Sugar Regulation of EHEC virulence

描述(申请人提供)：胃肠道(GI)细菌感知不同的环境信号，包括宿主激素和营养物质，作为差异基因调节和生态位适应的线索。虽然碳营养对肠道微生物区系定植的影响已经得到了广泛的研究，但碳源对入侵病原体对毒力因子调节的影响程度还没有完全确定。等电点试验表明，肠道病原菌肠出血性大肠杆菌(EHEC)通过测量糖源来调节其毒力基因的表达。具体地说，这种糖依赖的调节微调了肠细胞消退(Lee)致病岛的表达，这是形成肠细胞附着和消退(AE)损伤所必需的。糖酵解环境抑制Lee基因的表达。相反，在生糖环境中的生长会激活这些基因的表达。这种糖依赖调节的一部分是通过两种转录因子实现的：KdpE和CRA。CRA和KdpE相互作用，以代谢物依赖的方式最佳地直接激活Lee基因的表达。鉴于kdpE突变体在体内减弱，这种糖依赖的调节在哺乳动物宿主感染期间是关键。此外，一个新的双组分系统，称为FusKR(其中FusK是膜结合的组氨酸传感器激酶(HK)，FusR是反应调节因子(RR))，它可以感知岩藻糖，控制Lee基因的表达。这种岩藻糖感应系统是EHEC肠道定植所必需的。在粘液中生长过程中，胃肠道微生物区系中的重要成员--嗜糖杆菌为EHEC提供岩藻糖，调节其毒力基因的表达。然而，仍有几个问题有待解答，如KdpE、CRA和FusR转录因子之间的相互作用如何控制Lee基因的最佳表达？众所周知，FusK能感应岩藻糖，但这种传感器是否对其他糖源有反应，目前还缺乏广泛的研究。此外，目前尚不清楚FusK是否只对其同源RR FusR进行磷酸化，或者它是否也可以对其他非同源RR进行磷酸化。最后，这些复杂的糖感相互作用在哺乳动物感染过程中的意义，尽管由于kdpE和fusR突变体在体内的衰减而明显重要，但仍有待解决。因此，这项资助的具体目的是：目标1：揭示KdpE、CRA和FusR之间对Lee基因表达的机制相互作用。目的2：研究FusK磷酸信号转导通路。目的3：糖对哺乳动物感染过程中毒力的调节。

DESCRIPTION (provided by applicant): Gastrointestinal (GI) bacteria sense diverse environmental signals, including host hormones and nutrients, as cues for differential gene regulation and niche adaptation. Although the impact of carbon nutrition on the colonization of the gut by the microbiota has been extensively studied, the extent to which carbon sources affect the regulation of virulence factors by invading pathogens has not been fully defined. The PI has shown that the enteric pathogen enterohemorrhagic Escherichia coli (EHEC) gages sugar sources as an important cue to regulate expression of its virulence genes. Specifically, this sugar dependent regulation fine tunes the expression of the locus of enterocyte effacement (LEE) pathogenicity island necessary for the formation of attaching and effacing (AE) lesions on enterocytes. Glycolytic environments inhibit the expression of the LEE genes. Conversely, growth within a gluconeogenic environment activates expression of these genes. Part of this sugar-dependent regulation is achieved through two transcription factors: KdpE and Cra. Cra and KdpE interact to optimally directly activate expression of the LEE genes in a metabolite dependent fashion. This sugar dependent regulation is key during infection of the mammalian host, given that a kdpE mutant is attenuated in vivo. Additionally, a novel two component system, named FusKR (where FusK is a membrane bound histidine sensor kinase (HK), and FusR a response regulator (RR)) that senses fucose, controls expression of the LEE genes. This fucose-sensing system is required for robust EHEC intestinal colonization. During growth in mucus, the glycophagic prominent member of the GI microbiota, Bacteroides thetaiotaomicron, supplies fucose to EHEC, modulating its virulence gene expression. However, several questions remain answered, such as how does the interplay among the KdpE, Cra and FusR transcription factors controls optimal expression of the LEE genes? It is also known that FusK senses fucose, but an extensive investigation on whether this sensor is responsive to other sugar sources is lacking. Additionally, it is unknown whether FusK exclusively phosphorylates its cognate RR FusR, or whether it can also phosphorylate other non-cognate RRs. Finally, the implications of these complex sugar sensing interactions during mammalian infection, although clearly important due to the attenuation of kdpE and fusR mutants in vivo, remains to be addressed. Hence, the Specific Aims of this grant are: Aim 1: Unravel the mechanistic interactions among KdpE, Cra and FusR on LEE gene expression. Aim 2: Investigate the FusK phosphorelay signaling cascade. Aim 3: Sugar regulation of virulence during mammalian infection.