Jones Bradley S, Pareek Vikram, Hu Daniel D, Weaver Simon D, Syska Camille, Galfano Grace, Champion Matthew M, Champion Patricia A
bioRxiv. 2024 Jul 10:2024.07.05.602253. doi: 10.1101/2024.07.05.602253.
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 leveraged a nontubercular pathogen and an established model for hemolysis is a proxy for phagolytic activity. We generated strains 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 Δ strain. The Δ strain was attenuated in macrophage and infection models. Constitutive expression of either or which encode a KAT required for aminoglycoside resistance and a PDIM/PGL biosynthetic enzyme, rescued PDIM/PGL production and virulence of the Δ strain. Eis N-terminally acetylated PapA5 , 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 in . Our findings underscore the multifunctional nature of mycobacterial NATs and their connection to key virulence pathways.
Acetylation 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. is a well-established as a model to understand how causes tuberculosis. In this study we sought to identify conserved GNAT proteins required for early stages of mycobacterial infection. Using we determined that several GNAT proteins are required for the lytic activity of 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.
吞噬体裂解是宿主巨噬细胞感染分枝杆菌的一个关键方面。乙酰化是一种由N - 乙酰转移酶（NATs）酶介导的蛋白质修饰，它影响细菌的发病机制和生理功能。为了鉴定裂解活性所需的NATs，我们利用了一种非结核病原体，并建立了一个溶血模型作为吞噬溶解活性的替代指标。我们构建了保守NAT基因缺失的菌株，并筛选溶血活性。几种保守的赖氨酸乙酰转移酶（KATs）对溶血有作用。溶血由ESX - 1分泌系统和一种毒力脂质——结核硬脂酸二霉菌酸酯（PDIM）介导。对于几种菌株，通过添加ESX - 1基因座的第二个拷贝可恢复溶血活性。使用薄层色谱法（TLC），我们发现PDIM和酚糖脂（PGL）产生需要一种单一的NAT。MbtK是一种保守的KAT，是分枝杆菌铁载体合成和毒力所必需的。外源添加分枝杆菌铁载体J可补充Δ菌株中PDIM/PGL的产生。Δ菌株在巨噬细胞和感染模型中减毒。编码对氨基糖苷类耐药所需的KAT和一种PDIM/PGL生物合成酶的基因的组成型表达，挽救了Δ菌株的PDIM/PGL产生和毒力。Eis对PapA5进行N端乙酰化，支持脂质产生恢复的机制。总体而言，我们的研究建立了MbtK和Eis NATs之间以及结核分枝杆菌中铁摄取与PDIM和PGL合成之间的联系。我们的发现强调了分枝杆菌NATs的多功能性质及其与关键毒力途径的联系。
乙酰化是蛋白质N端、赖氨酸残基、抗生素和脂质的一种修饰。许多促进乙酰化的酶属于GNAT家族蛋白质。结核分枝杆菌是理解其如何导致结核病的一个成熟模型。在本研究中，我们试图鉴定分枝杆菌感染早期所需的保守GNAT蛋白。利用溶血模型，我们确定几种GNAT蛋白是结核分枝杆菌裂解活性所必需的。我们发现了铁摄取和抗微生物耐药性所需的乙酰转移酶与独特的分枝杆菌脂质PDIM和PGL产生之间以前未知的联系。我们的数据支持GNAT家族的乙酰转移酶相互关联，并且具有超出先前报道的活性。
