Alterations in the genetic capacity for cobamide biosynthesis have been identified as potentially critical in the evolution of Mycobacterium tuberculosis from a putative environmental ancestor. Moreover, recent studies have implicated cobamide biosynthesis pathway genes in the adaptation of the bacillus to intracellular pathogenesis. Although mycobacteria retain essential biochemical reactions that require cobamides, the specific role of these co-factors during tuberculosis (TB) disease remains unresolved. This thesis aimed to examine the production, uptake, and utilization of cobamides in mycobacteria using M. smegmatis as a model. To this end, the genetic capacity for de novo production and uptake of cobamide in host-associated and environmental mycobacteria was assessed, followed by direct validation in M. smegmatis. A combination of genetics, gene expression analysis, live-cell time-lapse microscopy and targeted metabolite and protein analysis via mass spectrometry (MS) was then employed to investigate cobamide riboswitch-dependent regulation of methionine biosynthesis in M. smegmatis. Results indicated that, in wild-type M. smegmatis, de novo cobamide biosynthesis ensured constitutive repression of metE, the gene encoding the mycobacterial cobalamin-independent methionine synthase. Owing to this repression, metH, a gene encoding the cobalamin-dependent methionine synthase, was found to be conditionally essential for bacillary replication in vitro. Drug susceptibility testing to investigate the link between cobamides and the intrinsic resistance to anti-folate antibiotics confirmed novel mycobacterial vulnerabilities in cobamide-related methionine metabolism, indicating that the outcomes of cobamidedependent regulation may have relevance to mycobacterial pathogenesis and drug discovery. In contrast to M. tuberculosis, which was previously shown to transport exogenous CNCbl readily, M. smegmatis poorly assimilated exogenous co-factor despite the presence of multiple putative cobamide transporters. However, uptake was enhanced in a mutant requiring CNCbl for growth. Elucidating the factors which regulate cobamide biosynthesis and co-factor utilization in M. smegmatis, an environmental mycobacterium, might provide a lens through which to consider the differential regulation and utilization of cobamides in M. tuberculosis, an obligate pathogen with a limited host range.

Mycobacteria is a genus characterized by its unique layer of mycomembrane, which enhances its pathogenicity causing notorious infections such as tuberculosis or leprosy in humans. Some pathogenic mycobacteria are part of the Mycobacterium tuberculosis complex (MTBC), while others are predominantly environmental and belong to the class of non-tuberculosis mycobacteria (NTM). Some of the NTMs are also opportunistic pathogens causing infections mostly in immunocompromised individuals. In this study, we focus on a recently discovered species of NTM known as M. riyadhense, originally isolated from a patient with TB-like symptoms in Riyadh. With prepublication access to the completely assembled and fully annotated genomes of M. riyadhense, we wanted to study the gene expression of M. riyadhense after establishing an infection model using a murine macrophage cell line. We performed transcriptomic analysis of M. riyadhense upon infection using RAW264.7 murine macrophages to determine the hallmarks of differentially expressed (DE) genes at early infection time points. Most DE genes observed belong to one of the crucial secretion systems known as ESX-1. Most genes were highly upregulated during 12-hour of infection, particularly esxA and esxB, which encode for ESAT-6 and CFP-10 secretion proteins. These substrates are essential for the virulence and pathogenicity of Mycobacterium tuberculosis (MTB). In addition, we observed downregulation of WhiB5, a transcriptional regulator that is a well-known controller of Mycobacterium tuberculosis virulence and reactivation, and regulates genes encoding the constituents of two type VII secretion systems, namely, ESX-2 and ESX-4. We have also identified other genes of yet unknown function that are highly upregulated during early infection needing functional characterization in future follow-up studies. Overall, we have established an in vitro cell infection model for M. riyadhense that can be used to study host pathogen cross talks during infection processes in tubercle bacilli.

Mycobacterium avium causes disseminated infection in humans with immunodeficiency, pulmonary infections in individuals with predisposing lung conditions (e.g., pneumoconiosis), and cervical lymphadenitis in children. Twenty-five to fifty percent of late stage AIDS patients are infected with M. avium. M. avium has been recovered from drinking water and strains from water share identical DNA fingerprints with isolates recovered from patients exposed to the water. Further, M. avium is resistant to chlorine, a disinfectant commonly used in municipal water supplies. Because of the slow growth of M. avium, measuring its susceptibility to disinfectants is laborious and reaction to a potential problem is delayed. Thus, there exists a need for a rapid test to measure the antimycobacterial disinfectant capability of chlorine containing water samples. The objective of this research was to develop a rapid and quantitative assay for the viability of mycobacteria using firefly luciferase as a reporter gene for disinfection survival studies. Derivatives of M. avium strains MD1 and A5, Mycobacterium smegmatis strain VT307 and Mycobacterium bovis BCG strain Pasteur carrying the firefly luciferase gene (pLUC10) were constructed. In pLUC10-carrying strains of M. avium strain A5 and M. smegmatis strain VT307, a direct correlation was shown between the quantity of light produced and the number of cells recovered as colony forming units. In disinfection studies of both pLUC10-carrying derivatives of M. avium strain A5 and M. smegmatis strain VT307, survival, as measured in colony forming units, correlated with survival in relative light units. Luciferase measurements appear to offer a method for rapid enumeration of mycobactericidal disinfection capacity of chlorinated water. / Master of Science