Quorum Sensing Dependent Interactions with Biofilms and Innate Immunity Defenses

群体感应与生物膜和先天免疫防御的相互作用

• 批准号: 9402029
• 负责人: ALEXANDER R HORSWILL
• 金额: --
• 依托单位: VA EASTERN COLORADO HEALTH CARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9402029
• 关键词: Acute; Address; Antibiotic Therapy; Antimicrobial Resistance; Aspartate; Biochemical; Biological Assay; Caspase; Cathepsin G; Catheters; Cells; Chronic; Cleaved cell; Clinical; Collaborations; Cytoplasmic Granules; Data; Development; Goals; Healthcare Systems; Host Defense; Human; Hypersensitivity; Image; Immune; Infection; Injury; Leukocyte Elastase; Life Style; Link; Mediating; Mediator of activation protein; Membrane Proteins; Methicillin Resistance; Methods; Microbial Biofilms; Modeling; Molecular; Molecular Genetics; Mus; Natural Immunity; Orthopedics; Outpatients; Pathway interactions; Patients; Peptide Hydrolases; Predisposition; Prevalence; Production; Property; Proteins; Proteomics; Publishing; Regulation; Regulator Genes; Reporting; Research; Research Personnel; Role; Seeds; Serine; Site; Staphylococcus aureus; Structure; Surface; System; Testing; Tissues; Toxin; Veterans; extracellular; glycosylation; high risk; implant material; improved; in vivo; innovation; insight; medical implant; neutrophil; novel; pathogen; prevent; programs; public health relevance; quorum sensing; service member; therapeutic development

 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.

 描述（由申请人提供）： 金黄色葡萄球菌是一种多功能病原体，会引起广泛的急性和慢性感染。慢性感染的重要决定因素是金黄色葡萄球菌在宿主组织或医疗植入物材料上开发生物膜的能力。生物膜因遗传性抗菌疗法和宿主免疫剥落性而产生的治疗问题是有问题的，并且这些问题因耐甲氧西林的金黄色葡萄球菌（MRSA）的增长而使这些问题更加复杂。我的研究计划的长期目标是了解金黄色葡萄球菌生物膜的开发途径，以改善治疗方法。我们的研究表明，法定感应系统（也称为辅助基因调节剂或“ AGR”）是生物膜生活方式的关键中介。为了使金黄色葡萄球菌留下生物膜和种子新位点，必须重新激活AGR系统以拆卸生物膜结构，一旦细胞分散，它们就会恢复对抗生素处理的敏感性。我们小组的最新研究确定了在该机制中受到一致调节的细胞外蛋白酶的重要作用。我们的中心假设是金黄色葡萄球菌具有由半胱氨酸蛋白酶介导的（称为葡萄球菌）的一致调节的生物膜分散途径。我们的初步发现还表明，生物膜抑制因素是由人类中性粒细胞产生的，这使我们假设宿主防御能力可以利用分散途径并破坏生物膜。对于特定的目标1，我们假设葡萄球生产的AGR和ROT调节控制生物膜分散体。为了评估这一假设并进一步定义了分散机制，我们将通过分子方法和生物膜测定法调查ROT阻遏物对AGR-蛋白酶调节环节的贡献； （ii）评估葡萄球菌在生物膜成熟和分散体中的作用； （iii）使用鼠类导管生物膜模型中的感染成像测试体内调节级联反应。我们开发了一种表面剃须蛋白质组学方法，以鉴定葡萄球裂解靶标。使用这种方法，我们发现丝氨酸 - 天冬氨酸重复（SDR）蛋白通过生物膜状态的金黄色葡萄球菌表面取出。我们假设葡萄球清除了SDR表面蛋白以促进生物膜扩散。为了检验这一假设，在特定的目标2中，我们将（i）对SDR蛋白在生物膜发育中的功能进行深入的表征； （ii）生化在SDR上定义了葡萄球菌A和B的作用方式； （iii）确定糖基化对SDR蛋白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过敏（与William Nauseef博士合作）。通过纯化，我们将蛋白酶组织蛋白酶G鉴定为主要的抗生物胶质膜剂，人类嗜中性粒细胞弹性酶（HNE）也显示出活性。我们假设中性粒细胞颗粒蛋白酶通过清洁SDR蛋白来抑制生物膜。为了检验这一假设，在特定目标3中，我们将（i）对SDR蛋白进行中性粒细胞蛋白酶加工研究； （ii）评估中性粒细胞途径抑制抗生物膜活动的影响； AD（iii）识别并表征组织蛋白酶G和HNE从生物膜表面释放蛋白质。对生物膜分散机制以及对这些机制的宿主调节的了解，将有助于开发治疗剂，这些治疗剂可以为金黄色葡萄球菌慢性感染提供创新的治疗方法。