N<i>-</i>acetyltransferases required for iron uptake and aminoglycoside resistance promote virulence lipid production in <i>Mycobacterium marinum</i>.

Abstract

Phagosomal lysis is essential for mycobacterial infection of macrophages. Acetylation is a protein modification mediated enzymatically by N-acetyltransferases (NATs) that impacts bacterial pathogenesis and physiology. To identify NATs required for lytic activity, we leveraged <i>Mycobacterium marinum,</i> a nontubercular pathogen and a model for <i>Mycobacterium tuberculosis. M. marinum</i> hemolysis is a proxy for phagolytic activity. We generated <i>M. marinum</i> strains with deletions in conserved NAT genes and screened for hemolytic activity. Several conserved lysine acetyltransferases (KATs) contributed to hemolysis, which is mediated by the ESX-1 secretion system and by the virulence lipid phthiocerol dimycocerosate (PDIM). Using thin-layer chromatography, we found that MbtK, a conserved acyl-transferase required for mycobactin siderophore synthesis and virulence, was required for PDIM and phenolic glycolipid (PGL) production. Exogenous addition of sodium propionate or Mycobactin J restored PDIM/PGL production in the Δ<i>mbtK</i> strain. The Δ<i>mbtK M. marinum</i> strain was attenuated in macrophage and <i>Galleria mellonella</i> infection models. Constitutive expression of either <i>eis</i> or <i>papA5,</i> which encode a KAT required for aminoglycoside resistance and a PDIM/PGL biosynthetic enzyme, rescued PDIM/PGL production, and virulence of the ∆<i>mbtK</i> strain. Eis N-terminally acetylated PapA5 in vitro, providing a plausible mechanism for restored lipid production. Overall, our study establishes connections between the MbtK and Eis NATs, and between iron uptake and PDIM and PGL synthesis in <i>M. marinum</i>. Our findings underscore the multifunctional nature of mycobacterial NATs and their connection to key virulence pathways.