Molecular strategies to quantify and control dormant and active state of mycobacteria

Persisters are drug tolerant, and are able to switch spontaneously between dormant phenotypic state and a proliferative, antibiotic-sensitive state. Therefore, to ensure clearance of the infection without relapse, conventional tuberculosis drug regimens are 6-9 months in length, which has the important downside that it leads to evolution of drug-resistance. A better understanding of mycobacterial persistence/dormancy is required to devise strategies targeted towards their control and elimination. However, due to the transient nature and low frequency of their formation, relevant research remains challenging. In this thesis, the formation, quantification, and resuscitation of mycobacteria persister cells from various stressors were studied. Chapter 1 provides an overview of the heterogeneous nature of TB infections and the challenges posed by drug-tolerant persisters. It further reviews the formidable challenge posed by the drug-tolerant subpopulation, commonly referred to as "Persisters”, in combatting tuberculosis. Additionally, it provides a brief overview of various in vitro models used to study persistence in mycobacteria, along with strategies to target persister cells. Chapter 2 examines persister-cell formation from an integrative biology perspective and tried to derive common principles of persister-cell formation that could be applicable across evolutionary-distinct microbes. Despite the diversity of microbial species, we argue that similar principles govern them at molecular, physiological, and evolutionary levels. We argued that persister-cells formation is actually a response event to such a sudden reduction in growth rate and, hence, occurring as a response to many stresses. Therefore, while resistance may involve specific mechanisms or mutations in particular genes, persistence, on the other hand, can be achieved simply through slow growth. Chapter 3 describes the development of a high-throughput tool for quantifying and characterizing slow- or non-growing fractions in mycobacterial populations. This approach utilizes a lipophilic, small fluorescent organic labeling probe, that can detect and visualize pathogenic and non-pathogenic mycobacterial species in a specific, sensitive and non-invasive and fast manner. Using this tool, we can quantitatively determine the growth rate of mycobacteria under various in vitro and ex vivo growth conditions. Moreover, this method facilitated the study of mycobacterial resuscitation dynamics from hypoxia-induced dormancy at both population and single-cell level. Chapter 4 reports the role of serine hydroxamate, a stringent response inducer, in forming persister-like cells in Mycobacterium smegmatis. These cells showed typical persister properties, including growth arrest, low ATP levels, increased antibiotic tolerance, and delayed recovery. We also found that the recovery rate from stress depends on the bacteria's prior growth rate and stress exposure duration and can be accelerated by external stimuli. In Chapter 5, we studied the adaptation response of M. smegmatis to a nutrient transition, specifically from glucose to gluconeogenic carbon sources. We observed a delay in adaption to new carbon source with association to antibiotic tolerance. Our study indicates the presence of a metabolic bottleneck causing the delay, underscoring the importance of mycobacterial metabolism in the speed of growth recovery on a new carbon source, and antibiotic tolerance. In Chapter 6, we characterized Q203 that targets cytochrome bcc complex of electron transport chain. We were intrigued by the low sensitivity of M. smegmatis to Q203, which is otherwise highly effective in killing both replicating and non-replicating M. tuberculosis. We showed that M. smegmatis employs a double-pronged strategy to overcome the effect of Q203, including both the increased production of the target enzyme cytochrome bcc and the induction of the alternative cytochrome bd respiratory branch. Additionally, we investigated the potential role of a putative second cytochrome bd isoform postulated for M. smegmatis. The findings in this thesis are further discussed in Chapter 7, along with the outlook to future experimental needs and approaches.