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.
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