Staphylococcal biofilm and disease

葡萄球菌生物膜和疾病

• 批准号: 7694663
• 负责人: KENNETH W. BAYLES
• 金额: $ 266.23万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7694663
• 关键词: Staphylococcal; biofilm; disease

DESCRIPTION (provided by applicant): Staphylococcus aureus has historically been a leading cause of nosocomial infections in humans worldwide. In the past several decades the emergence of methicillin-resistant S. aureus (MRSA) harboring multiple antibiotic resistance determinants has left relatively few therapeutic options available. Today, these strains have made their way into the community and now pose a very serious public health threat, causing more deaths per year than HIV-AIDS. The University of Nebraska Medical Center has committed to advancing research into the staphylococci with the hiring of several new researchers, and the concerted collaboration with regional investigators, who are all focused on this important pathogen. These researchers have expertise in varied aspects of the staphylococci including: biofilm development, gene regulation, physiology, and the immunology of staphylococcal infections. The hypothesis to be tested by this team in this application is that S. aureus biofilm formation involves complex developmental processes that affect the host immune response. The application includes four projects centered around staphylococcal biofilm and disease including: regulated cell death during biofilm development (Project 1, K. Bayles); effect of arginine metabolism on biofilm formation in the staphylococci (Project 2, P. Fey); the role of nuclease in biofilm development and disease (Project 3, A. Horswill); and innate immunity to S. aureus biofilm (Project 4, T. Kielian). The cores include: Biofilm Growth and Analysis Core (Core A, J. Bose); Bioimaging Core (Core B, T. Fritz); and Administrative Core (Core C, K. Bayles). Each project involves highly collaborative and synergistic research endeavors and relies heavily on the cores. These efforts will have a dramatic impact on our understanding of biofilm formation in the staphylococci and the effect it has on the host response. Ultimately, these studies will pave the way for novel therapeutic approaches for the treatment of staphylococcal infections. PROJECT 1: [Regulated cell death during biofilm development (Bayles, K)] PROJECT 1 DESCRIPTION (provided by applicant): Bacterial physiology has been extensively studied in the context of cell growth, but the molecular details by which bacteria undergo cell death and lysis have remained a near complete mystery. A growing body of recent evidence suggests that bacterial cell death and lysis involves active, genetically-encoded mechanisms that are critical to complex developmental processes such as sporulation and biofilm formation. The Staphylococcus aureus cid and Irg operons encode novel proteins that regulate bacterial death and lysis. CidA and LrgA proteins are proposed to be structurally and functionally similar to bacteriophageencoded holins and antihollns, and the ubiquitous distribution of these genes among bacteria suggests that they play a conserved physiological role. Recent studies have demonstrated an important biological role for CidA-mediated cell lysis during biofilm development, but the specific metabolic and environmental cues that regulate cid and /rg-mediated cell death and lysis within the context of biofilm growth remain ill-defined. Low oxygen growth and endogenous nitric oxide (NO) production have both been implicated as regulators of cell death and dispersal in biofilm of other bacteria, but the molecular mechanisms involved in these processes are not well understood. Preliminary data have suggested that growth under low oxygen conditions and NO are both potent signals that regulate cid and Irg expression. The scdA and NO-reductase {nor) genes, involved in the nitrosative stress response and NO metabolism, respectively, are also located in close proximity to lytSR-lrgAB in the clinical isolate UAMS-1. Thus, the central hypothesis of this application is that the transition into anaerobic metabolism during S. aureus biofilm growth is an important developmental signal in the control of Cid-/Lrg-mediated cell death and lysis. The specific aims of this project are 1) to study the transition to anaerobic metabolism during biofilm development and its effect on cid and Irg expression, 2) to examine the role of NO during biofilm development, and 3) to determine the molecular mechanism and role of LytSR-mediated regulation of cid and Irg expression during biofilm growth. Temporal and spatial patterns of aerobic and anaerobic metabolism within the biofilm will be determined using fluorescent reporter genes fused to aerobic and anaerobic promoters, and cid and Irg expression within these defined regions will be measured by laser capture microdissection microscopy (LCM) and real-time RT-PCR. The effect of NO donors and scavengers on cell death during biofilm development will be monitored using fluorescent dyes, and the effect of these compounds on cid and Irg expression will also be assessed. A detailed molecular characterization of the LytSR signal transduction cascade will also be performed to elucidate the role of this regulatory system during I0W-O2 growth and biofilm development. Collectively, these studies will reveal new insights into the molecular control of cell death and lysis during biofilm development.

