.R. (2005).
Giardia assemblage A infection and diarrhea in Bangladesh. J. Infect. Dis. 192,
2171–2173.
73
Hehl, A.B., Marti, M., and Köhler, P. (2000). Stage-specific expression and targeting of cyst wall protein-green fluorescent protein chimeras in Giardia. Mol. Biol. Cell 11, 1789–1800.
Hjort, K., Goldberg, A.V., Tsaousis, A.D., Hirt, R.P., and Embley, T.M. (2010).
Diversity and reductive evolution of mitochondria among microbial eukaryotes.
Philos. Trans. R. Soc. Lond. B. Biol. Sci. 365, 713–727.
Hofmann, A., Osman, A., Leow, C.Y., Driguez, P., McManus, D.P., and Jones,
M.K. (2010). Parasite annexins--new molecules with potential for drug and vaccine development. BioEssays News Rev. Mol. Cell. Dev. Biol. 32, 967–976.
Holberton, D.V. (1974). Attachment of Giardia-a hydrodynamic model based on
flagellar activity. J. Exp. Biol. 60, 207–221.
Homan, W.L., and Mank, T.G. (2001). Human giardiasis: genotype linked differences in clinical symptomatology. Int. J. Parasitol. 31, 822–826.
House, S.A., Richter, D.J., Pham, J.K., and Dawson, S.C. (2011). Giardia flagellar
motility is not directly required to maintain attachment to surfaces. PLoS
Pathog. 7, e1002167.
Humen, M.A., De Antoni, G.L., Benyacoub, J., Costas, M.E., Cardozo, M.I., Kozubsky, L., Saudan, K.-Y., Boenzli-Bruand, A., Blum, S., Schiffrin, E.J., et al.
(2005). Lactobacillus johnsonii La1 antagonizes Giardia intestinalis in vivo. Infect. Immun. 73, 1265–1269.
Iyer, L.M., Anantharaman, V., Wolf, M.Y., and Aravind, L. (2008). Comparative
genomics of transcription factors and chromatin proteins in parasitic protists and
other eukaryotes. Int. J. Parasitol. 38, 1–31.
Januschka, M.M., Erlandsen, S.L., Bemrick, W.J., Schupp, D.G., and Feely, D.E.
(1988). A comparison of Giardia microti and Spironucleus muris cysts in the
vole: an immunocytochemical, light, and electron microscopic study. J. Parasitol. 74, 452–458.
Jedelský, P.L., Doležal, P., Rada, P., Pyrih, J., Smíd, O., Hrdý, I., Sedinová, M.,
Marcinčiková, M., Voleman, L., Perry, A.J., et al. (2011). The minimal proteome in the reduced mitochondrion of the parasitic protist Giardia intestinalis.
PloS One 6, e17285.
Jenikova, G., Hruz, P., Andersson, M.K., Tejman-Yarden, N., Ferreira, P.C.D., Andersen, Y.S., Davids, B.J., Gillin, F.D., Svärd, S.G., Curtiss, R., et al. (2011).
Α1-giardin based live heterologous vaccine protects against Giardia lamblia infection in a murine model. Vaccine 29, 9529–9537.
Jerlström-Hultqvist, J., Franzén, O., Ankarklev, J., Xu, F., Nohýnková, E., Andersson, J.O., Svärd, S.G., and Andersson, B. (2010). Genome analysis and comparative genomics of a Giardia intestinalis assemblage E isolate. BMC Genomics
11, 543.
Jerlström-Hultqvist, J., Stadelmann, B., Birkestedt, S., Hellman, U., and Svärd, S.G.
(2012). Plasmid vectors for proteomic analyses in Giardia: purification of virulence factors and analysis of the proteasome. Eukaryot. Cell 11, 864–873.
Jiráková, K., Kulda, J., and Nohýnková, E. (2012). How nuclei of Giardia pass
through cell differentiation: semi-open mitosis followed by nuclear interconnection. Protist 163, 465–479.
Jørgensen, A., and Sterud, E. (2006). The marine pathogenic genotype of Spironucleus barkhanus from farmed salmonids redescribed as Spironucleus salmonicida n. sp. J. Eukaryot. Microbiol. 53, 531–541.
Jørgensen, A., and Sterud, E. (2007). Phylogeny of spironucleus (eopharyngia:
diplomonadida: hexamitinae). Protist 158, 247–254.
Kabnick, K.S., and Peattie, D.A. (1990). In situ analyses reveal that the two nuclei
of Giardia lamblia are equivalent. J. Cell Sci. 95 ( Pt 3), 353–360.
74
Kamikawa, R., Inagaki, Y., Tokoro, M., Roger, A.J., and Hashimoto, T. (2011).
Split introns in the genome of Giardia intestinalis are excised by spliceosomemediated trans-splicing. Curr. Biol. CB 21, 311–315.
Kane, A.V., Ward, H.D., Keusch, G.T., and Pereira, M.E. (1991). In vitro encystation of Giardia lamblia: large-scale production of in vitro cysts and strain and
clone differences in encystation efficiency. J. Parasitol. 77, 974–981.
Karanis, P., and Ey, P.L. (1998). Characterization of axenic isolates of Giardia intestinalis established from humans and animals in Germany. Parasitol. Res. 84,
442–449.
Karr, C.D., and Jarroll, E.L. (2004). Cyst wall synthase: Nacetylgalactosaminyltransferase activity is induced to form the novel Nacetylgalactosamine polysaccharide in the Giardia cyst wall. Microbiol. Read.
Engl. 150, 1237–1243.
Keeling, P.J., and Doolittle, W.F. (1997). Widespread and ancient distribution of a
noncanonical genetic code in diplomonads. Mol. Biol. Evol. 14, 895–901.
