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: 9150294
• 负责人: Carrie Shaffer
• 金额: $ 3.87万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 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 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9150294
• 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; Multi-Drug Resistance; 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; alpha Toxin; 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）：尿路致病性大肠杆菌（UPEC）是尿路感染（UTI）的主要原因，它已经进化出精致的机制来破坏宿主防御并在尿路的细胞内和细胞外生态位中持续存在。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，并最终导致细胞内细菌群落 (IBC) 的生物发生，其中包含紧密包裹在细胞外基质内的细菌。因此，控制细菌细胞内复制的决定因素的鉴定将增进我们对尿路感染的理解 发病机制，将描述病原体在感染过程中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（大肠菌素 V 产生辅助蛋白 (CvpA)）可消除细胞外生物膜形成，而不影响 1 型菌毛的产生。为了了解 CvpA 对 UPEC 发病机制的贡献，我在急性 UTI 小鼠模型中测试了 UPEC cvpA 缺失突变体，并确定 CvpA 的缺失会消除 UPEC 在急性膀胱感染期间形成 IBC 的能力，尽管体内浅表膀胱上皮细胞的粘附和随后侵袭的 WT 水平。我还发现 CvpA 缺陷突变体更容易被中性粒细胞杀死。唯一描述的 CvpA 功能是，在没有它的情况下，在细胞外环境中检测不到大肠杆菌素 V（一种小型成孔毒素）。尽管并非所有 UPEC 菌株（包括这些研究中使用的菌株 UTI89）都含有编码大肠菌素 V 结构和转运基因的质粒，但 CvpA 在大肠杆菌中是保守的，这表明 CvpA 可能具有其他功能，例如产生和/或分泌其他成孔毒素。我假设在感染过程中 (1) CvpA 调节 UPEC-宿主细胞相互作用，并可能参与 a) 抑制宿主免疫反应和/或 b) 进入宿主细胞后从液泡中逸出； (2) CvpA 功能的丧失会损害 UPEC 对抗菌攻击的防御能力。在本提案的目标 1 中，我将确定 CvpA 的丢失如何减弱 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、生化和质谱方法以及药敏测定来探讨 cvpA 缺失对 UPEC 毒素相关因子和膜完整性的影响。总的来说，这些研究将描述 CvpA 如何促进泌尿道定植，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。