Cell Surface Protein Anchoring and Function in Gram-Positive Bacteria

描述（由申请方提供）：细菌表面展示蛋白在发病机制中起关键作用，因为它们促进细菌粘附于宿主组织、获得必需营养素、逃避和抑制免疫应答以及宿主细胞进入。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质展示来发挥作用的新抗生素。研究将集中在金黄色葡萄球菌上，金黄色葡萄球菌是美国致命的医院和社区获得性感染的主要原因，每年杀死的人比任何其他传染病都多。S.金黄色葡萄球菌和其它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它们的细胞壁上。In S.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）酶展示。他们共同构建了铁调节表面决定簇（Isd）系统，该系统在感染期间从人类血红蛋白中积极收获必需的营养铁。分选酶和Isd蛋白是开发新的抗感染剂的主要目标，因为两者都有助于S。金黄色葡萄球菌致病性。在目标#1中，我们通过确定与专门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交叉桥肽，来获得对病原菌如何显示毒力因子的广泛的机理性见解。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NMR，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，因为它们可以防止细菌在其表面上显示毒力因子，使它们对宿主的免疫反应毫无防御能力。一旦它们的效力和选择性被优化，它们的治疗功效将使用S.金黄色葡萄球菌全身感染目标3中的研究将研究S。金黄色葡萄球菌使用分选酶连接的Isd蛋白从人血红蛋白中去除必需的营养铁，并将试图通过靶向氨基酸诱变来破坏这一过程。使用NMR和生物化学方法，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如何将血红素从位于细胞表面附近的上游血受体传递到位于膜中的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S.金黄色葡萄球菌的发病机制，并可能导致新的治疗方法来治疗细菌感染。 公共卫生相关性：金黄色葡萄球菌和其他细菌病原体利用表面展示的蛋白质来感染。我们将研究显示这些蛋白质的酶机制，以及它们如何从人类血红蛋白中提取必需的营养铁。我们还将继续开发几种有前途的小分子蛋白质展示抑制剂，这些抑制剂可以作为有效的抗感染剂来治疗由耐甲氧西林的S.金黄色葡萄球菌和其他多重耐药细菌。

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. PUBLIC HEALTH RELEVANCE: Staphylococcus aureus and other bacterial pathogens use surface displayed proteins to mount infections. We will study the enzymatic machinery that displays these proteins, and how they function to scavenge the essential nutrient iron from human hemoglobin. We will also continue to develop several promising small molecule protein display inhibitors that could function as potent anti-infective agents to treat infections caused methicillin-resistant strains of S. aureus, and other multi-drug resistant bacteria.