Homeostasis of N-α-terminal acetylation of EsxA correlates with virulence in Mycobacterium marinum

Abstract

The mycobacterial Esx-1 (ESAT-6 system 1) exporter translocates virulence factors across the cytoplasmic membrane to the cell wall, cell surface, and the bacteriological medium in vitro. The mechanisms underlying substrate targeting to distinct locations are unknown. Several Esx-1 substrates are N-α-terminally acetylated. The role of this rare modification in bacteria is unclear. We sought to identify genes required for Esx-1 substrate modification, transport, and localization. Pathogenic mycobacteria lyse Acanthamoeba castellanii in an Esx-1-dependent manner. We conducted a genetic screen to identify Mycobacterium marinum strains which failed to lyse amoebae. We identified a noncytotoxic M. marinum strain with a transposon insertion in a predicted N-α-terminal acetyltransferase not previously linked to mycobacterial pathogenesis. Disruption of this gene led to attenuation of virulence, failure to induce a type I interferon response during macrophage infection, and loss of hemolytic activity. The major Esx-1 substrates, EsxA and EsxB, were exported to the cell surface, but only low levels were released into the bacteriological medium. The balance of EsxA N-α-terminal acetylation was disrupted, resulting in a mycobacterial strain in which surface-associated EsxA was hyperacetylated. Genetic complementation completely restored Esx-1 function and the levels of N-α-terminally acetylated EsxA on the surface but restored only low levels of Esx-1 substrates in the bacteriological medium. Our results reveal a novel gene required for mycobacterial Esx-1 export. Our findings indicate that maintaining the homeostasis of Esx-1 substrate N-α-terminal acetylation is essential for Esx-1-mediated virulence. We propose an inverse correlation between EsxA acetylation and virulence.