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（由申请人提供）：尿路感染（UTI）的主要原因是尿路致病性大肠杆菌（UPEC），其进化出了破坏宿主防御的精巧机制，并在尿路内和细胞外小生境中持续存在。在细胞内位点内，UPEC经历与细胞外生物膜形成平行的发育过程，并在细胞内细菌群落（IBC）的生物发生中达到高潮，所述细胞内细菌群落（IBC）含有包裹在细胞外基质内的紧密包装的细菌。因此，确定控制细菌细胞内复制的决定因素将促进我们对UTI的理解 致病机制，将描绘病原体在感染期间占据细胞内位点的策略，并将阐明抗毒力治疗的新靶点。删除UPEC因子，大肠杆菌素V生产辅助蛋白（CvpA），消除细胞外生物膜的形成，而不影响1型皮利的生产。为了了解CvpA对UPEC发病机制的贡献，我在急性UTI的小鼠模型中测试了UPEC cvpA缺失突变体，并确定CvpA的缺失废除了UPEC在急性膀胱感染期间形成IBC的能力，尽管WT水平粘附于体内浅表膀胱上皮细胞并随后侵入。我还发现CvpA缺陷突变体更容易被中性粒细胞杀死。CvpA的唯一描述的功能是，在其不存在的情况下，大肠杆菌素V，一种小的成孔毒素，在细胞外环境中检测不到。虽然编码大肠杆菌素V结构和转运基因的质粒并不存在于所有的UPEC菌株（包括这些研究中使用的UTI 89菌株）中，但CvpA在E.这表明CvpA可能具有额外的功能，例如产生和/或分泌其他成孔毒素。我假设在感染期间（1）CvpA调节UPEC-宿主细胞相互作用，并且可能参与a）抑制宿主免疫应答和/或B）在进入宿主细胞后从空泡流出;和（2）CvpA功能的丧失损害UPEC对抗细菌攻击的防御。在本提案的目标1中，我将确定CvpA的缺失如何减弱UPEC毒力。这些研究将采用急性尿路感染的小鼠模型、免疫学测定和先进的全器官成像方法。在目标2中，我将使用经典的遗传学、生物化学和质谱方法以及敏感性测定来询问cvpA缺失对UPEC毒素相关因子和膜完整性的影响。总之，这些研究将描述CvpA如何促进尿路定植，将确定CvpA调节发病机制的机制，并将评估CvpA作为药物开发的细菌靶标。