Kent, M.., Ellis, J., Fournie, J.., Dawe, S.., Bagshaw, J.., and Whitaker, D.. (1992).
Systemic hexamitid (Protozoa: Diplomonadida) infection in seawater pen-reared
chinook salmon Oncorhynchus tshawytscha. Dis. Aquat. Organ. 1992, 81–89.
Kim, J., Lee, H.Y., Lee, M.-A., Yong, T.-S., Lee, K.-H., and Park, S.-J. (2013).
Identification of α-11 giardin as a flagellar and surface component of Giardia
lamblia. Exp. Parasitol. 135, 227–233.
Kim, K.-T., Mok, M.T.S., and Edwards, M.R. (2005). Protein kinase B from Giardia
intestinalis. Biochem. Biophys. Res. Commun. 334, 333–341.
Kolisko, M., Cepicka, I., Hampl, V., Leigh, J., Roger, A.J., Kulda, J., Simpson,
A.G.B., and Flegr, J. (2008). Molecular phylogeny of diplomonads and enteromonads based on SSU rRNA, alpha-tubulin and HSP90 genes: implications for
the evolutionary history of the double karyomastigont of diplomonads. BMC
Evol. Biol. 8, 205.
Konrad, C., Spycher, C., and Hehl, A.B. (2010). Selective Condensation Drives
Partitioning and Sequential Secretion of Cyst Wall Proteins in Differentiating
Giardia lamblia. PLoS Pathog 6, e1000835.
Koonin, E.V. (2010). The origin and early evolution of eukaryotes in the light of
phylogenomics. Genome Biol. 11, 209.
Kotloff, K.L., Nataro, J.P., Blackwelder, W.C., Nasrin, D., Farag, T.H., Panchalingam, S., Wu, Y., Sow, S.O., Sur, D., Breiman, R.F., et al. (2013). Burden and
aetiology of diarrhoeal disease in infants and young children in developing
countries (the Global Enteric Multicenter Study, GEMS): a prospective, casecontrol study. Lancet Lond. Engl. 382, 209–222.
Koudela, B., Nohýnková, E., Vítovec, J., Pakandl, M., and Kulda, J. (1991). Giardia
infection in pigs: detection and in vitro isolation of trophozoites of the Giardia
intestinalis group. Parasitology 102 Pt 2, 163–166.
Kulakova, L., Singer, S.M., Conrad, J., and Nash, T.E. (2006). Epigenetic mechanisms are involved in the control of Giardia lamblia antigenic variation. Mol.
Microbiol. 61, 1533–1542.
Lane, S., and Lloyd, D. (2002). Current trends in research into the waterborne parasite Giardia. Crit. Rev. Microbiol. 28, 123–147.
Lauwaet, T., Davids, B.J., Torres-Escobar, A., Birkeland, S.R., Cipriano, M.J., Preheim, S.P., Palm, D., Svärd, S.G., McArthur, A.G., and Gillin, F.D. (2007). Protein phosphatase 2A plays a crucial role in Giardia lamblia differentiation. Mol.
Biochem. Parasitol. 152, 80–89.
75
Lebbad, M., Petersson, I., Karlsson, L., Botero-Kleiven, S., Andersson, J.O., Svenungsson, B., and Svärd, S.G. (2011). Multilocus genotyping of human Giardia
isolates suggests limited zoonotic transmission and association between assemblage B and flatulence in children. PLoS Negl. Trop. Dis. 5, e1262.
Leitsch, D. (2015). Drug Resistance in the Microaerophilic Parasite Giardia lamblia.
Curr. Trop. Med. Rep. 2, 128–135.
Leitsch, D., Schlosser, S., Burgess, A., and Duchêne, M. (2012). Nitroimidazole
drugs vary in their mode of action in the human parasite Giardia lamblia. Int. J.
Parasitol. Drugs Drug Resist. 2, 166–170.
Li, W., Saraiya, A.A., and Wang, C.C. (2011). Gene regulation in Giardia lambia
involves a putative microRNA derived from a small nucleolar RNA. PLoS Negl.
Trop. Dis. 5, e1338.
Li, W., Saraiya, A.A., and Wang, C.C. (2013). Experimental verification of the
identity of variant-specific surface proteins in Giardia lamblia trophozoites.
mBio 4, e00321–00313.
Liao, J.-Y., Guo, Y.-H., Zheng, L.-L., Li, Y., Xu, W.-L., Zhang, Y.-C., Zhou, H.,
Lun, Z.-R., Ayala, F.J., and Qu, L.-H. (2014). Both endo-siRNAs and tRNAderived small RNAs are involved in the differentiation of primitive eukaryote
Giardia lamblia. Proc. Natl. Acad. Sci. U. S. A. 111, 14159–14164.
Lin, B.-C., Su, L.-H., Weng, S.-C., Pan, Y.-J., Chan, N.-L., Li, T.-K., Wang, H.-C.,
and Sun, C.-H. (2013). DNA topoisomerase II is involved in regulation of cyst
wall protein genes and differentiation in Giardia lamblia. PLoS Negl. Trop. Dis.
7, e2218.
Linden, K.G., Shin, G.-A., Faubert, G., Cairns, W., and Sobsey, M.D. (2002). UV
disinfection of Giardia lamblia cysts in water. Environ. Sci. Technol. 36, 2519–
2522.
Lizarbe, M.A., Barrasa, J.I., Olmo, N., Gavilanes, F., and Turnay, J. (2013). Annexin-phospholipid interactions. Functional implications. Int. J. Mol. Sci. 14, 2652–
2683.
