The Search for Hypermutability: The Search for Hypermutability: Determining Frequency of Determining Frequency of Mutation in Mutation in Mycobacterium Mycobacterium

smegmatis smegmatis mcmc22155155___________

Susan PuckettMentors: Dr. Digby Warner, Dr. Valerie MizrahiMRC/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

Source: World Health Organization (WHO) websitehttp://www.who.int/mediacentre/factsheets/fs104/en/index.htmlhttp://www.who.int/globalatlas/dataQuery/default.asp

Estimated Death Toll in 2005

USA 1,345 people

South Africa 101,989 people

World 1,600,000 people

TB Treatment and Drug Resistance

• BCG Vaccine, Antibiotics• MDR-TB

– Isoniazid, rifampicin resistant

• XDR-TB– Isoniazid, rifampicin resistant– 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. 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

• Colonies are smaller, grow more slowly• Consistently different than mc26 results on

TSA

mc2155 on TSA mc2155 on 7H10 mc26 on TSA mc26 on 7H10

Before plating, these were incubated in TSB at 30ºC, 100RPM, with TW80

mc2155 grows differently on TSA

• Sometimes, colonies do not appear at all. Perhaps longer incubation is needed.

Before plating, these were incubated in TSB at 30ºC, 100RPM, with TW80

mc2155 on TSA mc2155 on 7H10 mc26 on TSA mc26 on 7H10

mc2155 RIFR mutants grow on TSA

• Stationary phase cultures• Contamination problems with mc26

mc2155 on TSA (RIF) mc2155 on 7H10 (RIF)

Before plating, these were incubated in TSB at 30ºC, 100RPM, with TW80

TW80-less TSB encourages growth on TSA RIF plates

• Cultures under similar log phase conditions with TW80 had 0-5 mutants per TSA plate and 0-1 mutants per 7H10 plate

mc2155 on TSA (RIF) mc2155 on 7H10 (RIF) mc26 on TSA (RIF) mc26 on 7H10 (RIF)

Before plating, these were incubated in TSB at 30ºC, 100RPM, without TW80

CONTAMINATEDCONTAMINATED

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)