Mechanism of Action of Stenotrophomonas maltophilia proteases

嗜麦芽寡养单胞菌蛋白酶的作用机制

• 批准号: 8783561
• 负责人: Ashley Lynn DuMont
• 金额: $ 5.6万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8783561
• 关键词: A549; Accounting; Actins; Address; Alveolar; Amino Acid Sequence; Antibiotics; Apoptotic; Bacteremia; Biochemical Genetics; Biological; Bronchioles; Catalytic Domain; Cell Death; Cell Line; Cells; Cessation of life; Chemicals; Cleaved cell; Clinical; Community-Acquired Infections; Cystic Fibrosis; DNA Sequence Rearrangement; Data; Development; Disease; Endocarditis; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Family; Fellowship; Fractionation; Gram-Negative Bacteria; Hospitals; Human; Immunocompromised Host; Individual; Infection; Inflammation; Inflammatory; Inflammatory Response; Intensive Care Units; Laboratories; Location; Lung; Lung diseases; Lytic; Maps; Mediating; Medical Device; Modeling; Molecular; Multi-Drug Resistance; Mus; Mutagenesis; National Research Service Awards; Nature; Nosocomial pneumonia; Organism; Outcome; Pathogenesis; Patients; Peptide Hydrolases; Pneumonia; Predisposition; Prevalence; Production; Proteins; Resistance; Serine; Serine Protease; Serine Proteinase Inhibitors; Skin Tissue; Soft Tissue Infections; Specimen; Stenotrophomonas maltophilia; Streptococcus pneumoniae; Subtilisins; System; Temperature; Testing; Tissues; Type II Secretion System Pathway; United States; Urinary tract infection; Virulence; Virulence Factors; cell type; clinically relevant; cystic fibrosis patients; cytokine; cytotoxic; cytotoxicity; design; extracellular; inhibitor/antagonist; mutant; novel; pathogen; public health relevance; respiratory; response; treatment strategy

DESCRIPTION (provided by applicant): Stenotrophomonos maltophilia is an emerging opportunistic Gram-negative pathogen that is increasingly isolated from medical devices and clinical specimens. As one of the top 11 organisms isolated from intensive care unit (ICU) patients, the pathogen poses significant threat to immunocompromised individuals, and is commonly isolated from Cystic Fibrosis (CF) patients. In the debilitated host, S. maltophilia infection most commonly manifests as pneumonia, but the pathogen can also cause bacteremia, endocarditis, urinary tract infection, and skin and soft tissue infections. Treatment o S. maltophilia infection is difficult due to the pathogen's inherent resistance to multiple antibiotics. Currently, our understanding of the virulence mechanisms employed by S. maltophilia to cause disease is very limited. In murine models of pneumonia, S. maltophilia has been shown to grow and persist, elicit an inflammatory response, and cause tissue destruction in the lung. However, the virulence factors that mediate these effects remain to be determined. Our lab has recently identified a type II secretion system in the S. maltophilia strain K279a that mediates cell rounding, actin rearrangement, detachment, and ultimately cell death in the human lung epithelial cell line A549. Preliminary data generated using mutagenesis indicate that the type II secreted serine proteases StmPr1 and StmPr2, which share 40% amino acid sequence identity, contribute to the observed rounding and cell death in A549 cells. Additionally, StmPr1 and StmPr2 are entirely responsible for the serine protease activity observed in cell-free supernatants collected from strain K279a. Both the stmPr1 and stmPr2 loci are found in in the majority of S. maltophilia isolates from CF patients, bolstering the potential clinical relevance o these proteases. Using biochemical and genetic approaches, this proposal aims to i) determine whether or not StmPr1 and StmPr2 are directly involved and sufficient to cause cell rounding; and determine if it is the serine protease activity of StmPr1 and StmPr2 that is responsible for A549 cell rounding, ii) elucidate the mechanism of protease-mediated A549 cell rounding by identifying host targets, and iii) investigate how cell rounding contributes to S. maltophilia pathogenesis by investigating protease-mediated cytotoxic effects and pro-inflammatory responses in a variety of lung cells. Together these aims will further our understanding of protease-mediated disease mechanisms, and will be informative for the development of novel treatment strategies centered on S. maltophilia proteases.

描述（由申请人提供）：嗜性嗜性嗜性菌株是一种新兴的机会性革兰氏阴性病原体，越来越多地与医疗设备和临床标本分离出来。作为从重症监护病房（ICU）患者中分离出的前11种生物之一，该病原体对免疫功能低下的个体构成了重大威胁，通常是从囊性纤维化（CF）患者中分离出来的。在衰弱的宿主中，麦芽葡萄球菌感染最常见于肺炎，但病原体也会引起菌血症，心内膜炎，尿路感染以及皮肤和软组织感染。由于病原体对多种抗生素的固有抗性，因此很难治疗麦芽菌感染。目前，我们对麦芽链球菌引起疾病的毒力机制的理解非常有限。在肺炎的鼠模型中，已证明一个嗜嗜嗜性链球菌会生长并持续存在，引起炎症反应，并在肺部造成组织破坏。但是，介导这些作用的毒力因素仍有待确定。我们的实验室最近确定了麦芽链球菌菌株K279a中的II型分泌系统，该系统介导了人类肺上皮细胞系A549中细胞圆形，肌动蛋白重排，分离和最终的细胞死亡。使用诱变产生的初步数据表明，具有40％氨基酸序列身份的II型分泌丝氨酸蛋白酶STMPR1和STMPR2有助于A549细胞中观察到的圆形和细胞死亡。另外，STMPR1和STMPR2完全负责在K279a菌株收集的无细胞上清液中观察到的丝氨酸蛋白酶活性。 STMPR1和STMPR2基因座都在CF患者的大多数麦芽菌分离株中都发现，从而增强了这些蛋白酶的潜在临床相关性。使用生化和遗传方法，该建议的目的是i）确定STMPR1和STMPR2是否直接参与并且足以引起细胞舍入；并确定负责A549细胞舍入的STMPR1和STMPR2的丝氨酸蛋白酶是否活性，ii）ii）通过鉴定宿主靶标阐明蛋白酶介导的A549细胞圆形的机制，以及iiii）研究细胞圆形如何通过研究麦芽葡萄球菌病原体来通过研究蛋白酶介导的蛋白酶介导的细胞毒性和反应效应效应，以促进蛋白酶病原体。这些目标共同将进一步了解蛋白酶介导的疾病机制，并将为以麦芽链球菌蛋白酶为中心的新型治疗策略提供信息。