Role of N-alpha acetylation in mycobacterial secretion and virulence

描述（由申请人提供）：70 - 90%的真核蛋白质是N-乙酰化的。有七种已确定的N ²-乙酰化原核蛋白质。ESAT-6（早期分泌抗原，6kDa）是唯一的N-乙酰化细菌毒力因子。ESAT-6由ESX-1（ESAT-6系统1）分泌，是革兰氏阳性和分枝杆菌病原体中的关键毒力决定因子。细菌蛋白质的N <$-乙酰化（N <$-Ac）在发病机制中的作用尚未研究。ESX-1底物需要彼此输出（偶联分泌）。偶联分泌的机制尚不清楚。结果是理解细菌如何引起疾病的根本差距。长期目标是了解分枝杆菌发病机制的分子机制。总的目标是确定N-Ac如何促进分枝杆菌的发病机制。核心假设是ESAT-6的N <$-Ac通过直接介导ESX-1系统的蛋白质输出而导致发病。该假设基于申请人的初步数据，其显示改变ESAT-6的N-Ac使ESX-1输出解偶联并导致衰减。因此，提出了以下具体目标。1)确定N-Ac如何促进ESX-1输出2）确定M. marinum促进ESAT-6乙酰化3）鉴定M.结核（M. tb）。在目标1下，将使用分子、生物化学和蛋白质组学方法研究ESAT-6的N ²-Ac和偶联分泌之间的联系。将通过鉴定分枝杆菌中所有的N <$-乙酰化蛋白质来确定N <$-Ac在分枝杆菌中的范围。marinum和M. tb结核using运用proteomics蛋白质组学.在目标2下，将使用生物化学在体外研究ESAT-6的N <$-Ac机制。一种互补的遗传学方法将鉴定ESAT-6 N <$-乙酰转移酶中的残基，该酶是ESAT-6的N <$-Ac和与ESX-1装置相互作用所需的。最后，根据目标3，在M。marinum系统将用于人M. ESAT-6的N-Ac所需的tb。N <$-Ac在M.将对结核病进行调查。拟议的工作将提供第一个例子，如何N ²-Ac促进细菌的发病机制，并将导致更好地了解ESX分泌的分子机制。该提案具有创新性，因为偶联分泌是ESX-1领域最大的悬而未决的问题之一，正在通过关注N ²-Ac（细菌中一种可能被忽视的蛋白质修饰）来解决。本文提出的研究的意义在于鉴定和表征了第一个报道的具有非偶联ESX-1底物分泌的分枝杆菌突变株，这将导致分枝杆菌突变株的垂直步骤。 ESX-1字段。此外，这一提议代表了理解N-Ac如何促进细菌蛋白质分泌和发病机制的第一步，这可能会开创一个新的研究领域。

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.