Lopez, A.B., Sener, K., Jarroll, E.L., and van Keulen, H. (2003). Transcription regulation is demonstrated for five key enzymes in Giardia intestinalis cyst wall polysaccharide biosynthesis. Mol. Biochem. Parasitol. 128, 51–57.
Lujan, H.D. (2011). Mechanisms of adaptation in the intestinal parasite Giardia
lamblia. Essays Biochem. 51, 177–191.
Luján, H.D., Mowatt, M.R., Conrad, J.T., Bowers, B., and Nash, T.E. (1995). Identification of a novel Giardia lamblia cyst wall protein with leucine-rich repeats.
Implications for secretory granule formation and protein assembly into the cyst
wall. J. Biol. Chem. 270, 29307–29313.
Luján, H.D., Mowatt, M.R., Byrd, L.G., and Nash, T.E. (1996). Cholesterol starvation induces differentiation of the intestinal parasite Giardia lamblia. Proc. Natl.
Acad. Sci. U. S. A. 93, 7628–7633.
Ma’ayeh, S.Y., and Brook-Carter, P.T. (2012). Representational difference analysis
identifies specific genes in the interaction of Giardia duodenalis with the murine
intestinal epithelial cell line, IEC-6. Int. J. Parasitol. 42, 501–509.
Mai, Z., Ghosh, S., Frisardi, M., Rosenthal, B., Rogers, R., and Samuelson, J.
(1999). Hsp60 is targeted to a cryptic mitochondrion-derived organelle (“crypton”) in the microaerophilic protozoan parasite Entamoeba histolytica. Mol.
Cell. Biol. 19, 2198–2205.
Makiuchi, T., and Nozaki, T. (2014). Highly divergent mitochondrion-related organelles in anaerobic parasitic protozoa. Biochimie 100, 3–17.
76
Marti, M., Li, Y., Schraner, E.M., Wild, P., Köhler, P., and Hehl, A.B. (2003a). The
secretory apparatus of an ancient eukaryote: protein sorting to separate export
pathways occurs before formation of transient Golgi-like compartments. Mol.
Biol. Cell 14, 1433–1447.
Marti, M., Regös, A., Li, Y., Schraner, E.M., Wild, P., Müller, N., Knopf, L.G., and
Hehl, A.B. (2003b). An ancestral secretory apparatus in the protozoan parasite
Giardia intestinalis. J. Biol. Chem. 278, 24837–24848.
Martincová, E., Voleman, L., Pyrih, J., Žárský, V., Vondráčková, P., Kolísko, M.,
Tachezy, J., and Doležal, P. (2015). Probing the Biology of Giardia intestinalis
Mitosomes Using In Vivo Enzymatic Tagging. Mol. Cell. Biol. 35, 2864–2874.
McCaffery, J.M., and Gillin, F.D. (1994). Giardia lamblia: ultrastructural basis of
protein transport during growth and encystation. Exp. Parasitol. 79, 220–235.
Midlej, V., Meinig, I., de Souza, W., and Benchimol, M. (2013). A new set of carbohydrate-positive vesicles in encysting Giardia lamblia. Protist 164, 261–271.
Millet, C.O.M., Cable, J., and Lloyd, D. (2010). The diplomonad fish parasite Spironucleus vortens produces hydrogen. J. Eukaryot. Microbiol. 57, 400–404.
Millet, C.O.M., Lloyd, D., Coogan, M.P., Rumsey, J., and Cable, J. (2011). Carbohydrate and amino acid metabolism of Spironucleus vortens. Exp. Parasitol.
129, 17–26.
Millet, C.O.M., Williams, C.F., Hayes, A.J., Hann, A.C., Cable, J., and Lloyd, D.
(2013). Mitochondria-derived organelles in the diplomonad fish parasite Spironucleus vortens. Exp. Parasitol. 135, 262–273.
Monis, P.T., Andrews, R.H., Mayrhofer, G., and Ey, P.L. (2003). Genetic diversity
within the morphological species Giardia intestinalis and its relationship to host
origin. Infect. Genet. Evol. J. Mol. Epidemiol. Evol. Genet. Infect. Dis. 3, 29–
38.
Monis, P.T., Caccio, S.M., and Thompson, R.C.A. (2009). Variation in Giardia:
towards a taxonomic revision of the genus. Trends Parasitol. 25, 93–100.
Morf, L., Spycher, C., Rehrauer, H., Fournier, C.A., Morrison, H.G., and Hehl, A.B.
(2010). The Transcriptional Response to Encystation Stimuli in Giardia lamblia
Is Restricted to a Small Set of Genes. Eukaryot. Cell 9, 1566–1576.
Morrison, H.G., McArthur, A.G., Gillin, F.D., Aley, S.B., Adam, R.D., Olsen, G.J.,
Best, A.A., Cande, W.Z., Chen, F., Cipriano, M.J., et al. (2007). Genomic minimalism in the early diverging intestinal parasite Giardia lamblia. Science 317,
1921–1926.
Muhsen, K., and Levine, M.M. (2012). A systematic review and meta-analysis of the
association between Giardia lamblia and endemic pediatric diarrhea in developing countries. Clin. Infect. Dis. Off. Publ. Infect. Dis. Soc. Am. 55 Suppl 4,
S271–S293.
Muller, E. (1889). Ett fynd af cercomonas intestinalis i jejunum från människa MÜLLER - 2009 - Nordiskt Medicinskt Arkiv - Wiley Online Library. Nord.
Med. Ark. 1889, 1–13.
Müller, M. (1993). The hydrogenosome. J. Gen. Microbiol. 139, 2879–2889.
