An N-acetyltransferase required for EsxA N-terminal protein acetylation and virulence in Mycobacterium marinum.
An N-acetyltransferase required for EsxA N-terminal protein acetylation and virulence in Mycobacterium marinum.
复制标题
海分枝杆菌中 EsxA N 末端蛋白乙酰化和毒力所需的 N-乙酰转移酶。
DOI：
10.1101/2023.03.14.532585
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Champion,PatriciaA
中科院分区：
文献类型：
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作者：
Collars,OwenA;Jones,BradleyS;Hu,DanielD;Weaver,SimonD;Champion,MatthewM;Champion,PatriciaA
蛋白质N端乙酰化是一种普遍存在的翻译后修饰，广泛影响着高等生物的多种细胞过程。细菌蛋白质也是N-末端乙酰化的，但对细菌中这种修饰的机制和后果知之甚少。我们先前定量了致病性分枝杆菌中广泛存在的N-末端蛋白乙酰化（C。R.汤普森，M。M.冠军和P.A. Champion，J Proteome Res 17（9）：3246-3258，2018，https：//doi：10.1021/acs.jproteome.8b00373）。主要毒力因子EsxA（ESAT-6，早期分泌抗原，6 kDa）是在细菌中鉴定的第一个N-末端乙酰化的蛋白质之一。EsxA在分枝杆菌病原体中是保守的，包括结核分枝杆菌和海分枝杆菌，一种在外温动物中引起结核样疾病的非结核分枝杆菌物种。然而，负责EsxA N-末端乙酰化的酶一直难以捉摸。在这里，我们使用遗传学，分子生物学和质谱为基础的蛋白质组学，以证明MMAR_1839（更名为Emp 1，ESX-1修饰蛋白，1）是假定的N-乙酰转移酶（NAT），只负责EsxA乙酰化在海分枝杆菌。我们证明，ERD_3144，在M.结核病Erdman，在功能上等同于Emp 1。我们确定了至少22个额外的蛋白质，需要Emp 1的乙酰化，证明这种假定的NAT不是专用于EsxA。最后，我们发现emp 1的缺失导致M. marinum引起巨噬细胞溶解。总的来说，这项研究确定了在分枝杆菌中N-末端乙酰化所需的NAT，并提供了对EsxA和其他蛋白质在巨噬细胞中分枝杆菌毒力中N-末端乙酰化的需求的了解。
N-terminal protein acetylation is a ubiquitous post-translational modification that broadly impacts diverse cellular processes in higher organisms. Bacterial proteins are also N-terminally acetylated, but the mechanisms and consequences of this modification in bacteria are poorly understood. We previously quantified widespread N-terminal protein acetylation in pathogenic mycobacteria (C. R. Thompson, M. M. Champion, and P.A. Champion, J Proteome Res 17(9): 3246–3258, 2018, https://doi:10.1021/acs.jproteome.8b00373). The major virulence factor EsxA (ESAT-6, Early secreted antigen, 6kDa) was one of the first N-terminally acetylated proteins identified in bacteria. EsxA is conserved in mycobacterial pathogens, including Mycobacterium tuberculosis and Mycobacterium marinum, a non-tubercular mycobacterial species that causes tuberculosis-like disease in ectotherms. However, enzyme responsible for EsxA N-terminal acetylation has been elusive. Here, we used genetics, molecular biology, and mass-spectroscopy based proteomics to demonstrate that MMAR_1839 (renamed Emp1, ESX-1 modifying protein, 1) is the putative N-acetyl transferase (NAT) solely responsible for EsxA acetylation in Mycobacterium marinum. We demonstrated that ERD_3144, the orthologous gene in M. tuberculosis Erdman, is functionally equivalent to Emp1. We identified at least 22 additional proteins that require Emp1 for acetylation, demonstrating that this putative NAT is not dedicated to EsxA. Finally, we showed that loss of emp1 resulted in a significant reduction in the ability of M. marinum to cause macrophage cytolysis. Collectively, this study identified a NAT required for N-terminal acetylation in Mycobacterium and provided insight into the requirement of N-terminal acetylation of EsxA and other proteins in mycobacterial virulence in the macrophage.