Cell Surface Protein Anchoring and Function in Gram-Positive Bacteria

革兰氏阳性细菌的细胞表面蛋白锚定和功能

基本信息

批准号：
8437143
负责人：
Robert Thompson Clubb
金额：
$ 35.36万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-06-01 至 2017-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8437143
关键词：
Active Sites Amino Acids Anti-Infective Agents Antibiotics Bacillus anthracis Bacteria Bacterial Adhesion Bacterial Infections Bacterial Proteins Binding Binding Sites Biochemical Biological Assay Blood-Borne Pathogens Catalysis Cell Surface Proteins Cell Wall Cell surface Cells Community-Acquired Infections Complex Development Drug Design Enzyme Inhibitor Drugs Enzyme Inhibitors Enzymes Exhibits Goals Gram-Positive Bacteria Growth Harvest Heme Hemoglobin Hospitals Human Immune response In Vitro Infection Infectious Agent Iron Lead Learning Life Lipids Membrane Membrane Proteins Methicillin Resistance Methods Molecular Multi-Drug Resistance Mutagenesis Mutation Nosocomial Infections Nutrient Pathogenesis Peptides Peptidyltransferase Play Positioning Attribute Process Proteins Research Research Personnel Signal Transduction Site Sorting - Cell Movement Staphylococcus aureus Structure Surface Synthesis Chemistry System Systemic infection Testing Therapeutic Agents Tissues Treatment Efficacy United States Variant Virulence Factors Work analog base combinatorial chemistry computational chemistry drug resistant bacteria improved in vivo inhibitor/antagonist innovation insight killings methicillin resistant Staphylococcus aureus mouse model novel therapeutics pathogen pathogenic bacteria prevent protein function receptor research study resistant strain screening small molecule sortase uptake

项目摘要

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.

描述（由申请人提供）：表面在细菌上显示蛋白质在发病机理中起关键作用，因为它们促进细菌粘附到宿主组织，获得必需营养素，逃避和抑制免疫反应和宿主细胞的进入。我们将研究革兰氏阳性细菌病原体在感染过程中如何显示和利用毒力因子，并寻求通过抑制细菌蛋白显示的新抗生素来开发新的抗生素。研究将集中于金黄色葡萄球菌，这是美国致命医院和社区获得感染的主要原因，杀死每年比任何其他感染毒剂的人多。金黄色葡萄球菌和其他革兰氏阳性细菌使用分子酶转肽酶将毒力因子共价附着在其细胞壁上。在金黄色葡萄球菌中，表面蛋白通过排序酶A（SA-SRTA）和排序酶B（SA-SRTB）酶显示。他们共同构建铁调节的表面决定因素（ISD）系统，该系统在感染过程中积极收获人类血红蛋白的基本营养铁。排序酶和ISD蛋白是开发新抗感染剂的主要靶标，因为这两者都有助于金黄色葡萄球菌的发病机理。在AIM＃1中，我们通过确定与特殊合成的分类信号类似物结合的代表性酶的结构以及使用新开发的体内经肽酶测定法以探索排序酶识别跨桥肽的结构来探索分类方式识别跨桥肽。在AIM＃2中，我们发现的几种有前途的分类酶抑制剂将使用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，因为它们会防止细菌在其表面表现出毒力因子，从而使它们无法防御宿主的免疫反应。一旦优化了其效力和选择性，将使用金黄色葡萄球菌全身感染的小鼠模型评估其治疗功效。 AIM＃3中的研究将研究金黄色葡萄球菌如何使用分子酶附着的ISD蛋白来清除人类血红蛋白的必需营养铁，并将尝试通过靶向的氨基酸诱变来破坏这一过程。使用NMR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，以及如何从位于层表中的蜂窝表面附近的上游输血器中的SA-SRTB靶蛋白ISDC继电器Heme heme heme heme heme。总的来说，这项研究将增加我们对金黄色葡萄球菌发病机理的分子基础的理解，并可能导致新的治疗细菌感染。