Müller, J., Ley, S., Felger, I., Hemphill, A., and Müller, N. (2008). Identification of
differentially expressed genes in a Giardia lamblia WB C6 clone resistant to
nitazoxanide and metronidazole. J. Antimicrob. Chemother. 62, 72–82.
Nageshan, R.K., Roy, N., Hehl, A.B., and Tatu, U. (2011). Post-transcriptional repair of a split heat shock protein 90 gene by mRNA trans-splicing. J. Biol.
Chem. 286, 7116–7122.
Nash, T.E., and Aggarwal, A. (1986). Cytotoxicity of monoclonal antibodies to a
subset of Giardia isolates. J. Immunol. Baltim. Md 1950 136, 2628–2632.
77
Nash, T.E., Herrington, D.A., Losonsky, G.A., and Levine, M.M. (1987). Experimental human infections with Giardia lamblia. J. Infect. Dis. 156, 974–984.
Nash, T.E., Herrington, D.A., Levine, M.M., Conrad, J.T., and Merritt, J.W. (1990).
Antigenic variation of Giardia lamblia in experimental human infections. J. Immunol. Baltim. Md 1950 144, 4362–4369.
Nash, T.E., Luján, H.T., Mowatt, M.R., and Conrad, J.T. (2001). Variant-specific
surface protein switching in Giardia lamblia. Infect. Immun. 69, 1922–1923.
Nixon, J.E.J., Wang, A., Morrison, H.G., McArthur, A.G., Sogin, M.L., Loftus, B.J.,
and Samuelson, J. (2002). A spliceosomal intron in Giardia lamblia. Proc. Natl.
Acad. Sci. U. S. A. 99, 3701–3705.
Nohria, A., Alonso, R.A., and Peattie, D.A. (1992). Identification and characterization of gamma-giardin and the gamma-giardin gene from Giardia lamblia. Mol.
Biochem. Parasitol. 56, 27–37.
Nohynková, E., Tumová, P., and Kulda, J. (2006). Cell division of Giardia intestinalis: flagellar developmental cycle involves transformation and exchange of
flagella between mastigonts of a diplomonad cell. Eukaryot. Cell 5, 753–761.
Novarina, D., Amara, F., Lazzaro, F., Plevani, P., and Muzi-Falconi, M. (2011).
Mind the gap: keeping UV lesions in check. DNA Repair 10, 751–759.
Olson, M.E., O’Handley, R.M., Ralston, B.J., McAllister, T.A., and Thompson,
R.C.A. (2004). Update on Cryptosporidium and Giardia infections in cattle.
Trends Parasitol. 20, 185–191.
Palm, D., Weiland, M., McArthur, A.G., Winiecka-Krusnell, J., Cipriano, M.J.,
Birkeland, S.R., Pacocha, S.E., Davids, B., Gillin, F., Linder, E., et al. (2005).
Developmental changes in the adhesive disk during Giardia differentiation. Mol.
Biochem. Parasitol. 141, 199–207.
Papanastasiou, P., Hiltpold, A., Bommeli, C., and Köhler, P. (1996). The release of
the variant surface protein of Giardia to its soluble isoform is mediated by the
selective cleavage of the conserved carboxy-terminal domain. Biochemistry
(Mosc.) 35, 10143–10148.
Papanastasiou, P., McConville, M.J., Ralton, J., and Köhler, P. (1997). The variantspecific surface protein of Giardia, VSP4A1, is a glycosylated and palmitoylated protein. Biochem. J. 322 ( Pt 1), 49–56.
Paredez, A.R., Assaf, Z.J., Sept, D., Timofejeva, L., Dawson, S.C., Wang, C.-J.R.,
and Cande, W.Z. (2011). An actin cytoskeleton with evolutionarily conserved
functions in the absence of canonical actin-binding proteins. Proc. Natl. Acad.
Sci. U. S. A. 108, 6151–6156.
Pathuri, P., Nguyen, E.T., Svärd, S.G., and Luecke, H. (2007). Apo and calciumbound crystal structures of Alpha-11 giardin, an unusual annexin from Giardia
lamblia. J. Mol. Biol. 368, 493–508.
Pathuri, P., Nguyen, E.T., Ozorowski, G., Svärd, S.G., and Luecke, H. (2009). Apo
and calcium-bound crystal structures of cytoskeletal protein alpha-14 giardin
(annexin E1) from the intestinal protozoan parasite Giardia lamblia. J. Mol. Biol. 385, 1098–1112.
Paull, G.C., and Matthews, R.A. (2001). Spironucleus vortens, a possible cause of
hole-in-the-head disease in cichlids. Dis. Aquat. Organ. 45, 197–202.
Perry, D.A., Morrison, H.G., and Adam, R.D. (2011). Optical map of the genotype
A1 WB C6 Giardia lamblia genome isolate. Mol. Biochem. Parasitol. 180, 112–
114.
Pimenta, P.F., da Silva, P.P., and Nash, T. (1991). Variant surface antigens of Giardia lamblia are associated with the presence of a thick cell coat: thin section and
label fracture immunocytochemistry survey. Infect. Immun. 59, 3989–3996.
78
Ponts, N., Fu, L., Harris, E.Y., Zhang, J., Chung, D.-W.D., Cervantes, M.C., Prudhomme, J., Atanasova-Penichon, V., Zehraoui, E., Bunnik, E.M., et al. (2013).
Genome-wide mapping of DNA methylation in the human malaria parasite
Plasmodium falciparum. Cell Host Microbe 14, 696–706.
Poppe, T., Mo, T.., and Iversen, L. (1992). Disseminated hexamitosis in sea-caged
Atlantic salmon Salmo salar. Dis. Aquat. Organ. 1992, 91–97.
