Cell Surface Protein Anchoring and Function in Gram-Positive Bacteria

描述(申请人提供)：细菌表面展示的蛋白质在致病过程中起关键作用，因为它们促进细菌与宿主组织的黏附，获得必要的营养，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在感染过程中如何显示和利用毒力因子，并寻求开发通过抑制细菌蛋白显示起作用的新抗生素。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比其他任何感染源都多。金黄色葡萄球菌和其他革兰氏阳性细菌使用排序酶转肽酶将毒力因子共价结合到细胞壁上。在金黄色葡萄球菌中，表面蛋白由分解酶A(Sa-SrtA)和分解酶B(Sa-SrtB)显示。他们共同构建铁调节表面决定因子(ISD)系统，该系统在感染期间从人类血红蛋白中主动获取必要的营养铁。糖苷酶和ISD蛋白是开发新的抗感染药物的主要靶点，因为它们都与金黄色葡萄球菌的致病机制有关。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，以及通过使用新开发的体内转肽酶试验来探索分类酶如何识别跨桥肽，从而获得了对病原菌如何显示毒力因子的广泛的机制洞察。在Aim#2中，我们发现的几种有前途的索酸酶抑制剂将利用核磁共振、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一种潜在的创新方法，因为它们将防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，它们的治疗效果将通过金黄色葡萄球菌全身感染的小鼠模型进行评估。AIM#3中的研究将研究金黄色葡萄球菌如何使用附着在索尔特酶上的ISD蛋白从人类血红蛋白中清除必要的营养铁，并将试图通过定向氨基酸突变来破坏这一过程。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如何将位于细胞表面附近的上游血色素受体中的血红素传递到位于膜上的IsdDEF血红素转运体复合体。总之，这项研究将增加我们对金黄色葡萄球菌致病分子基础的了解，并可能导致治疗细菌感染的新疗法。

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.