Determining the role of the CvpA protein in uropathogenic E. coli virulence

确定 CvpA 蛋白在尿路致病性大肠杆菌毒力中的作用

• 批准号: 8980943
• 负责人: Carrie Shaffer
• 金额: $ 3.49万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2016-04-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8980943
• 关键词: Accounting; Acute; Address; Adherence; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Attenuated; Bacteria; Bacterial Infections; Biochemical; Biogenesis; Biological Assay; Bladder; Cell Communication; Cell membrane; Cells; Communities; Community-Acquired Infections; Data; Defect; Development; Developmental Process; Disease; Drug resistance; Drug-sensitive; Epithelial; Epithelial Cells; Escherichia coli; Escherichia coli Infections; Extracellular Matrix; Flow Cytometry; Genes; Genetic; Growth; Health Care Costs; Hemolysin; Histology; Hospitals; Host Defense; Image; Immune; Immune response; In Vitro; Infection; Infective cystitis; Inflammatory; Invaded; Literature; Mass Spectrum Analysis; Mediating; Membrane; Methodology; Microbial Biofilms; Microscopy; Modeling; Multi-Drug Resistance; Mus; Organ; Pathogenesis; Peptide Antibiotics; Pharmaceutical Preparations; Pilum; Plasmids; Predisposition; Process; Production; Proteins; Proteomics; Recruitment Activity; Recurrence; Research; Resistance profile; Role; Severities; Signal Transduction; Site; Staging; Surface; Testing; Therapeutic; Toxin; United States; Urinary tract; Urinary tract infection; Uropathogenic E. coli; Vacuole; Virulence; Woman; assault; attenuation; bacteriocin; base; colicin; community-acquired UTI; cytokine; drug development; extracellular; fitness; in vivo; killings; mouse model; mutant; neutrophil; new therapeutic target; novel; pathogen; prevent; protein function; public health relevance; receptor; response; stressor; therapeutic target

 DESCRIPTION (provided by applicant): Uropathogenic Escherichia coli (UPEC), the primary cause of urinary tract infections (UTIs), have evolved exquisite mechanisms to subvert host defenses and to persist within the urinary tract in both intra- and extracellular niches. Within th intracellular site, UPEC undergo a developmental process that parallels extracellular biofilm formation and culminates in the biogenesis of intracellular bacterial communities (IBCs) that contain tightly packed bacteria encased within an extracellular matrix. Thus, identification of determinants that control bacterial intracellular replication will advance our understanding of UTI pathogenesis, will delineate strategies utilized by pathogens to occupy intracellular sites during infection, and will elucidate novel targets for anti-virulence therapy. Deletion of a UPEC factor, Colicin V production Accessory protein (CvpA), abolishes extracellular biofilm formation without influencing production of type 1 pili. Aiming to understand the contribution of CvpA to UPEC pathogenesis, I tested a UPEC cvpA deletion mutant in a murine model of acute UTI and determined that loss of CvpA abrogated the ability of UPEC to form IBCs during acute bladder infection despite WT levels of adherence to and subsequent invasion of superficial bladder epithelial cells in vivo. I also discovered that CvpA-deficient mutants are more susceptible to killing by neutrophils. The only described function for CvpA is that in its absence, colicin V, a small pore-forming toxin is not detected in the extracellular milieu. Although the plasmids that encode the colicin V structural and transport genes are not harbored by all UPEC strains (including strain UTI89, used in these studies), CvpA is conserved among E. coli, suggesting that CvpA has additional functions possibly such as the production and/or secretion of other pore- forming toxins. I hypothesize that during infection (1) CvpA modulates UPEC-host cell interactions and may be involved in a) dampening the host immune response and/or b) egressing from the vacuole upon entry into the host cells; and (2) loss of CvpA function impairs UPEC defenses against anti-bacterial assaults. In Aim 1 of this proposal, I will determine how loss of CvpA attenuates UPEC virulence. These studies will employ murine models of acute urinary tract infection, immunological assays, and advanced whole organ imaging methodologies. In Aim 2, I will interrogate the effects of cvpA deletion on UPEC toxin- associated factors and membrane integrity, using classical genetic, biochemical, and mass spectrometry approaches, and susceptibility assays. In total, these studies will delineate how CvpA promotes colonization of the urinary tract, will identify the mechanism by which CvpA modulates pathogenesis, and will evaluate CvpA as a bacterial target for drug development.

 描述(申请人提供)：尿路致病性大肠杆菌(UPEC)，尿路感染(UTIs)的主要原因，已经进化出精细的机制来颠覆宿主的防御并在尿路内细胞内和细胞外的壁龛内持续存在。在细胞内，UPEC经历一个与细胞外生物膜形成平行的发育过程，最终形成细胞内细菌群落(IBCs)，其中包含紧密包裹在细胞外基质中的细菌。因此，识别控制细菌细胞内复制的决定因素将促进我们对UTI的理解 将描述病原体在感染过程中占据细胞内位置的策略，并将阐明抗毒力治疗的新靶点。UPEC因子Colicin V产生辅助蛋白(CVPA)的缺失取消了细胞外生物膜的形成，而不影响1型菌毛的产生。为了了解CVPA在UPEC发病机制中的作用，我在急性尿路感染小鼠模型中测试了UPEC CVPA缺失突变体，并确定CVPA缺失会削弱UPEC在急性膀胱感染期间形成IBCs的能力，尽管WT水平在体内黏附和随后侵袭浅表膀胱上皮细胞。我还发现，缺乏CVPA的突变体更容易被中性粒细胞杀死。CVPA唯一被描述的功能是，当它缺乏Colicin V时，细胞外环境中不会检测到一种小孔形成毒素。虽然并不是所有的UPEC菌株(包括研究中使用的菌株UTI89)都含有编码Colicin V结构和运输基因的质粒，但CVPA在大肠杆菌中是保守的，这表明CVPA可能具有额外的功能，例如产生和/或分泌其他形成孔的毒素。我推测，在感染过程中(1)CVPA调节UPEC-宿主细胞的相互作用，可能参与a)抑制宿主免疫反应和/或b)进入宿主细胞时从空泡中排出；以及(2)CVPA功能丧失损害UPEC对抗细菌攻击的防御。在这项提案的目标1中，我将确定CVPA的缺失如何减弱UPEC的毒力。这些研究将使用急性尿路感染的小鼠模型、免疫学分析和先进的整体器官成像方法。在目标2中，我将使用经典的遗传、生化和质谱学方法以及敏感性分析来询问CVPA缺失对UPEC毒素相关因子和膜完整性的影响。总之，这些研究将描绘CVPA如何促进尿路定植，将确定CVPA调节发病机制，并将评估CVPA作为药物开发的细菌靶点。