NEW INSIGHTS INTO THE MYCOBACTERIUM TUBERCULOSIS ESX-1 TYPE VII SECRETION SYSTEM THROUGH ITS RECONSTITUTION IN DIVERSE BCG STRAINS AND MYCOBACTERIUM SMEGMATIS

Mycobacterium tuberculosis (M. tb) is the causative agent of human tuberculosis (TB), an infectious disease that recently regained its status as the leading cause of death from a single infectious agent worldwide. Members of the Mycobacterium tuberculosis complex (MTBC), including M. tb and M. bovis, rely on the type VII secretion system (T7SS) ESX-1 to export key virulence effector proteins that drive host-pathogen interactions and tuberculosis immunopathogenesis. The ESX-1 system, which is encoded by genes in the esx-1 locus and the distal espACD operon, enables the orchestrated, energy-dependent export of highly immunodominant and virulence effector proteins EsxA, EsxB, EspA, EspB, and EspC, whose secretion is tightly co-regulated and interdependent. Despite advances in our understanding of ESX-1 composition and mechanism, many aspects of its regulation and functional architecture remain unresolved, partly due to the biosafety constraints in working with and the slow growth of the MTBC. To address these knowledge gaps, I employed a genetic complementation approach and reconstituted the M. tb ESX-1 system in the live attenuated tuberculosis vaccine M. bovis bacillus Calmette-Guérin (BCG), which is comprised of at least 12 genetically distinct strains as well as in the surrogate mycobacterial model M. smegmatis for detailed analyses. As such, in chapter 3, I took advantage of the genetic diversity of different BCG in which the ESX-1 system is inactive due to a shared genomic deletion called the Region of Difference 1 (RD1) that affects core esx-1 genes to show that restoration of the deleted RD1 genes in diverse BCG strains yields variable outcomes, with some strains exhibit partial or no recovery of ESX-1 secretion activity, while in others, low intracellular ATP-dependent regulation of ESX-1 remains impaired. These observations suggest that additional genetic variations outside RD1, possibly including strain-specific deletions, duplications, and single-nucleotide polymorphisms, are essential for ESX-1 activity or involved in its regulatory control. By leveraging prior knowledge in the research literature on ESX-1 and my data from my study described in chapter 3, I sought to assemble the entire M. tb ESX-1 system in the fast-growing, non-pathogenic surrogate model mycobacteria, Mycobacterium smegmatis, to dissect the molecular determinants of ESX-1 assembly and secretion in chapter 4 of my thesis. This strategy led to the serendipitous discovery of rv3860 as a new transcriptional regulator of the ESX-1 system. Moreover, MSX-1, the resulting M. smegmatis strain engineered to express the M. tb ESX-1 system, was found to provide protection against M. tb challenge in mice without sensitizing them to tuberculin or purified protein derivative (PPD). Taken together, the findings of my work emphasize the value of exploring the relationship between BCG strain diversity and the restoration of ESX-1 function and its assembly in M. smegmatis to identify novel molecular factors essential to optimal ESX-1 function and regulation.