Role of N-alpha acetylation in mycobacterial secretion and virulence

描述（由申请人提供）：所有真核蛋白的70-90%是N¿-乙酰化的。有7种已确定的N -乙酰化原核蛋白。ESAT-6（早期分泌抗原，6kDa）是N -乙酰化细菌毒力因子的唯一例子。ESAT-6是由ESX-1 （ESAT-6系统1）分泌的，ESX-1是革兰氏阳性和分枝杆菌病原体的关键毒力决定因素。细菌蛋白的N¿-乙酰化（N¿-Ac）在发病机制中的作用尚未被研究。ESX-1底物相互需要输出（偶联分泌）。偶联分泌的机制尚不清楚。结果是在理解细菌如何引起疾病方面出现了根本性的空白。长期目标是了解分枝杆菌发病机制的分子机制。总体目标是确定N¿-Ac如何促进分枝杆菌发病。核心假设是ESAT-6的N¿-Ac通过直接介导ESX-1系统的蛋白质输出而参与发病机制。该假设是基于申请人的初步数据，表明改变ESAT-6的N¿-Ac会使ESX-1的输出不耦合并导致衰减。因此，提出了以下具体目标。1)确定N¿-ac如何促进ESX-1的输出2)确定海洋分枝杆菌中的新基因如何促进ESAT-6乙酰化3)确定结核分枝杆菌（M. tb）中ESX-1相关的N¿-乙酰转移酶。在Aim 1下，将使用分子、生化和蛋白质组学方法研究ESAT-6的N¿-Ac与偶联分泌之间的联系。通过使用蛋白质组学鉴定m.m arinum和m.t tb中所有的N′-乙酰化蛋白，将解决分枝杆菌中N′-Ac的范围。目的2：利用体外生物化学方法研究ESAT-6的N¿-Ac作用机制。一种互补的遗传方法将鉴定ESAT-6的N¿-乙酰转移酶的残基，这是ESAT-6的N¿-Ac和ESX-1装置相互作用所必需的。最后，在Aim 3下，在海洋分枝杆菌系统中生成的工具将用于鉴定ESAT-6的N¿-Ac所需的人类结核分枝杆菌基因。N¿-Ac在结核分枝杆菌ESX-1分泌和毒力中的作用将被研究。提出的工作将提供N¿-Ac如何促进细菌发病机制的第一个例子，并将导致对ESX分泌的分子机制的更好理解。这一提议是创新的，因为偶联分泌是ESX-1领域最大的突出问题之一，人们正在关注细菌中可能被忽视的蛋白质修饰N¿-Ac。本文提出的研究意义在于首次报道的ESX-1底物分泌解偶联的分枝杆菌突变株的鉴定和表征，这将导致该领域的垂直发展

DESCRIPTION (provided by applicant): 70-90% of all eukaryotic proteins are N¿-acetylated. There are seven established N¿-acetylated prokaryotic proteins. ESAT-6 (Early secreted antigen, 6kDa) is the only example of an N¿-acetylated bacterial virulence factor. ESAT-6 is secreted by ESX-1 (ESAT-6 system 1), a key virulence determinant in both Gram-positive and mycobacterial pathogens. The role of N¿-acetylation (N¿-Ac) of bacterial proteins in pathogenesis has not been investigated. The ESX-1 substrates require each other for export (coupled secretion). The mechanisms underlying coupled secretion are not known. The result is a fundamental gap in understanding how bacteria cause disease. The long-term goal is to understand the molecular mechanisms that underlie mycobacterial pathogenesis. The overall objective is to identify how N¿-Ac promotes mycobacterial pathogenesis. The central hypothesis is that N¿-Ac of ESAT-6 contributes to pathogenesis by directly mediating protein export by the ESX-1 system. The hypothesis is based on the applicant's preliminary data which show that altering N¿-Ac of ESAT-6 uncouples ESX-1 export and leads to attenuation. Therefore, the following specific aims have been proposed. 1) Establish how N¿-Ac promotes ESX-1 export 2) Determine how a novel gene in M. marinum pro- motes ESAT-6 acetylation 3) Identify the ESX-1-associated N¿-acetyltransferase in M. tuberculosis (M. tb). Under Aim 1, the link between N¿-Ac of ESAT-6 and coupled secretion will be investigated using molecular, biochemical and proteomic approaches. The scope of N¿-Ac in Mycobacterium will be addressed by identifying all of the N¿-acetylated proteins in M. marinum and M. tb using proteomics. Under Aim 2, the mechanism of N¿-Ac of ESAT-6 will be studied in vitro using biochemistry. A complementary genetic approach will identify residues in the putative ESAT-6 N¿-acetyltransferase required for N¿-Ac of ESAT-6 and for interaction with the ESX-1 apparatus. Finally, under the Aim 3, the tools generated in the M. marinum system will be used to identify the gene in human M. tb required for N¿-Ac of ESAT-6. The role of N¿-Ac in ESX-1 secretion and virulence in M. tb will be investigated. The proposed work will provide the first example of how N¿-Ac promotes bacterial pathogenesis and will result in an improved understanding of the molecular mechanisms of ESX secretion. This proposal is innovative because coupled secretion, one of the biggest outstanding questions in the ESX-1 field, is being approached by focusing on N¿-Ac, a likely overlooked protein modification in bacteria. The significance of the research proposed here is the identification and characterization of the first reported mycobacterial mutant strain with uncoupled ESX-1 substrate secretion, which will result in a vertical step in the ESX-1 field. Moreover, this proposal represents the primary step in understanding how N¿-Ac promotes bacterial protein secretion and pathogenesis, which will likely initiate a new field of study.