Expression control mechanisms of LEE pathogenic factors by AAA proteases in Enterohaemorrhagic and Enteropathogenic Eschericha coli

1. 肠出血性大肠杆菌（EHEC）和肠致病性大肠杆菌（EPEC）引起肠上皮细胞的组织病理学病变，称为附着和消退（A/E）病变。这两种菌株都携带一个独特的染色体DNA区域，称为肠细胞消失位点（LEE），该区域编码III型分泌系统（TTSS）和毒力蛋白。我们最近报道了AAA蛋白酶（CIpXP, Lon）和分子伴侣控制鼠伤寒沙门菌的2个ttss:(i)鞭毛生物发生和（ii）致病性岛1的表达。我们发现EHEC和EPEC clpPX突变体强烈地破坏了TTSS分泌的毒力蛋白，并抑制了所有LEE启动子的转录。EHEC中rpoS突变增强了所有LEE启动子的转录和毒力蛋白的分泌，并能部分抑制clpPX突变的缺陷。这些数据表明，EHEC ClpXP蛋白酶是LEE表达的正调控因子，并且这种调控通过2种途径发生：o^<S->依赖性途径和os非依赖性途径。最近发现了2个LEE编码的调节因子grla （LEE激活因子的全局调节因子）和GrlR （Grl抑制因子）。Iyoda等人报道，ClpXP蛋白酶通过调节肠出血性大肠杆菌中GrlR的水平来控制leepromoter的表达(J.Bacteriol。2005. 187: 4086 - 94)。然而，Gr1A、GrlR和AAA蛋白酶表达leepromoter的控制机制尚不清楚。因此，我们利用非致病性大肠杆菌建立了一个检测系统，通过测量LEE2、LEE3和lee5启动子的活性来分析这些蛋白之间的相互作用。我们的数据显示，Gr1A激活le （lee编码调节因子）表达需要参与到ler停止密码子下游131个碱基对的结构域中。目前，我们正在详细分析Gr1R、Gr1A和AAA蛋白酶对LEE表达的调控机制。少

1. Enterohemorrhagic Escherichia coli (EHEC) and Enteropathogenic E. coli (EPEC) cause histopathological lesions of the intestinal epithelial cells that are known as attaching and effacing (A/E) lesions. Both strains carry a unique region of chromosomal DNA known as the locus for enterocyte effacement (LEE) that encodes a type III secretion system (TTSS) and virulence proteins. We have recently reported that AAA proteases (CIpXP, Lon) and molecular chaperones control 2 TTSSs in Salmonella Typhimurium: (i) flagellum biogenesis and (ii) the expression of the Pathogenicity Island 1. We demonstrated that the EHEC and EPEC clpPX mutants strongly impaired the secretion of virulence proteins by TTSS and repressed transcription from all the LEE promoters. The rpoS mutation in EHEC enhanced transcription from all the LEE promoters and the secretion of virulence proteins, and it could partially suppress the defects of the clpPX mutation. These data indicate that the EHEC ClpXP protease is a posi … More tive regulator for LEE expression, and this regulation occurs via 2 pathways: the o^<S->dependent and os-independent pathways.2. Recently, 2 LEE-encoded regulators-GrlA (global regulator of LEE activator) and GrlR (Grl repressor)-were identified. Iyoda et al. reported that ClpXP protease controls the expression of LEEpromoters via the regulation of GrlR levels in EHEC (J.Bacteriol. 2005. 187: 4086-94). However, the control mechanisms underlying the expression of LEEpromoters by Gr1A, GrlR, and AAA proteases remain unclear. Therefore, we established an assay system using nonpathogenic E. coli that enables analysis of the interaction among these proteins by measuring the activities of the LEE2, LEE3, and LEE5promoters. Our data revealed that the activation of ler (LEE-encoded regulator) expression by Gr1A entailed the participation of the domain that exists for a length of 131 base pairs downstream from the ler stop codon. Presently, we are analyzing the LEE expression control mechanisms exerted by Gr1R, Gr1A, and AAA proteases in detail. Less