Shin S, Castanie-Cornet M P, Foster J W, Crawford J A, Brinkley C, Kaper J B
Center for Vaccine Development and Department of Microbiology and Immunology, University of Maryland School of Medicine, 685 W. Baltimore St., Baltimore, MD 21201, USA.
Mol Microbiol. 2001 Sep;41(5):1133-50. doi: 10.1046/j.1365-2958.2001.02570.x.
Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhoea in a number of developing countries and is the prototype of pathogenic bacteria that cause attaching and effacing (A/E) intestinal lesions. A chromosomal pathogenicity island, termed the locus of enterocyte effacement (LEE), contains all the genes necessary for the A/E phenotype as well as genes for a type III secretion system and intimate adhesion. Genes in the LEE and genes involved in the synthesis of bundle-forming pili (BFP) are positively regulated by the plasmid-encoded regulator (Per) and comprise the per regulon. In order to identify factors that control the per regulon, we screened an EPEC genomic library for clones that modulate the expression of per. A plasmid clone that decreased the expression of per was isolated using a lacZ reporter gene fused to the per promoter. Subcloning revealed that YhiX, a putative AraC/XylR family transcriptional regulator, was the effector of per repression. Through downregulation of per, a plasmid overproducing YhiX reduced the synthesis of intimin, BfpA, Tir, and CesT, factors important for EPEC virulence. yhiX is located downstream of gadA, which encodes glutamate decarboxylase, an enzyme involved in acid resistance of E. coli. YhiX was found to be an activator of gadA, and the cloned yhiX gene increased production of glutamate decarboxylases (GAD) and activated the transcription of the gadA and gadB promoters. Therefore, yhiX was renamed gadX. Analysis of a gadX mutant grown in the different culture media with acidic and alkaline pH showed that regulation of perA, gadA and gadB by GadX was altered by the external pH and the culture media condition. Under conditions in which EPEC infects cultured epithelial cells, GadX negatively regulated perA expression, and the derepression in the gadX mutant increased translocation of Tir into epithelial cells relative to wild-type EPEC. DNA mobility shift experiments showed that purified GadX protein bound to the perA, gadA and gadB promoter regions in vitro, indicating that GadX is a transcriptional regulator of these genes. On the basis of these results, we propose that GadX may be involved in the appropriate expression of genes required for acid resistance and virulence of EPEC. Our data are consistent with a model in which environmental changes resulting from passage from the stomach to the proximal small intestine induce the functional effect of GadX on per and GAD expression in order to prevent inappropriate expression of the products of these two systems.
肠致病性大肠杆菌（EPEC）是许多发展中国家婴幼儿腹泻的主要病因，是导致肠细胞黏附与脱落（A/E）性肠道病变的病原菌原型。一个名为肠细胞脱落位点（LEE）的染色体致病岛包含了A/E表型所需的所有基因以及一个III型分泌系统和紧密黏附相关的基因。LEE中的基因以及参与束状菌毛（BFP）合成的基因受质粒编码的调节因子（Per）正调控，构成了per调节子。为了鉴定控制per调节子的因子，我们在EPEC基因组文库中筛选能够调节per表达的克隆。使用与per启动子融合的lacZ报告基因分离出一个降低per表达的质粒克隆。亚克隆显示YhiX，一个假定的AraC/XylR家族转录调节因子，是per抑制的效应物。通过下调per，过量表达YhiX的质粒降低了intimin、BfpA、Tir和CesT的合成，这些都是对EPEC毒力很重要的因子。yhiX位于gadA下游，gadA编码谷氨酸脱羧酶，一种参与大肠杆菌耐酸性的酶。发现YhiX是gadA的激活剂，克隆的yhiX基因增加了谷氨酸脱羧酶（GAD）的产生并激活了gadA和gadB启动子的转录。因此，yhiX被重新命名为gadX。对在不同pH值的酸性和碱性培养基中生长的gadX突变体的分析表明，GadX对perA、gadA和gadB的调节受到外部pH值和培养基条件的影响。在EPEC感染培养的上皮细胞的条件下，GadX负调控perA表达，相对于野生型EPEC，gadX突变体中的去抑制增加了Tir向上皮细胞的转运。DNA迁移率变动实验表明，纯化的GadX蛋白在体外与perA、gadA和gadB启动子区域结合,表明GadX是这些基因的转录调节因子。基于这些结果，我们提出GadX可能参与EPEC耐酸性和毒力所需基因的适当表达。我们的数据与一个模型一致，即在从胃进入近端小肠过程中环境变化诱导GadX对per和GAD表达的功能效应，以防止这两个系统产物的不适当表达。