描述（由申请方提供）：金黄色葡萄球菌历来是全球人类医院感染的主要原因。在过去的几十年中，耐甲氧西林的S。携带多种抗生素抗性决定簇的金黄色葡萄球菌（MRSA）留下了相对较少的可用治疗选择。今天，这些菌株已经进入社区，现在构成了非常严重的公共卫生威胁，每年造成的死亡人数超过艾滋病毒/艾滋病。内布拉斯加大学医学中心致力于推进对葡萄球菌的研究，聘请了几名新的研究人员，并与区域研究人员进行了协调合作，他们都专注于这种重要的病原体。这些研究人员在葡萄球菌的各个方面都有专业知识，包括：生物膜发育，基因调控，生理学和葡萄球菌感染的免疫学。在这个应用程序中，这个团队要测试的假设是S。金黄色葡萄球菌生物膜的形成涉及影响宿主免疫应答的复杂发育过程。该应用程序包括四个项目围绕葡萄球菌生物膜和疾病，包括：生物膜发展过程中的细胞死亡（项目1，K。Bayles）;精氨酸代谢对葡萄球菌中生物膜形成的影响（项目2，P. Fey）;核酸酶在生物膜形成和疾病中的作用（项目3，A.马的意志）;和先天免疫S。金黄色葡萄球菌生物膜（Project 4，T. Kielian）。核心包括：生物膜生长和分析核心（核心A，J. Bose）;生物成像核心（核心B，T. Fritz）;和管理核心（核心C，K。Bayles）。每个项目都涉及高度协作和协同的研究工作，并严重依赖于核心。这些努力将对我们理解葡萄球菌中生物膜的形成及其对宿主反应的影响产生巨大影响。最终，这些研究将为治疗葡萄球菌感染的新治疗方法铺平道路。 项目1：[生物膜发育过程中的调节性细胞死亡（Bayles，K）] 项目1描述（由申请人提供）：细菌生理学已在细胞生长的背景下进行了广泛研究，但细菌进行细胞死亡和裂解的分子细节仍然是一个近乎完整的谜。最近越来越多的证据表明，细菌细胞死亡和裂解涉及活跃的遗传编码机制，这些机制对复杂的发育过程（如孢子形成和生物膜形成）至关重要。金黄色葡萄球菌cid和Irg操纵子编码调节细菌死亡和裂解的新蛋白。CidA和LrgA蛋白被认为在结构和功能上与噬菌体编码的holins和antiholins相似，并且这些基因在细菌中的普遍分布表明它们发挥保守的生理作用。最近的研究表明，在生物膜发育过程中，CidA介导的细胞裂解具有重要的生物学作用，但在生物膜生长的背景下，调节cid和/或rg介导的细胞死亡和裂解的特定代谢和环境线索仍然不明确。低氧生长和内源性一氧化氮（NO）的产生都被认为是其他细菌生物膜中细胞死亡和分散的调节因子，但这些过程中涉及的分子机制还不清楚。初步数据表明，在低氧条件下生长和NO都是调节cid和Irg表达的有效信号。分别参与亚硝化应激反应和NO代谢的scdA和NO-还原酶（nor）基因也位于临床分离株UAMS-1中的lytSR-lrgAB附近。因此，本申请的中心假设是S.金黄色葡萄球菌生物膜生长是控制Cid-/Lrg-介导的细胞死亡和裂解的重要发育信号。本项目的具体目的是1）研究生物膜发育过程中向厌氧代谢的转变及其对cid和Irg表达的影响，2）研究NO在生物膜发育过程中的作用，3）确定生物膜生长过程中LytSR介导的cid和Irg表达调控的分子机制和作用。使用与需氧和厌氧启动子融合的荧光报告基因确定生物膜内需氧和厌氧代谢的时间和空间模式，并通过激光捕获显微切割显微镜（LCM）和实时RT-PCR测量这些限定区域内的cid和Irg表达。将使用荧光染料监测NO供体和清除剂对生物膜发育期间细胞死亡的影响，并且还将评估这些化合物对cid和Irg表达的影响。还将进行LytSR信号转导级联的详细分子表征，以阐明该调节系统在I 0 W-O2生长和生物膜发育过程中的作用。总的来说，这些研究将揭示生物膜发育过程中细胞死亡和裂解的分子控制的新见解。