DR ADHESINS IN E COLI RENAL VS BLADDER TROPISM

大肠杆菌肾向性与膀胱向性中的 DR 粘附素

• 批准号: 6298555
• 负责人: MARGARET DAS
• 金额: $ 4.02万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6298555
• 关键词: CHO cells; Escherichia coli; Escherichia coli infections; adhesin; bacteria infection mechanism; kidney infection; laboratory mouse; molecular cloning; pilus; protein purification; receptor; site directed mutagenesis; urinary bladder; urinary tract infection

Escherichia coli expressing Dr adhesin cause pyelonephritis and related AFA-III cause cystitis. This study seeks to identify the receptor-binding site of DraE and provide insights into the mechanism of renal vs bladder tropism. Aim 1. To identify and characterize domains of Dr fimbriae that interact with DAF. We propose that N-terminal surface exposed residues of DraE form an assembled epitope that bind DAF. The hydrophilic domain Val30 to Pro42 will be mutated by alanine-scanning mutagenesis and mutants analyzed for binding and invasion of DAF+ CHO cells. Aim 2. To investigate the pathogenic mechanism that determines kidney vs bladder tropism of Dr+ E. coli. DraE and AWE differ in three amino acids (D54N, T90M, I113T). Dr+E. coli infect the kidneys while AFA-III+ E. coli infect the bladder. Amino acids of DraE will be replaced with those of Afa3E and renal tropism analysed using isogenic mutants of E. coli HI 11128 in mice. Aim 3. To study attachment and cell invasion by DraE and Afa3E adhesins. DraE is more invasive than Afa3E. We will study invasion using polystyrene beads coated with purified DraE mutants, immunogold staining and electron microscopy. Resolving the binding domain will enable design of peptide inhibitors against the common receptor preventing bacterial dispersion and cell invasion.

表达DR粘附素的大肠埃希菌可引起肾盂肾炎，相关的AFA-III可引起膀胱炎。本研究试图确定DRE的受体结合部位，并为肾与膀胱的趋向性机制提供见解。目的1.鉴定和鉴定DR菌毛与DAF相互作用的结构域。我们认为DRAE的N端表面暴露残基形成了一个与DAF结合的组装表位。亲水区Val30至Pro42将通过丙氨酸扫描突变，并分析突变体与DAF+CHO细胞的结合和侵袭。目的2.探讨决定DR+大肠埃希菌肾与膀胱嗜性的致病机制。DRE和AWE在三个氨基酸(D54N、T90M、I113T)上存在差异。DR+大肠杆菌感染肾脏，而AFA-III+大肠杆菌感染膀胱。DRE的氨基酸将被Afa3E取代，并在小鼠中使用E.ColiHI 11128的等基因突变体进行肾脏趋向性分析。目的3.研究DRAE和Afa3E黏附素对细胞的黏附和侵袭作用。DRE比Afa3E更具侵袭性。我们将使用包被纯化的DRAE突变体的聚苯乙烯微珠、免疫金染色法和电子显微镜来研究侵袭性。分解结合结构域将使针对共同受体的多肽抑制剂的设计成为可能，防止细菌扩散和细胞入侵。