Poxleitner, M.K., Carpenter, M.L., Mancuso, J.J., Wang, C.-J.R., Dawson, S.C., and
Cande, W.Z. (2008). Evidence for karyogamy and exchange of genetic material
in the binucleate intestinal parasite Giardia intestinalis. Science 319, 1530–
1533.
Prucca, C.G., Slavin, I., Quiroga, R., Elías, E.V., Rivero, F.D., Saura, A., Carranza,
P.G., and Luján, H.D. (2008). Antigenic variation in Giardia lamblia is regulated by RNA interference. Nature 456, 750–754.
Rada, P., Doležal, P., Jedelský, P.L., Bursac, D., Perry, A.J., Šedinová, M., Smíšková, K., Novotný, M., Beltrán, N.C., Hrdý, I., et al. (2011). The core components
of organelle biogenesis and membrane transport in the hydrogenosomes of
Trichomonas vaginalis. PloS One 6, e24428.
Ramesh, M.A., Malik, S.-B., and Logsdon, J.M. (2005). A phylogenomic inventory
of meiotic genes; evidence for sex in Giardia and an early eukaryotic origin of
meiosis. Curr. Biol. CB 15, 185–191.
Regoes, A., Zourmpanou, D., León-Avila, G., van der Giezen, M., Tovar, J., and
Hehl, A.B. (2005). Protein import, replication, and inheritance of a vestigial mitochondrion. J. Biol. Chem. 280, 30557–30563.
Reiner, D.S., McCaffery, J.M., and Gillin, F.D. (2001). Reversible interruption of
Giardia lamblia cyst wall protein transport in a novel regulated secretory pathway. Cell. Microbiol. 3, 459–472.
Reiner, D.S., Hetsko, M.L., Meszaros, J.G., Sun, C.-H., Morrison, H.G., Brunton,
L.L., and Gillin, F.D. (2003). Calcium signaling in excystation of the early diverging eukaryote, Giardia lamblia. J. Biol. Chem. 278, 2533–2540.
Reiner, D.S., Ankarklev, J., Troell, K., Palm, D., Bernander, R., Gillin, F.D., Andersson, J.O., and Svärd, S.G. (2008). Synchronisation of Giardia lamblia: identification of cell cycle stage-specific genes and a differentiation restriction point.
Int. J. Parasitol. 38, 935–944.
Ringqvist, E., Palm, J.E.D., Skarin, H., Hehl, A.B., Weiland, M., Davids, B.J.,
Reiner, D.S., Griffiths, W.J., Eckmann, L., Gillin, F.D., et al. (2008). Release of
metabolic enzymes by Giardia in response to interaction with intestinal epithelial cells. Mol. Biochem. Parasitol. 159, 85–91.
Ringqvist, E., Avesson, L., Söderbom, F., and Svärd, S.G. (2011). Transcriptional
changes in Giardia during host-parasite interactions. Int. J. Parasitol. 41, 277–
285.
Riordan, C.E., Langreth, S.G., Sanchez, L.B., Kayser, O., and Keithly, J.S. (1999).
Preliminary evidence for a mitochondrion in Cryptosporidium parvum: phylogenetic and therapeutic implications. J. Eukaryot. Microbiol. 46, 52S – 55S.
Rivero, F.D., Saura, A., Prucca, C.G., Carranza, P.G., Torri, A., and Lujan, H.D.
(2010). Disruption of antigenic variation is crucial for effective parasite vaccine.
Nat. Med. 16, 551–557, 1p following 557.
Robbins, P.W., and Samuelson, J. (2005). Asparagine linked glycosylation in Giardia. Glycobiology 15, 15G – 16G.
Robertson, L.J., Hanevik, K., Escobedo, A.A., Mørch, K., and Langeland, N. (2010).
Giardiasis--why do the symptoms sometimes never stop? Trends Parasitol. 26,
75–82.
79
Roger, A.J., Svärd, S.G., Tovar, J., Clark, C.G., Smith, M.W., Gillin, F.D., and
Sogin, M.L. (1998). A mitochondrial-like chaperonin 60 gene in Giardia
lamblia: evidence that diplomonads once harbored an endosymbiont related to
the progenitor of mitochondria. Proc. Natl. Acad. Sci. U. S. A. 95, 229–234.
Roxström-Lindquist, K., Ringqvist, E., Palm, D., and Svärd, S. (2005). Giardia
lamblia-induced changes in gene expression in differentiated Caco-2 human intestinal epithelial cells. Infect. Immun. 73, 8204–8208.
Roxström-Lindquist, K., Jerlström-Hultqvist, J., Jørgensen, A., Troell, K., Svärd,
S.G., and Andersson, J.O. (2010). Large genomic differences between the morphologically indistinguishable diplomonads Spironucleus barkhanus and Spironucleus salmonicida. BMC Genomics 11, 258.
Roy, S.W., Hudson, A.J., Joseph, J., Yee, J., and Russell, A.G. (2012). Numerous
fragmented spliceosomal introns, AT-AC splicing, and an unusual dynein gene
expression pathway in Giardia lamblia. Mol. Biol. Evol. 29, 43–49.
Russell, A.G., Shutt, T.E., Watkins, R.F., and Gray, M.W. (2005). An ancient
spliceosomal intron in the ribosomal protein L7a gene (Rpl7a) of Giardia
lamblia. BMC Evol. Biol. 5, 45.
Ryan, U., and Cacciò, S.M. (2013). Zoonotic potential of Giardia. Int. J. Parasitol.
43, 943–956.
Sagolla, M.S., Dawson, S.C., Mancuso, J.J., and Cande, W.Z. (2006). Threedimensional analysis of mitosis and cytokinesis in the binucleate parasite Giardia intestinalis. J. Cell Sci. 119, 4889–4900.
