Quorum Sensing Dependent Interactions with Biofilms and Innate Immunity Defenses

 描述(由申请人提供)： 金黄色葡萄球菌是一种多功能病原体，可引起广泛的急性和慢性感染。慢性感染的一个重要决定因素是金黄色葡萄球菌在宿主组织或医用植入材料上形成生物膜的能力。由于生物膜对抗菌素治疗和宿主免疫防御的固有耐药性，生物膜在治疗中存在问题，而耐甲氧西林金黄色葡萄球菌(MRSA)水平的增长加剧了这些问题。我的研究计划的长期目标是了解金黄色葡萄球菌生物被膜的发育途径，以便改进治疗方法。我们的研究表明，群体感应系统，也被称为辅助基因调节器或“AGR”，是生物膜生活方式的关键调节因子。对于金黄色葡萄球菌来说，要离开生物膜并播种新的位置，必须重新激活agr系统来分解生物膜结构，一旦细胞分散，它们就会重新对抗生素治疗敏感。我们小组最近的研究已经确定了AGR调节的胞外蛋白水解酶在这一机制中的重要作用。我们的中心假设是金黄色葡萄球菌有一个由半胱氨酸蛋白酶(称为葡萄球菌)介导的agr调节的生物膜扩散途径。我们的初步发现还表明，生物膜抑制因子是由人类中性粒细胞产生的，这使我们假设宿主防御可以进入扩散途径并破坏生物膜。对于特定的目标1，我们假设金黄色葡萄球菌酶生产的AGR和Rot调控控制生物膜的扩散。为了评估这一假说并进一步确定扩散机制，我们将(I)通过分子方法和生物膜分析来研究腐烂抑制物对农业蛋白水解酶调节环节的贡献；(Ii)评估葡萄球菌蛋白在生物膜成熟和扩散中的作用；以及(Iii)在小鼠导管生物膜模型中使用感染成像来测试体内调节级联的保守性。我们开发了一种表面剃毛蛋白质组学方法来鉴定葡萄球菌酶A的切割靶标。利用这种方法，我们发现金黄色葡萄球菌表面的丝氨酸-天冬氨酸重复序列(SDR)蛋白被葡萄球菌A以生物被膜的状态去除。我们假设葡萄球菌切割SDR表面蛋白以促进生物膜的扩散。为了验证这一假说，在特定目标2中，我们将(I)深入表征SDR蛋白质在生物膜发育中的功能；(Ii)生化确定葡萄球菌酶A和B对SDR的作用模式；以及(Iii)确定糖基化对SDR蛋白质加工和生物膜功能的影响。最后，我们发现金黄色葡萄球菌生物膜对中性粒细胞颗粒高度敏感(与William Nauseef博士合作)。通过纯化，我们确定组织蛋白酶G是主要的抗生物被膜物质，人中性粒细胞弹性蛋白酶(HNE)也显示了活性。我们假设中性粒细胞颗粒蛋白水解酶通过裂解SDR蛋白来抑制生物膜。为了验证这一假设，在特定的目标3中，我们将(I)对SDR蛋白进行中性粒细胞蛋白酶处理研究；(Ii)评估中性粒细胞途径抑制对抗生物膜活性的影响；(Iii)鉴定和表征从生物膜表面释放的组织蛋白酶G和HNE蛋白。对生物被膜扩散机制和宿主对这些机制的调控的深入了解，将有助于开发能够为金黄色葡萄球菌慢性感染提供创新治疗的治疗方法。

 DESCRIPTION (provided by applicant): Staphylococcus aureus is a versatile pathogen that causes a broad spectrum of acute and chronic infections. An important determinant of the chronic infections is the ability of S. aureus to develop a biofilm on host tissue or medical implant material. Biofilms are problematic for treatment due to their inherent resistance to antimicrobial therapies and host immune defenses, and these problems are compounded by the growing levels of methicillin-resistant S. aureus (MRSA). The long-term goal of my research program is to understand S. aureus biofilm development pathways in order to improve treatment approaches. Our studies have demonstrated that the quorum-sensing system, also called the accessory gene regulator or "agr", is a key mediator of the biofilm lifestyle. For S. aureus to leave a biofilm and seed new sites, the agr system has to be reactivated to disassemble the biofilm structure, and once cells have dispersed, they regain susceptibility to antibiotic treatment. Recent studies in our group have identified a prominent role for agr-regulated extracellular proteases in this mechanism. Our central hypothesis is that S. aureus has an agr-regulated biofilm dispersal pathway that is mediated by cysteine proteases (called Staphopains). Our preliminary findings also demonstrate that biofilm inhibitory factors are produced by human neutrophils, leading us to hypothesize that host defenses can tap into the dispersal pathway and destroy biofilms. For Specific Aim 1, we hypothesize that agr and Rot regulation of Staphopain production controls biofilm dispersal. To evaluate this hypothesis and further define the dispersal mechanism, we will (i) investigate the contribution of Rot repressor to the agr-protease regulatory link through molecular approaches and biofilm assays; (ii) assess the role of Staphopains in biofilm maturation and dispersal; and (iii) test the conservation of the regulatory cascade in vivo using infection imaging in a murine catheter biofilm model. We developed a surface shaving proteomic method to identify Staphopain A cleavage targets. Using this method, we discovered that the Serine-Aspartate-Repeat (Sdr) proteins are removed by Staphopain A from the S. aureus surface in a biofilm state. We hypothesize that the Staphopains cleave the Sdr surface proteins to promote biofilm dispersal. To test this hypothesis, in Specific Aim 2 we will (i) perform an in-depth characterization of the function of Sdr proteins in biofilm development; (ii) biochemically define the mode of action of Staphopain A and B on the Sdr's; and (iii) determine the impact of glycosylation on Sdr protein processing and biofilm function. Finally, we discovered that S. aureus biofilms are hypersensitive to neutrophil granules (in collaboration with Dr. William Nauseef). Through purification, we identified the protease Cathepsin G as the primary anti-biofilm agent, and human neutrophil elastase (HNE) also showed activity. We hypothesize that neutrophil granule proteases inhibit biofilms by cleaving the Sdr proteins. To test this hypothesis, in Specific Aim 3 we will (i) perform neutrophil protease processing studies on the Sdr proteins; (ii) assess the impact of neutrophil pathway inhibition on anti-biofilm activities; ad (iii) identify and characterize Cathepsin G and HNE released proteins from the surface of biofilms. An improved understanding of biofilm dispersal mechanisms, and host modulation of these mechanisms, will aid the development of therapeutics that can provide innovative treatments for S. aureus chronic infections.