Cell Surface Protein Anchoring and Function in Gram-Positive Bacteria

描述（由申请人提供）：细菌表面显示的蛋白质在发病过程中起关键作用，因为它们促进细菌粘附到宿主组织，获取必需营养物质，逃避和抑制免疫反应和宿主细胞进入。我们将研究革兰氏阳性细菌病原体在感染过程中如何展示和利用毒力因子，并寻求开发通过抑制细菌蛋白展示而起作用的新抗生素。研究将集中在金黄色葡萄球菌上，这是美国致命的医院和社区获得性感染的主要原因，每年导致的死亡人数超过任何其他传染病。金黄色葡萄球菌和其他革兰氏阳性细菌利用分选酶转肽酶将毒力因子共价附着在细胞壁上。在金黄色葡萄球菌中，表面蛋白由分选酶A （Sa-SrtA）和分选酶B （Sa-SrtB）酶显示。他们共同构建了铁调控的表面决定因子（Isd）系统，该系统在感染期间积极地从人血红蛋白中获取必需的营养铁。分类酶和Isd蛋白是开发新的抗感染药物的主要目标，因为它们都有助于金黄色葡萄球菌的发病机制。在目标#1中，我们通过确定与特殊合成的分选信号类似物结合的代表性酶的结构，并通过使用新开发的体内转肽酶测定来探索分选酶如何识别过桥肽，从而获得对致病菌如何显示毒力因子的广泛机制见解。在目标#2中，我们发现的几种有前途的分选酶抑制剂将使用核磁共振、计算和合成化学方法进一步开发。这些分子是治疗致命感染的潜在创新方法，因为它们可以阻止细菌在其表面显示毒力因子，使它们对宿主的免疫反应毫无抵抗力。一旦它们的效力和选择性得到优化，它们的治疗效果将使用金黄色葡萄球菌全身感染的小鼠模型进行评估。目标3的研究将研究金黄色葡萄球菌如何利用附着在Isd蛋白上的分选酶清除人类血红蛋白中必需的营养铁，并将尝试通过靶向氨基酸诱变来破坏这一过程。利用核磁共振和生化方法，我们将阐明Sa- SrtA靶蛋白IsdH从血红蛋白中提取血红素的机制，以及Sa- srtb靶蛋白IsdC如何将血红素从靠近细胞表面的上游血红素受体传递到位于膜上的IsdDEF血红素转运蛋白复合物。总的来说，这项研究将增加我们对金黄色葡萄球菌发病机制的分子基础的理解，并可能导致新的治疗方法来治疗细菌感染。

DESCRIPTION (provided by applicant): Surface displayed proteins on bacteria play key roles in pathogenesis as they promote bacterial adhesion to host tissues, acquisition of essential nutrients, evasion and suppression of the immune response and host cell entry. We will study how Gram-positive bacterial pathogens display and utilize virulence factors during infections, and seek to develop new antibiotics that work by inhibiting bacterial protein display. Research will concentrate on Staphylococcus aureus, a leading cause of lethal hospital- and community-acquired infections in the United States that kill more people than any other infectious agent annually. S. aureus and other Gram- positive bacteria covalently attach virulence factors to their cell wall using sortase transpeptidase enzymes. In S. aureus, surface proteins are displayed by the sortase A (Sa-SrtA) and sortase B (Sa-SrtB) enzymes. They work together to construct the Iron-regulated surface determinant (Isd) system that actively harvests the essential nutrient iron from human hemoglobin during infections. Sortases and Isd proteins are prime targets for the development of new anti-infective agents as both contribute to S. aureus pathogenesis. In aim #1, we obtain broad mechanistic insight into how pathogenic bacteria display virulence factors by determining structures of representative enzymes bound to specially synthesized sorting signal analogs, and by using a newly developed in vivo transpeptidase assay to explore how sortases recognize the cross-bridge peptide. In aim #2, several promising sortase inhibitors we have discovered will be further developed using NMR, computational and synthetic chemistry methods. These molecules are a potentially innovative approach to treat lethal infections, as they would prevent bacteria from displaying virulence factors on their surface, rendering them defenseless against the host's immune response. Once their potency and selectivity have been optimized, their therapeutic efficacy will be evaluated using a mouse model of S. aureus systemic infection. Research in aim #3 will study how S. aureus uses sortase attached Isd proteins to scavenge the essential nutrient iron from human hemoglobin, and will attempt to disrupt this process through targeted amino acid mutagenesis. Using NMR and biochemical methods we will elucidate the mechanism through which the Sa- SrtA target protein IsdH extracts heme from hemoglobin, and how the Sa-SrtB target protein IsdC relays heme from upstream hemoreceptors positioned near the cell surface to the IsdDEF heme transporter complex located in the membrane. Collectively, this research will increase our understanding of the molecular basis of S. aureus pathogenesis and it could lead to new therapeutics to treat bacterial infections.