The Search for Hypermutability: Determining Frequency of Mutation in Mycobacterium smegmatis mc2155 ___________ Susan Puckett Mentors: Dr. Digby Warner, Dr. Valerie Mizrahi MRC/NHLS/WITS Molecular Mycobacteriology Research Unit, School of Pathology, University of the Witwatersrand and National Health Laboratory Service, Johannesburg, South Africa Funded by the Howard Hughes Medical Institute

Background: Tuberculosis (TB) Contagious, airborne disease caused by Mycobacterium tuberculosis (MTB) Coughing, weakness, weight loss, fever, death Over 1/3 of the world’s population infected, with 5-10% projected to become infectious at some point Estimated Death Toll in 2005 USA 1,345 people South Africa 101,989 people World 1,600,000 people Source: World Health Organization (WHO) website http://www.who.int/mediacentre/factsheets/fs104/en/index.html http://www.who.int/globalatlas/dataQuery/default.asp

TB Treatment and Drug Resistance BCG Vaccine, Antibiotics MDR-TB Isoniazid, rifampicin resistant XDR-TB Fluoroquinolone resistant Resistant to at least one of three second-line drugs capreomycin, kanamycin, and amikacin All drug resistance is chromosomally encoded

Understanding Mutation Problem of drug resistance caused by mutations in genes required for antibiotic effectiveness Link between mutagenesis and drug resistance: Boshoff, H., Reed, M., Barry, C., V. Mizrahi. 2003. DnaE2 Polymerase Contributes to In Vivo Survival and the Emergence of Drug Resistance in Mycobacterium tuberculosis. Cell 113: 183-193.

Questions to Answer Do mutations occur more frequently under certain conditions? Is there a hypermutable state in Mycobacterium? Mycobacterium smegmatis (MSM) Not pathogenic, genetically similar, faster grower Hypothesis: Stress-induced hypermutability Targeting mutation during a hypermutable state could be an effective way of combating TB

Karunakaran, P. , and J. Davies. 2000 Karunakaran, P., and J. Davies. 2000. Genetic Antagonism and Hypermutability in Mycobacterium smegmatis. J. Bacteriol. 182: 3331-3335. FIG. 4. Growth phase-dependent hypermutability in M. smegmatis, shown as frequencies of the appearance of resistant mutants

Experiment Test MSM strains mc2155 and mc26 using method outlined in the paper, with different types of media Incubate cultures and plate on rifampicin plates during log phase and stationary phase over a period of several days Expectations: -- More RifR mutants in stationary phase than in log phase -- mc2155 and mc26 generate similar numbers of RifR mutants -- Process is growth medium independent

Results with 7H9, 7H10 First tested with 7H9 and 7H10, media commonly used to grow MSM and MTB. With and without TW80. Only a few RifR colonies per plate after plating 0.4 ml undiluted culture NO increase in frequencies of mutation in stationary phase vs. log phase (mutation frequency ~10-8)

Results with TSB and TSA Later tested with tryptic soy broth (TSB) and tryptic soy agar (TSA), the media used in the paper. No TW80. Log phase: a few mutants and a haze of smaller colonies on rifampicin plates Stationary phase: a lawn of growth on rifampicin plates Seems to correlate with paper

Final TSB experiment mc2155 and mc26 grown in TSB at 30ºC with TW80 7H10 and TSA used for plating Simultaneous experiment: sample from starting culture diluted with new media every day and incubated at same conditions as main culture

Results Contamination issues, plates reflect trends as seen in last experiment with TSB Simultaneous experiment: contaminated However, certain plates show interesting results

mc2155 grows differently on TSA Before plating, these were incubated in TSB at 30ºC, 100RPM, with TW80 mc2155 on TSA mc2155 on 7H10 mc26 on TSA mc26 on 7H10 Colonies are smaller, grow more slowly Consistently different than mc26 results on TSA

mc2155 grows differently on TSA Before plating, these were incubated in TSB at 30ºC, 100RPM, with TW80 mc2155 on TSA mc2155 on 7H10 mc26 on TSA mc26 on 7H10 Sometimes, colonies do not appear at all. Perhaps longer incubation is needed.

mc2155 RIFR mutants grow on TSA Before plating, these were incubated in TSB at 30ºC, 100RPM, with TW80 mc2155 on TSA (RIF) mc2155 on 7H10 (RIF) Stationary phase cultures Contamination problems with mc26

TW80-less TSB encourages growth on TSA RIF plates Before plating, these were incubated in TSB at 30ºC, 100RPM, without TW80 CONTAMINATED mc2155 on TSA (RIF) mc2155 on 7H10 (RIF) mc26 on TSA (RIF) mc26 on 7H10 (RIF) Cultures under similar log phase conditions with TW80 had 0-5 mutants per TSA plate and 0-1 mutants per 7H10 plate

Conclusion MSM behaves differently under different conditions Media: TSB vs. 7H9, TSA vs. 7H10 Presence or lack of TW80 Greater number of RIFR mutants with TSB, TSA mc2155 behaves differently than mc26 on TSA but not on 7H10 Frequency of mutation: more testing needed Comparable results to Karunakaran and Davies paper

Analysis If stationary phase hypermutability occurs, it is only under certain growth conditions Other explanations: competitive advantage Clustering of cells in TW80-less media could act like biofilm, allowing RIF susceptible cells to survive Perkins, A., W. Nicholson. 2007. Uncovering New Metabolic Capabilities of Bacillus sublitis Using Phenotype Profiling of Rifampicin-Resistant rpoB Mutants. J. Bact. 190: 807-814. Wrande, M., Roth, J., D. Hughes. 2008. Accumulation of Mutants in “Aging” Bacterial Colonies is Due to Growth Under Selection, Not Stress-Induced Mutagenesis. Proc. Natl. Acad. Sci. USA . 105: 11863-11868

Significance Hypermutability is questionable due to error catastrophe Significance: cannot assume hypermutability in MTB from in vitro studies, since there is so much variability Emphasizes need for better understanding of in vivo conditions in order to mimic them in vitro

Acknowledgements Dr. Digby Warner Dr. Valerie Mizrahi Dr. Kevin Ahern Mizrahi lab University of the Witwatersrand Howard Hughes Medical Institute (HHMI)