The role of GraRS in resistance to cationic antimicrobial peptides in S. aureus

描述（由申请人提供）：金黄色葡萄球菌是一种高度适应性的人类病原体。这种适应的一个结果是它能够产生抗性阳离子抗菌肽（CAPs），这是宿主先天免疫的重要组成部分。在仔细研究金黄色葡萄球菌基因组时，我们发现了四个双组分调控系统，每个系统都与ABC转运体系统相邻。其中一个系统GraRS调节邻近的VraFG外排系统，该系统由atp酶和膜渗透酶组成。graR或vraG的突变导致对诸如RP-1（来自血小板的mp -1的同系物）和hNP-1（来自中性粒细胞）、吲哚霉素和LL37等cap的耐药性降低。作为血管内病原体，金黄色葡萄球菌可能会遇到来自血小板和中性粒细胞（RP-1和hNP-1）的cap，我们将在本提案中重点讨论。根据诱导研究和微阵列分析，金黄色葡萄球菌暴露于选择性CAP也会诱导graRS和vraFG以及其他与CAP抗性相关的基因，包括mprF、dltABCD和另一组ABC转运基因vraDE。然而，这些基因对金黄色葡萄球菌中GraRS介导的CAP抗性的确切贡献尚未确定（Aim I）。此外，与表皮葡萄球菌的graRS和vraFG的表达可以被任何cap诱导相比，金黄色葡萄球菌的诱导更具选择性，只对少数cap有反应。基于金黄色葡萄球菌和表皮葡萄球菌之间激酶传感器GraS序列的差异，我们建议确定GraS中介导特异性cap识别以诱导下游基因的确切残基（Aim II）。除了细菌因素外，我们还对合成cap的结构及其结合和诱导graRS表达的能力感兴趣（Aim III）。最后，将在兔感染性心内膜炎的相关模型中探讨GraRS中这些发现的体内相关性。总之，这些研究将引出金黄色葡萄球菌对相关宿主cap先天耐药的科学依据，从而为设计减轻对阳离子抗菌肽耐药的策略提供平台。

DESCRIPTION (provided by applicant): S. aureus is a highly adaptive human pathogen. One result of this adaptation is its ability to develop resistance cationic antimicrobial peptides (CAPs), which are an important part of innate immunity in the hosts. In perusing the S. aureus genomes, we found four two component regulatory systems each of lie adjacent to an ABC transporter system. One of the system, GraRS, regulates the adjacent VraFG efflux system comprising an ATPase and a membrane permease. Mutations in graR or vraG have led to a reduction in resistance to CAPs such as RP-1 (a congener to PMP-1 from platelets) and hNP-1 (from neutrophils), indolicidin and LL37. As an intravascular pathogen, S. aureus likely encounters CAPs from platelets and neutrophils (RP-1 and hNP-1), which we will focus on in this proposal. The exposure of S. aureus to selective CAP has also led to induction of graRS and vraFG and other genes relevant CAP resistance including mprF, dltABCD and also another set of ABC transporter genes vraDE, based on induction studies and microarray analysis. However, the exact contribution of these genes to CAP resistance mediated by GraRS in S. aureus is not defined (Aim I). In addition, in contrast to S. epidermidis where the expression of graRS and vraFG can be induced by any CAPs, the induction in S. aureus is more selective, responding to only a few CAPs. Based on the difference in the sequence of the kinase sensor GraS between S. aureus and S. epidermidis, we propose to define the exact residues within GraS that mediate this recognition of specific CAPs for induction of downstream genes (Aim II). Besides the bacterial factors, we are also interested in the structure of synthetic CAPs and their ability to bind and induce expression of graRS (Aim III). Finally, the in vivo correlate of these finding in GraRS will be explored in a relevant rabbit model of infective endocarditis. Together, these studies will elicit the scientific basis of innate resistance to relevant host CAPs in S. aureus, thus providing a platform to devise strategies to alleviate resistance to cationic antimicrobial peptides.