N-acetyl-transferases required for iron uptake and aminoglycoside resistance promote virulence lipid production inM. marinum

Abstract

Phagosomal lysis is a key aspect of mycobacterial infection of host 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 leveragedMycobacterium marinum,a nontubercular pathogen and an established model forM. tuberculosis. M. marinumhemolysis is a proxy for phagolytic activity. We generatedM. marinumstrains with deletions in conserved NAT genes and screened for hemolytic activity. Several conserved lysine acetyltransferases (KATs) contributed to hemolysis. Hemolysis is mediated by the ESX-1 secretion system and by phthiocerol dimycocerosate (PDIM), a virulence lipid. For several strains, the hemolytic activity was restored by the addition of second copy of the ESX-1 locus. Using thin-layer chromatography (TLC), we found a single NAT required for PDIM and phenolic glycolipid (PGL) production. MbtK is a conserved KAT required for mycobactin siderophore synthesis and virulence. Mycobactin J exogenously complemented PDIM/PGL production in the ΔmbtKstrain. The ΔmbtK M. marinumstrain was attenuated in macrophage andGalleria mellonellainfection models. Constitutive expression of eithereisorpapA5,which encode a KAT required for aminoglycoside resistance and a PDIM/PGL biosynthetic enzyme, rescued PDIM/PGL production and virulence of the ΔmbtKstrain. Eis N-terminally acetylated PapA5in vitro, supporting a 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 inM. marinum. Our findings underscore the multifunctional nature of mycobacterial NATs and their connection to key virulence pathways.Significance StatementAcetylation is a modification of protein N-termini, lysine residues, antibiotics and lipids. Many of the enzymes that promote acetylation belong to the GNAT family of proteins.M. marinumis a well-established as a model to understand howM. tuberculosiscauses tuberculosis. In this study we sought to identify conserved GNAT proteins required for early stages of mycobacterial infection. UsingM. marinum,we determined that several GNAT proteins are required for the lytic activity ofM. marinum.We uncovered previously unknown connections between acetyl-transferases required for iron uptake and antimicrobial resistance, and the production of the unique mycobacterial lipids, PDIM and PGLOur data support that acetyl-transferases from the GNAT family are interconnected, and have activities beyond those previously reported.