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Identification and Characterization of a Novel Uropathogenic Escherichia coli-Associated Fimbrial Gene Cluster

机译：新型致病性大肠杆菌相关纤维基因簇的鉴定与表征

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Recently, we identified a fimbrial usher gene in uropathogenic Escherichia coli strain CFT073 that is absent from an E. coli laboratory strain. Analysis of the CFT073 genome indicates that this fimbrial usher gene is part of a novel fimbrial gene cluster, aufABCDEFG. Analysis of a collection of pathogenic and commensal strains of E. coli and related species revealed that the auf gene cluster was significantly associated with uropathogenic E. coli isolates. For in vitro expression analysis of the auf gene cluster, RNA was isolated from CFT073 bacteria grown to the exponential or stationary phase in Luria-Bertani broth and reverse transcriptase PCR (RT-PCR) with oligonucleotide primers specific to the major subunit, aufA, was performed. We found that aufA is expressed in CFT073 only during the exponential growth phase; however, no expression of AufA protein was observed by Western blotting, indicating that under these conditions, the expression of the auf gene cluster is low. To determine if the auf gene cluster is expressed in vivo, RT-PCR was performed on bacteria from urine samples of mice infected with CFT073. Out of three independent experiments, we were able to detect expression of aufA at least once at 4, 24, and 48 h of infection, indicating that the auf gene cluster is expressed in the murine urinary tract. Furthermore, antisera from mice infected with CFT073 reacted with recombinant AufA in an enzyme-linked immunosorbent assay. To identify the structure encoded by the auf gene cluster, a recombinant plasmid containing the auf gene cluster under the T7 promoter was introduced into the E. coli BL-21 (AI) strain. Immunogold labeling using AufA antiserum revealed the presence of amorphous material extending from the surface of BL-21 cells. No hemagglutination or cellular adherence properties were detected in association with expression of AufA. Deletion of the entire auf gene cluster had no effect on the ability of CFT073 to colonize the kidney, bladder, or urine of mice. In addition, no significant histological differences between the parent and aufC mutant strain were observed. Therefore, Auf is a uropathogenic E. coli-associated structure that plays an uncertain role in the pathogenesis of urinary tract infections.
机译：最近，我们在 E中缺失了尿路致病性大肠杆菌菌株CFT073中的纤维诱导基因。大肠杆菌实验室菌株。对CFT073基因组的分析表明，该纤维原位基因是新型纤维基因簇 aufABCDEFG 的一部分。对 E的致病和普通菌株的分析。大肠菌及相关物种表明， auf 基因簇与尿毒症性 E显着相关。大肠杆菌分离株。为了对 auf 基因簇进行体外表达分析，从生长在Luria-Bertani肉汤中的指数期或固定期的CFT073细菌中分离RNA，并使用特异于寡核苷酸的引物进行逆转录PCR（RT-PCR）。主要亚基 aufA 被执行。我们发现 aufA 仅在指数生长阶段才在CFT073中表达。然而，通过蛋白质印迹法未观察到AufA蛋白的表达，表明在这些条件下， auf 基因簇的表达较低。为了确定 auf 基因簇是否在体内表达，对感染了CFT073的小鼠尿液样本中的细菌进行了RT-PCR。在三个独立的实验中，我们能够在感染的第4、24和48小时至少检测一次 aufA 的表达，这表明 auf 基因簇已表达在鼠尿道中。此外，来自CFT073感染小鼠的抗血清在酶联免疫吸附试验中与重组AufA反应。为了鉴定 auf 基因簇编码的结构，将在T7启动子下包含 auf 基因簇的重组质粒引入 E。大肠杆菌BL-21（AI）菌株。使用AufA抗血清的免疫金标记显示存在从BL-21细胞表面延伸的无定形物质。没有检测到与AufA的表达相关的血凝或细胞粘附特性。删除整个 基因簇对CFT073在小鼠的肾脏，膀胱或尿液中定殖的能力没有影响。另外，在亲本和 aufC 突变株之间没有观察到明显的组织学差异。因此，Auf是尿毒症性大肠杆菌。大肠埃希菌相关结构在尿路感染的发病机理中起不确定作用。

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