Role of N-alpha acetylation in mycobacterial secretion and virulence

描述(申请人提供)：70-90%的真核蛋白质是N？乙酰化的。已建立的N？乙酰化原核蛋白有7种。ESAT-6(早期分泌抗原，6 kDa)是唯一一个N？乙酰化细菌毒力因子的例子。ESAT-6由ESX-1(ESAT-6系统1)分泌，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底物需要彼此输出(耦合分泌)。耦合分泌的机制尚不清楚。其结果是在理解细菌如何导致疾病方面存在根本性的差距。长期目标是了解分枝杆菌致病的分子机制。总体目标是确定N？-ac如何促进分枝杆菌的发病。中心假说是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。这一假设是基于申请人的初步数据，这些数据表明，改变ESAT-6的N？-ac会解除ESX-1的出口，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输出2)确定海洋分枝杆菌ESAT-6乙酰化的新基因如何识别结核分枝杆菌(M.TB)中与ESX-1相关的N？乙酰转移酶。在目标1下，将使用分子、生物化学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海洋分枝杆菌和结核分枝杆菌中的所有N？乙酰化蛋白，将解决分枝杆菌中N？-ac的范围。在目标2下，将利用生物化学方法在体外研究ESAT-6的N？-Ac的作用机制。一种互补的遗传方法将确定ESAT-6的N？-ac以及与ESX-1装置相互作用所需的假定ESAT-6 N？-乙酰转移酶中的残基。最后，在目标3下，海洋分枝杆菌系统中产生的工具将用于确定人类结核分枝杆菌中ESAT-6的N？ac所需的基因。将研究N？-ac在结核分枝杆菌ESX-1分泌和毒力中的作用。这项拟议的工作将提供N？ac如何促进细菌致病的第一个例子，并将导致对ESX分泌的分子机制的更好理解。这一建议是创新的，因为耦合分泌是ESX-1领域最大的悬而未决的问题之一，人们正在通过关注N？ac来解决这一问题，N？ac是细菌中一种可能被忽视的蛋白质修饰。这项研究的意义是鉴定和鉴定了第一个报道的具有ESX-1底物非偶联分泌的分支杆菌突变株，这将导致在 ESX-1字段。此外，这一建议代表了理解N？-ac如何促进细菌蛋白质分泌和致病机理的第一步，这可能会开启一个新的研究领域。

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.