Investigations of the Virulence Mechanisms of Mycobacterium marinum

Mycobacterium tuberculosis 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. Mycobacterium marinum, a related environmental pathogen that uses many of the same conserved virulence mechanisms as M. tb. serves as a mycobacterial disease model. Using M. marinum 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 M. marinum 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 M. marinum and M. tb. 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 M. marinum as a model we have gained new insights into protein homeostasis, virulence regulation and virulence factor secretion, which will inform future studies in M. tuberculosis.