Saraiya, A.A., and Wang, C.C. (2008). snoRNA, a novel precursor of microRNA in
Giardia lamblia. PLoS Pathog. 4, e1000224.
Saraiya, A.A., Li, W., and Wang, C.C. (2011). A microRNA derived from an apparent canonical biogenesis pathway regulates variant surface protein gene expression in Giardia lamblia. RNA N. Y. N 17, 2152–2164.
Saraiya, A.A., Li, W., Wu, J., Chang, C.H., and Wang, C.C. (2014). The microRNAs in an ancient protist repress the variant-specific surface protein expression by targeting the entire coding sequence. PLoS Pathog. 10, e1003791.
Saric, M., Vahrmann, A., Niebur, D., Kluempers, V., Hehl, A.B., and Scholze, H.
(2009). Dual acylation accounts for the localization of {alpha}19-giardin in the
ventral flagellum pair of Giardia lamblia. Eukaryot. Cell 8, 1567–1574.
Savioli, L., Smith, H., and Thompson, A. (2006). Giardia and Cryptosporidium join
the “Neglected Diseases Initiative.” Trends Parasitol. 22, 203–208.
Schneider, R.E., Brown, M.T., Shiflett, A.M., Dyall, S.D., Hayes, R.D., Xie, Y.,
Loo, J.A., and Johnson, P.J. (2011). The Trichomonas vaginalis hydrogenosome
proteome is highly reduced relative to mitochondria, yet complex compared
with mitosomes. Int. J. Parasitol. 41, 1421–1434.
Schofield, P.J., Edwards, M.R., Matthews, J., and Wilson, J.R. (1992). The pathway
of arginine catabolism in Giardia intestinalis. Mol. Biochem. Parasitol. 51, 29–
36.
Scott, K.G.-E., Yu, L.C.H., and Buret, A.G. (2004). Role of CD8+ and CD4+ T
lymphocytes in jejunal mucosal injury during murine giardiasis. Infect. Immun.
72, 3536–3542.
Shin, G.A., Linden, K.G., Arrowood, M.J., and Sobsey, M.D. (2001). Low-pressure
UV inactivation and DNA repair potential of Cryptosporidium parvum oocysts.
Appl. Environ. Microbiol. 67, 3029–3032.
Simpson, A.G.B. (2003). Cytoskeletal organization, phylogenetic affinities and
systematics in the contentious taxon Excavata (Eukaryota). Int. J. Syst. Evol.
Microbiol. 53, 1759–1777.
80
Singer, S.M., and Nash, T.E. (2000). The role of normal flora in Giardia lamblia
infections in mice. J. Infect. Dis. 181, 1510–1512.
Singer, S.M., Yee, J., and Nash, T.E. (1998). Episomal and integrated maintenance
of foreign DNA in Giardia lamblia. Mol. Biochem. Parasitol. 92, 59–69.
Singer, S.M., Elmendorf, H.G., Conrad, J.T., and Nash, T.E. (2001). Biological
selection of variant-specific surface proteins in Giardia lamblia. J. Infect. Dis.
183, 119–124.
Skarin, H., Ringqvist, E., Hellman, U., and Svärd, S.G. (2011). Elongation factor 1alpha is released into the culture medium during growth of Giardia intestinalis
trophozoites. Exp. Parasitol. 127, 804–810.
Slavin, I., Saura, A., Carranza, P.G., Touz, M.C., Nores, M.J., and Luján, H.D.
(2002). Dephosphorylation of cyst wall proteins by a secreted lysosomal acid
phosphatase is essential for excystation of Giardia lamblia. Mol. Biochem. Parasitol. 122, 95–98.
Smíd, O., Matusková, A., Harris, S.R., Kucera, T., Novotný, M., Horváthová, L.,
Hrdý, I., Kutejová, E., Hirt, R.P., Embley, T.M., et al. (2008). Reductive evolution of the mitochondrial processing peptidases of the unicellular parasites
trichomonas vaginalis and giardia intestinalis. PLoS Pathog. 4, e1000243.
Solaymani-Mohammadi, S., and Singer, S.M. (2010). Giardia duodenalis: the double-edged sword of immune responses in giardiasis. Exp. Parasitol. 126, 292–
297.
Soltys, B.J., Falah, M., and Gupta, R.S. (1996). Identification of endoplasmic reticulum in the primitive eukaryote Giardia lamblia using cryoelectron microscopy
and antibody to Bip. J. Cell Sci. 109 ( Pt 7), 1909–1917.
Sonda, S., Stefanic, S., and Hehl, A.B. (2008). A sphingolipid inhibitor induces a
cytokinesis arrest and blocks stage differentiation in Giardia lamblia. Antimicrob. Agents Chemother. 52, 563–569.
Sonda, S., Morf, L., Bottova, I., Baetschmann, H., Rehrauer, H., Caflisch, A., Hakimi, M.-A., and Hehl, A.B. (2010). Epigenetic mechanisms regulate stage differentiation in the minimized protozoan Giardia lamblia. Mol. Microbiol. 76,
48–67.
Spycher, C., Herman, E.K., Morf, L., Qi, W., Rehrauer, H., Aquino Fournier, C.,
Dacks, J.B., and Hehl, A.B. (2013). An ER-directed transcriptional response to
unfolded protein stress in the absence of conserved sensor-transducer proteins in
Giardia lamblia. Mol. Microbiol. 88, 754–771.
Stadelmann, B., Merino, M.C., Persson, L., and Svärd, S.G. (2012). Arginine consumption by the intestinal parasite Giardia intestinalis reduces proliferation of
intestinal epithelial cells. PloS One 7, e45325.
