Abstract
Treatment of tuberculosis requires multiple drugs and takes 6-9 months. The lengthy treatment has been mainly attributed to the pre-existing population of bacteria that are tolerant to the antibiotics, but not genetically resistant, known as persisters. Persisters are a small fraction of the bacterial population that survive in the presence of the antibiotic. They can be detected by the presence of a bimodal time–kill curve that deviates from the simple decay expected from a uniform bacterial population. The generation of bacterial persisters from normal cells has been extensively studied in E. coli. Mechanisms of switching to persistence include activation of Toxin-Antitoxin (TA) genes and switching to slow growth. However, much less is known about mycobacteria persisters and the factors involved in generating persisters. The overall aim of this study was to characterise mycobacterial persisters in order to better understand the factors influencing their generation and how they survive exposure to antibiotics, with the long-term aim of stimulating new approaches to tuberculosis control. Growth parameters that are associated with persistence were identified by tracking individual cells of M. smegmatis and measuring parameters such as birth and division size and division time for individual cells. In this study, it was discovered that mycobacterial subpopulations utilise different growth control models to maintain their size homeostasis that depends on their birth size. Whilst smaller cells follow a timer model of cell division in which cells divide after a particular time from birth, longer cells were more compatible with an adder model in which cells add a fixed length before division. I was also found that growth parameters, such as birth and division length, were inherited from between mother to daughters. It was attempted to measure the relationship between persistence and growth rate by engineering strains of M. smegmatis whose growth rate could be controlled by adjusting the expression level of the essential gene leuD. The results allowed us to propose a laboratory approach to select and quantify drug-tolerant M. smegmatis persisters in batch cultures using rifampicin (RIF) and streptomycin (STM). Finally, it was intended to identify genes that affected the persisters frequency in M. tuberculosis by transposon mutagenesis. A transposon mutant library of M. tuberculosis was exposed to antibiotics under conditions that selected for drug-tolerant persisters. Examination of genes affecting persister frequency to rifampicin were found to be completely different from those affecting persister frequency to streptomycin, indicating that different mechanisms are involved in persistence to these antibiotics. Genes identified after RIF selection were mainly associated with cell wall and cell processes and metabolism and respiration functions, whereas several toxin genes were identified after STM treatment. These results indicate that the mechanism behind persisters is drug-specific and highly coordinated. In addition, there was a marked redundant functionality within the identified genes. Overall, findings of this study have opened a new insight into mycobacterial persisters, and further investigation of the results presented in this project could contribute to advancement in the development of therapies to control this mycobacterial sub-population and reduce treatment length.