Mycobacterium tuberculosis (Mtb) poses a global health catastrophe that has been compounded by the emergence of highly drug resistant Mtb strains. We used whole genome sequencing (WGS) to directly compare the accumulation of mutations in Mtb isolated from cynomolgus macaques with active, latent and early reactivation disease. Based on the distribution of single nucleotide polymorphisms (SNPs) observed, we calculated the mutation rates for these disease states. Our data suggest that during latency, Mtb acquires a similar number of chromosomal mutations as would be expected to emerge in a logarithmically growing culture over the same period of time despite reduced bacterial replication during latent infection. The pattern of polymorphisms suggests that the mutational burden in vivo is due to oxidative
DNA damage. We next sought to determine why some strains of Mtb are preferentially associated with high-level drug resistance. We demonstrate that Mtb strains from the East Asian lineage acquire drug resistances in vitro more quickly than Mtb strains from the Euro-American lineage. Their higher drug resistance rate in vitro reflects a higher basal mutation. Moreover, the in vitro mutation rate correlates well with the bacterial mutation rate in humans as determined by whole genome sequencing of clinical isolates. Finally, using an agent-based model, we show that the observed differences in mutation rate predict a significantly higher probability of multi-drug resistance in patients infected with East Asian lineage strains of Mtb. Lastly, we sought to determine the mechanisms Mtb uses to proofread nascently
polymerized DNA. Through fluctuation analysis of deletion mutants of two potential \(polIII\epsilon\) homologs, we demonstrate that neither is responsible for the maintenance of DNA replication fidelity. To explore the possibility that one of these homologs, Rv3711c, participates in an unknown redundant pathway, we used transposon capture and sequence (TraCS) to identify genes conditionally essential in an Rv3711c deletion mutant. Our analysis suggests that while Rv3711c does not participate in proofreading, it may act in an alternative novel DNA repair pathway. Taken together, our fluctuation analysis and TraCS data suggest that mycobacteria do not use canonical methods of proofreading to maintain genomic fidelity.
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/9817661 |
| Date | 05 October 2013 |
| Creators | Ford, Christopher Burton |
| Contributors | Fortune, Sarah Merritt |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Rights | open |
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