Stäger, S., Gottstein, B., Sager, H., Jungi, T.W., and Müller, N. (1998). Influence of
antibodies in mother’s milk on antigenic variation of Giardia lamblia in the murine mother-offspring model of infection. Infect. Immun. 66, 1287–1292.
Stefanic, S., Morf, L., Kulangara, C., Regös, A., Sonda, S., Schraner, E., Spycher,
C., Wild, P., and Hehl, A.B. (2009). Neogenesis and maturation of transient
Golgi-like cisternae in a simple eukaryote. J. Cell Sci. 122, 2846–2856.
Sterud, E., Poppe, T., and Bornø, G. (2003). Intracellular infection with Spironucleus barkhanus (Diplomonadida: Hexamitidae) in farmed Arctic char Salvelinus
alpinus. Dis. Aquat. Organ. 56, 155–161.
Su, L.-H., Pan, Y.-J., Huang, Y.-C., Cho, C.-C., Chen, C.-W., Huang, S.-W.,
Chuang, S.-F., and Sun, C.-H. (2011). A novel E2F-like protein involved in
transcriptional activation of cyst wall protein genes in Giardia lamblia. J. Biol.
Chem. 286, 34101–34120.
81
Sulemana, A., Paget, T.A., and Jarroll, E.L. (2014). Commitment to cyst formation
in Giardia. Microbiol. Read. Engl. 160, 330–339.
Sun, C.-H., Palm, D., McArthur, A.G., Svärd, S.G., and Gillin, F.D. (2002). A novel
Myb-related protein involved in transcriptional activation of encystation genes
in Giardia lamblia. Mol. Microbiol. 46, 971–984.
Sun, C.-H., McCaffery, J.M., Reiner, D.S., and Gillin, F.D. (2003). Mining the Giardia lamblia genome for new cyst wall proteins. J. Biol. Chem. 278, 21701–
21708.
Sun, C.-H., Su, L.-H., and Gillin, F.D. (2006). Novel plant-GARP-like transcription
factors in Giardia lamblia. Mol. Biochem. Parasitol. 146, 45–57.
Svärd, S.G., Meng, T.C., Hetsko, M.L., McCaffery, J.M., and Gillin, F.D. (1998).
Differentiation-associated surface antigen variation in the ancient eukaryote Giardia lamblia. Mol. Microbiol. 30, 979–989.
Takishita, K., Kolisko, M., Komatsuzaki, H., Yabuki, A., Inagaki, Y., Cepicka, I.,
Smejkalová, P., Silberman, J.D., Hashimoto, T., Roger, A.J., et al. (2012). Multigene phylogenies of diverse Carpediemonas-like organisms identify the closest
relatives of “amitochondriate” diplomonads and retortamonads. Protist 163,
344–355.
Teodorovic, S., Walls, C.D., and Elmendorf, H.G. (2007). Bidirectional transcription
is an inherent feature of Giardia lamblia promoters and contributes to an abundance of sterile antisense transcripts throughout the genome. Nucleic Acids Res.
35, 2544–2553.
Thirion, J., Wattiaux, R., and Jadot, M. (2003). The acid phosphatase positive organelles of the Giardia lamblia trophozoite contain a membrane bound cathepsin C
activity. Biol. Cell Auspices Eur. Cell Biol. Organ. 95, 99–105.
Touz, M.C., Conrad, J.T., and Nash, T.E. (2005). A novel palmitoyl acyl transferase
controls surface protein palmitoylation and cytotoxicity in Giardia lamblia. Mol.
Microbiol. 58, 999–1011.
Touz, M.C., Rópolo, A.S., Rivero, M.R., Vranych, C.V., Conrad, J.T., Svard, S.G.,
and Nash, T.E. (2008). Arginine deiminase has multiple regulatory roles in the
biology of Giardia lamblia. J. Cell Sci. 121, 2930–2938.
Tovar, J., Fischer, A., and Clark, C.G. (1999). The mitosome, a novel organelle
related to mitochondria in the amitochondrial parasite Entamoeba histolytica.
Mol. Microbiol. 32, 1013–1021.
Tovar, J., León-Avila, G., Sánchez, L.B., Sutak, R., Tachezy, J., van der Giezen, M.,
Hernández, M., Müller, M., and Lucocq, J.M. (2003). Mitochondrial remnant
organelles of Giardia function in iron-sulphur protein maturation. Nature 426,
172–176.
Troeger, H., Epple, H.-J., Schneider, T., Wahnschaffe, U., Ullrich, R., Burchard, G.D., Jelinek, T., Zeitz, M., Fromm, M., and Schulzke, J.-D. (2007). Effect of
chronic Giardia lamblia infection on epithelial transport and barrier function in
human duodenum. Gut 56, 328–335.
Tůmová, P., Hofstetrová, K., Nohýnková, E., Hovorka, O., and Král, J. (2007). Cytogenetic evidence for diversity of two nuclei within a single diplomonad cell of
Giardia. Chromosoma 116, 65–78.
Vahrmann, A., Sarić, M., Koebsch, I., and Scholze, H. (2008). alpha14-Giardin
(annexin E1) is associated with tubulin in trophozoites of Giardia lamblia and
forms local slubs in the flagella. Parasitol. Res. 102, 321–326.
Wampfler, P.B., Tosevski, V., Nanni, P., Spycher, C., and Hehl, A.B. (2014). Proteomics of secretory and endocytic organelles in Giardia lamblia. PloS One 9,
e94089.
82
Wang, C.-H., Su, L.-H., and Sun, C.-H. (2007). A novel ARID/Bright-like protein
involved in transcriptional activation of cyst wall protein 1 gene in Giardia
lamblia. J. Biol. Chem. 282, 8905–8914.
