RCV02 (cadA deficient) v wild-type enterohemorrhagic E. coli (EHEC). Escherichia coli

Adherence of pathogenic Escherichia coli strains to intestinal epithelia is essential for infection. For enterohemorrhagic E. coli (EHEC) serotype O157:H7, we have previously demonstrated that multiple factors govern this pathogen’s adherence to HeLa cells (39). One of these factors is CadA, a lysine decarboxylase, and this protein has been proposed to negatively regulate virulence in several enteric pathogens. In the case of EHEC strains, CadA modulates expression of the intimin, an outer membrane adhesin involved in pathogenesis. Here, we experimentally inactivated cadA in O157:H7 strain 86-24 to investigate the role of this gene in EHEC adhesion to tissue culture monolayers, global gene expression patterns, and colonization of the infant rabbit intestine. As expected, the cadA mutant did not possess lysine decarboxylation activity and was hyper-adherent to tissue-culture cells. Adherence of the cadA mutant was nearly 2-fold greater than that of the wt and complementation of the cadA defect reduced adherence back to wt levels. Furthermore, the cadA mutant affected the expression of intimin protein. Disruption of the eae gene (encoding the intimin protein) in the cadA mutant significantly reduced its adherence to tissue-culture cells. However, adherence of the cadA eae double mutant was greater than that of an 86-24 eae mutant, suggesting that the enhanced adherence of the cadA mutant is not entirely attributable to enhanced expression of intimin in this background. Gene array analysis revealed that the cadA mutation significantly altered EHEC gene expression patterns; expression of 1332 genes was down-regulated and 132 genes up-regulated in the mutant compared to the wild type strain. Interestingly, the gene expression variation shows an EHEC-biased gene alteration including intergenic regions. Two putative adhesins: flagella and F9 fimbriae were up-regulated in the cadA mutant, suggestive of their association with adherence in absence of the Cad regulatory mechanism. Remarkably, in the infant rabbit model, the cadA mutant out-competed the wild type strain in the ileum but not in the cecum or mid-colon, raising the possibility that CadA negatively regulates EHEC pathogenicity in a tissue-specific fashion. Overall design: Arrays used as a starting point for further examination of the effects of the cadA deficiency. A limited number of biologically significant phenotypes and gene expression profiles were examined using qRT-PCR

致病性大肠杆菌（pathogenic Escherichia coli）菌株黏附肠上皮细胞，是其感染宿主的必要前提。针对肠出血性大肠杆菌（enterohemorrhagic Escherichia coli, EHEC）血清型O157:H7，本课题组既往已证实多种因子可调控该病原菌对海拉细胞（HeLa cells）的黏附能力[39]。其中一种调控因子为赖氨酸脱羧酶（lysine decarboxylase）CadA，已有研究表明该蛋白可对多种肠道病原菌的毒力产生负调控作用。就EHEC菌株而言，CadA可调控紧密素（intimin）的表达——该外膜黏附素（outer membrane adhesin）参与病原菌致病过程。
本研究通过实验手段敲除O157:H7菌株86-24中的cadA基因，旨在探究该基因在EHEC黏附组织培养单层细胞（tissue culture monolayers）、调控全局基因表达谱（global gene expression patterns）以及介导幼兔肠道（infant rabbit intestine）定植中的功能。实验结果与预期一致：cadA突变体（cadA mutant）丧失赖氨酸脱羧活性，且对组织培养细胞的黏附能力显著增强。该突变体的黏附水平较野生型（wild type, wt）菌株提升近2倍；通过互补实验（complementation）恢复cadA功能后，其黏附水平可回落至野生型标准。此外，cadA突变体可影响紧密素蛋白的表达。在cadA突变体中敲除编码紧密素的eae基因（eae gene）后，其对组织培养细胞的黏附能力显著降低。但cadA eae双突变体（double mutant）的黏附能力仍强于86-24菌株的eae单突变体，提示cadA突变体黏附能力增强并非完全由紧密素表达上调所介导。
基因芯片（gene array）分析结果显示，cadA突变显著改变了EHEC的基因表达模式：相较于野生型菌株，突变体中共有1332个基因下调（down-regulated）表达，132个基因上调（up-regulated）表达。值得注意的是，该基因表达变化呈现EHEC特异性的调控特征，且覆盖基因间区（intergenic regions）。两个潜在黏附因子——鞭毛（flagella）与F9菌毛（F9 fimbriae）在cadA突变体中表达上调，提示在CadA调控机制缺失的情况下，这两个因子可能参与病原菌的黏附过程。
尤为关键的是，在幼兔感染模型中，cadA突变体在回肠（ileum）中的定植能力优于野生型菌株，但在盲肠（cecum）或结肠中段（mid-colon）中未观察到该优势，这提示CadA可通过组织特异性方式负调控EHEC的致病能力。
实验整体设计：本研究以基因芯片分析为基础，进一步探究cadA缺失的生物学效应。通过定量实时聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）对有限数量的具有生物学意义的表型及基因表达谱进行验证。