Secretion of cAMP in Mycobacterium Under
Various Conditions
Kevin D. Reynolds and Michaela A. Gazdik
School of Natural Sciences and Mathematics, Ferrum College
Background Information
Figure 2.
Diagram of the
cAMP assay
used in these
experiments
E. coli
M. smeg
3000
2500
14000
cAMP/OD
12000
10000
8000
6000
2000
1500
1000
4000
500
2000
0
0
4
8
24
48
4
7
24
Time (hrs)
3500
12000
cAMP Level (pmol cAMP/OD)
E. coli
M. smeg
10000
8000
6000
4000
2000
E. coli
3000
M. smeg
2500
2000
1500
1000
500
0
EC
0
EC
IC
pH 5.5
EC
IC
EC
Starvation
IC
IC
Control
Effects of Room Temperature on cAMP Amounts
25000
E. coli
20000
The higher levels of cAMP in M. smegmatis compared
to E. coli are likely resulting from the fact that M.
smegmatis had nine more cyclases than E. coli. The
fact that our results showed M. smegmatis decreases
cAMP secretion under low pH is interesting, as
previous reports have suggested that M. bovis and
Mtb increase cAMP secretion under these conditions.
M. smegmatis is generally found in the soil, which
could explain the high secretion levels at room
temperature. These test will be repeated along with
others as new results appear.
References
•Abdel Motaal, A., I. Tews, J. E. Schultz, and J. U. Linder. 2006. Fatty acid
regulation of adenylyl cyclase Rv2212 from Mycobacterium tuberculosis
H37Rv. Febs J 273:4219-28.
•Akhter, Y., S. Yellaboina, A. Farhana, A. Ranjan, N. Ahmed, and S. E.
Hasnain. 2008. Genome scale portrait of cAMP-receptor protein (CRP)
regulons in mycobacteria points to their role in pathogenesis. Gene 407:14858.
•Cummings, K. J. 2007. Tuberculosis control: challenges of an ancient and
ongoing epidemic. Public Health Rep 122:683-92.
•McCue, L. A., K. A. McDonough, and C. E. Lawrence. 2000. Functional
classification of cNMP-binding proteins and nucleotide cyclases with
implications for novel regulatory pathways in Mycobacterium tuberculosis.
Genome Res 10:204-219.
•Botsford, J. L., and J. G. Harman. 1992. Cyclic AMP in prokaryotes. Microbiol
Rev 56:100-122.
Control
*Note: Extracellular (EC) levels of cAMP were measured after the cells
had been pelleted and taking an OD reading on the supernatant.
Intracellular (IC) levels of cAMP were measured by taking OD reading on
the supernatant after the cells had been washed, and lysed.
• M. Ssmegmatis consistently produces and/or
secretes four to six times more cAMP than E. coli.
under standard growth conditions, with the
exception of the seven hour time point.
• Low pH had opposite effects on M. smegmatis and
E. coli., causing M. smegmatis to decrease the
amount of cAMP secreted approximately five fold
while E. coli. Increased its intracellular levels three
fold.
• Room temperature led to three times higher levels
of secreted cAMP in M. smegmatis but showed no
effect on cAMP levels of E. coli.
• Two hour starvation had no effect on the cAMP
levels of either M. smegmatis or E. coli.
• All results need to be repeated to determine
reliability and gather statistical data
Discussion
Effects of Starvation on cAMP Amounts
Effect of pH on cAMP Levels
14000
48
Time (hrs)
cAMP Level (pmol cAMP/OD)
cAMP levels were measured using a cAMP ELISA
purchased from R&D Biosystems. A pictorial
representation of the cAMP kit is seen below.
E. coli
M. smeg
16000
cAMP Level (pmol cAMP/OD)
For this study, we are using M. smegmatis as our
model organism, as it is a fast-grower and a nonpathogenic species. We are also using E. coli as the
model prokaryotic organism. Each bacterium was
grown in culture, M.smeg was grown in TSA broth
and E. coli was grown in LB broth. Standards were
obtained by growing each bacterium for four hours,
eight hours, twenty-four hours and forty-eight hours.
The bacteria were then grown for twenty four hours
and placed under various conditions, including low
pH, starvation, and room temperature.
3500
18000
Figure 1. cAMP
structure
Methods
Results
Intracellular cAMP Standards
20000
cAMP Levels (pmol/OD)
Mycobacterium species are unique, as they contain
many more annotated adenylate cyclases (ACs),
which is the enzyme responsible for 3’,5’ cyclic
adenosine monophosphate (cAMP) production, than
most other microbes.
cAMP mediated gene
regulation is important for Mycobacterium
tuberculoses (Mtb) and evidence suggests that
secreted cAMP plays a role in Mtb-host interactions,
thus characterization of mycobacterial cAMP
secretion
mechanisms
will
improve
our
understanding of how this bacterium establishes
infection.
Mtb is a pathogen of importance to public health.
Despite the current treatments and vaccines,
tuberculoses is one of the leading causes of death
among infectious diseases, killing about two million
people every year.
cAMP is an important secondary messenger that
controls a wide range of cellular responses.
Extracellular cAMP Standards
M. smeg
15000
Acknowledgements
10000
5000
0
EC
IC
Room Temperature
EC
IC
Control
This work is supervised by Dr. Michaela Gazdik
Anti-microbial studies will be performed in
collaboration with Dr. Michaela Gazdik
Funding for this project has been provided by NIH.