Ward, W., Alvarado, L., Rawlings, N.D., Engel, J.C., Franklin, C., and McKerrow,
J.H. (1997). A primitive enzyme for a primitive cell: the protease required for
excystation of Giardia. Cell 89, 437–444.
Ware, M.W., Augustine, S.A.J., Erisman, D.O., See, M.J., Wymer, L., Hayes, S.L.,
Dubey, J.P., and Villegas, E.N. (2010). Determining UV inactivation of Toxoplasma gondii oocysts by using cell culture and a mouse bioassay. Appl. Environ. Microbiol. 76, 5140–5147.
Weber, C., Marchat, L.A., Guillen, N., and López-Camarillo, C. (2009). Effects of
DNA damage induced by UV irradiation on gene expression in the protozoan
parasite Entamoeba histolytica. Mol. Biochem. Parasitol. 164, 165–169.
Weeratunga, S.K., Osman, A., Hu, N.-J., Wang, C.K., Mason, L., Svärd, S., Hope,
G., Jones, M.K., and Hofmann, A. (2012). Alpha-1 giardin is an annexin with
highly unusual calcium-regulated mechanisms. J. Mol. Biol. 423, 169–181.
Weiland, M.E.-L., Palm, J.E.D., Griffiths, W.J., McCaffery, J.M., and Svärd, S.G.
(2003). Characterisation of alpha-1 giardin: an immunodominant Giardia
lamblia annexin with glycosaminoglycan-binding activity. Int. J. Parasitol. 33,
1341–1351.
Weiland, M.E.-L., McArthur, A.G., Morrison, H.G., Sogin, M.L., and Svärd, S.G.
(2005). Annexin-like alpha giardins: a new cytoskeletal gene family in Giardia
lamblia. Int. J. Parasitol. 35, 617–626.
Wenman, W.M., Meuser, R.U., Nyugen, Q., Kilani, R.T., el-Shewy, K., and Sherburne, R. (1993). Characterization of an immunodominant Giardia lamblia protein antigen related to alpha giardin. Parasitol. Res. 79, 587–592.
Wiesehahn, G.P., Jarroll, E.L., Lindmark, D.G., Meyer, E.A., and Hallick, L.M.
(1984). Giardia lamblia: autoradiographic analysis of nuclear replication. Exp.
Parasitol. 58, 94–100.
Williams, C.W., and Elmendorf, H.G. (2011). Identification and analysis of the
RNA degrading complexes and machinery of Giardia lamblia using an in silico
approach. BMC Genomics 12, 586.
Williams, B.A.P., Hirt, R.P., Lucocq, J.M., and Embley, T.M. (2002). A mitochondrial remnant in the microsporidian Trachipleistophora hominis. Nature 418,
865–869.
Williams, C.F., Lloyd, D., Poynton, S., Jørgensen, A., Millet, C.O.M., and Cable, J.
(2011). Spironucleus species: Economically-Important Fish Pathogens and Enigmatic Single-Celled Eukaryotes. 2011.
Woessner, D.J., and Dawson, S.C. (2012). The Giardia median body protein is a
ventral disc protein that is critical for maintaining a domed disc conformation
during attachment. Eukaryot. Cell 11, 292–301.
Wood, A.M., and Smith, H.V. (2005). Spironucleosis (Hexamitiasis, Hexamitosis)
in the ring-necked pheasant (Phasianus colchicus): detection of cysts and description of Spironucleus meleagridis in stained smears. Avian Dis. 49, 138–
143.
Worgall, T.S., Davis-Hayman, S.R., Magana, M.M., Oelkers, P.M., Zapata, F., Juliano, R.A., Osborne, T.F., Nash, T.E., and Deckelbaum, R.J. (2004). Sterol and
fatty acid regulatory pathways in a Giardia lamblia-derived promoter: evidence
for SREBP as an ancient transcription factor. J. Lipid Res. 45, 981–988.
Xu, F., Jerlström-Hultqvist, J., Einarsson, E., Astvaldsson, A., Svärd, S.G., and
Andersson, J.O. (2014). The genome of Spironucleus salmonicida highlights a
fish pathogen adapted to fluctuating environments. PLoS Genet. 10, e1004053.
83
Yee, J., Tang, A., Lau, W.-L., Ritter, H., Delport, D., Page, M., Adam, R.D., Müller,
M., and Wu, G. (2007). Core histone genes of Giardia intestinalis: genomic organization, promoter structure, and expression. BMC Mol. Biol. 8, 26.
Yichoy, M., Duarte, T.T., De Chatterjee, A., Mendez, T.L., Aguilera, K.Y., Roy, D.,
Roychowdhury, S., Aley, S.B., and Das, S. (2011). Lipid metabolism in Giardia:
a post-genomic perspective. Parasitology 138, 267–278.
Yu, L.Z., Birky, C.W., and Adam, R.D. (2002). The two nuclei of Giardia each have
complete copies of the genome and are partitioned equationally at cytokinesis.
Eukaryot. Cell 1, 191–199.
Zhang, H., Guo, F., Zhou, H., and Zhu, G. (2012). Transcriptome analysis reveals
unique metabolic features in the Cryptosporidium parvum Oocysts associated
with environmental survival and stresses. BMC Genomics 13, 647.
84
Acta Universitatis Upsaliensis
Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Science and Technology 1303
Editor: The Dean of the Faculty of Science and Technology
A doctoral dissertation from the Faculty of Science and
Technology, Uppsala University, is usually a summary of a
number of papers. A few copies of the complete dissertation
are kept at major Swedish research libraries, while the
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Dissertations from the Faculty of Science and Technology.
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2015