Sugar Regulation of EHEC virulence

描述（由申请人提供）：胃肠道（GI）细菌感知不同的环境信号，包括宿主激素和营养素，作为差异基因调控和生态位适应的线索。虽然碳营养对肠道微生物定植的影响已被广泛研究，但碳源对入侵病原体调节毒力因子的影响程度尚未完全确定。PI已经表明肠道病原体肠出血性大肠杆菌（EHEC）将糖源作为调节其毒力基因表达的重要线索。具体而言，这种糖依赖性调节微调肠上皮细胞上形成附着和消退（AE）损伤所必需的肠上皮细胞消退（LEE）致病岛位点的表达。糖酵解环境抑制LEE基因的表达。相反地，在一个致突变环境中的生长激活了这些基因的表达。这种糖依赖性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。这种糖依赖性调节在哺乳动物宿主感染期间是关键的，因为kdpE突变体在体内是减毒的。此外，一种新的双组分系统，名为FusKR（其中FusK是膜结合组氨酸传感器激酶（HK），FusR是反应调节剂（RR）），传感岩藻糖，控制LEE基因的表达。这种岩藻糖传感系统是肠出血性大肠杆菌肠道定植所必需的。在粘液中生长期间，GI微生物群的主要嗜糖成员多形拟杆菌向EHEC提供岩藻糖，调节其毒力基因表达。然而，有几个问题仍然没有答案，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？也知道FusK传感岩藻糖，但缺乏对这种传感器是否对其他糖源有反应的广泛研究。此外，尚不清楚FusK是否仅磷酸化其同源RR FusR，或是否也可以磷酸化其他非同源RR。最后，哺乳动物感染过程中这些复杂的糖传感相互作用的影响，虽然显然是重要的，由于衰减的kdpE和fusR突变体在体内，仍然有待解决。因此，本基金的具体目标是：目标1：揭示KdpE，Cra和FusR对LEE基因表达的相互作用机制。目的2：研究FusK磷酸化介导的信号级联反应。目的3：哺乳动物感染过程中糖对毒力的调节。

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.