Investigations of the Virulence Mechanisms of <i>Mycobacterium marinum</i>

<p><i>Mycobacterium tuberculosis </i>causes Tuberculosis (TB), the leading cause of death globally from a single infectious disease. TB eradication will require improved control strategies necessitating new insights into the basic biology of mycobacteria. <i>Mycobacterium</i> <i>marinum</i>, a related environmental pathogen that uses many of the same conserved virulence mechanisms as <i>M. tb. </i>serves as a mycobacterial disease model. Using <i>M. marinum</i> we studied the modification, regulation and transport of mycobacterial virulence factors through biochemical, molecular and genetic approaches. N-terminal acetylation is a protein modification that remains poorly understood in bacteria. Many proteins involved in mycobacterial disease are Nt-acetylated and mycobacteria have numerous potential N-acetyltransferase (NAT) enzymes. The identities of which NAT enzymes Nt-acetylate which proteins remain largely unknown. We developed a technique to screen NAT enzymes for Nt-acetylation activity. We identified NAT enzymes that Nt-acetylated the virulence factor EsxA and demonstrated that the loss of one of these enzymes alters virulence. In a complementary approach, we generated a collection of <i>M. marinum</i> strains with deletions of conserved NAT genes. Through this approach, we identified a previously unknown genetic interaction between two NAT genes and built tools for its further characterization. Virulence in <i>M. marinum </i>and <i>M. tb. </i>requires the secretion of virulence factors by the ESX-1 (ESAT-6-system-1) protein export system. Expression of the secreted virulence factors is a regulated process dependent upon an intact ESX-1 system. We investigated the mechanisms of this regulation and identified the transcriptional repressor EspM which is required for repressing virulence factor expression in the absence of a functional ESX-1 system. It was thought that virulence factors (substrates) secreted by the ESX-1 were interdependent for secretion with loss of one substrate abolishing the secretion of others. We built a series of substrate deletion strains and characterized their ESX-1 secretion. We determined that substrates are not strictly interdependent for secretion, consistent with our previous work. Together our work illustrates multiple dynamic processes involved in mycobacterial virulence. By utilizing <i>M. marinum</i> as a model we have gained new insights into protein homeostasis, virulence regulation and virulence factor secretion, which will inform future studies in <i>M. tuberculosis.</i></p><p></p><p></p>