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Identification of Host Factors Required for the Cytolytic Activity of Hemolysin from Uropathogenic Escherichia coli

尿路病原性大肠杆菌溶血素溶细胞活性所需宿主因子的鉴定

基本信息

批准号：
9322674
负责人：
Laura C Ristow
金额：
$ 5.92万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-01 至 2019-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9322674
关键词：
Accounting Adhesions Amino Acid Sequence Bacterial Infections Binding Biological Models Bladder Bordetella pertussis CRISPR/Cas technology Cattle Cell Death Cell Line Cells Cessation of life Complement Complex Country Data Diagnosis Direct Costs Dose Escherichia coli Eukaryotic Cell Exotoxins Family Family member Generations Genes Genetic Genome Glycoside Hydrolases Golgi Apparatus Gram-Negative Bacteria Hemolysin Hemorrhage Hospitals Human Human Cell Line ITGB2 gene In Vitro Individual Infection Integration Host Factors Integrin Binding Integrin beta Chains Integrins Intestines Kidney Knock-out Leukocytes Libraries Low Birth Weight Infant Lung diseases Lymphocyte Lymphocyte Function-Associated Antigen-1 Macrophage-1 Antigen Mannheimia haemolytica Mediating Meninges Mouse Cell Line Mus Mutate Mutation Peptide Hydrolases Pertussis Premature Birth Prevention Prevention Protocols Proteins Pyelonephritis Recurrence Reporting Research Resistance Risk Role Sepsis Signal Pathway Signal Transduction Site Site-Directed Mutagenesis Species Specificity Symbiosis Testing Therapeutic Therapeutic Intervention Time Toxin Treatment outcome U937 Cells Ureter Urinary tract Urinary tract infection Uropathogenic E. coli Virulence Factors Visit Work alpha Toxin base catheter associated UTI cell growth regulation cell type community-acquired UTI cytotoxic cytotoxicity design genome-wide glycosylation glycosyltransferase humanized mouse improved in vivo insight interest member microbiota monocyte mouse model mutant overexpression pathogen pathogenic Escherichia coli prevent prototype receptor signal peptide peptidase therapeutic target urinary

项目摘要

Project Summary Uropathogenic Escherichia coli (UPEC) are the causative agent in over 80% of urinary tract infections (UTIs). On average, 8-9 million people are treated for uncomplicated UTIs and over 1 million people are diagnosed with hospital-acquired UTIs annually in the US. Complications of UPEC infections include recurrent UTIs, ascending infection into the ureters and kidneys resulting in pyelonephritis, and in severe cases, urosepsis and death. Identifying and characterizing the mechanistic functions of UPEC virulence factors will lead to improved prevention protocols and treatment for complicated UTIs. The hemolysin exotoxin (HlyA) is an important UPEC virulence factor. HlyA is expressed in 31-48% of E. coli recovered from uncomplicated UTIs, while in strains isolated from cases of pyelonephritis or urosepsis, 50-78% of strains express HlyA. At high doses, HlyA is cytotoxic to a wide range of cell types and species, although the mechanism for initiating cell death remains unclear. The proposed research will test the hypothesis that a host receptor or signaling pathway is subverted by HlyA to cause cell death. A genome-wide selection of a CRISPR/Cas9 mutated library of eukaryotic cells was performed to identify host factors that are required for HlyA-mediated cell death. Two hits were identified, the integrin b2 subunit and signal peptide peptidase-like protein 3 (SPPL3), a protease involved in regulation of cellular glycosylation. This research plan is designed to validate the top candidates by discrete CRISPR/Cas9 mediated genome targeting to disrupt genes of interest. Preliminary data in cell lines with the subunits of b2 family integrins targeted for disruption individually reveals that none of the individual alpha subunits of b2 integrins are necessary for HlyA cytotoxicity. The sufficiency of the four possible b2 family members for HlyA cytotoxicity will be assess in this proposal. Based on the receptor range for HlyA, the interaction of HlyA with b2 family integrins will be characterized using site-directed mutagenesis targeting regions of interest. The role of SPPL3 in HlyA mediated cytotoxicity will be validated by genetic disruption using CRISPR/Cas9. A protease- deficient form of SPPL3, which cannot regulate glycosylation, will be used to complement SPPL3-deficient cells to investigate additional roles for SPPL3 in HlyA cytotoxicity. The glycosylation state of b2 family integrins will be examined in SPPL3 deficient and overexpressing cell lines. Finally, the receptor-mediated activity of HlyA will be examined in a species specific manner. Human cells expressing b2 receptors are 100-fold more sensitive to HlyA cytotoxic activity than mouse cells expressing b2 receptors. If the interaction is species specific, in vivo work in the mouse model cannot accurately assess the role of HlyA in human UPEC infections and the generation of a humanized mouse model will be warranted. This work will provide important insight into the mechanistic function of HlyA-mediated cytotoxicity and provide the groundwork for therapeutic intervention in complicated UTIs.

项目总结