Sugar Regulation of EHEC virulence

描述（申请人提供）：胃肠道（GI）细菌感知多种环境信号，包括宿主激素和营养物质，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群定植的影响已被广泛研究，但碳源通过入侵病原体影响毒力因子调节的程度尚未完全确定。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。具体来说，这种糖依赖性调节精细调节肠细胞粘附和清除（AE）病变形成所需的肠细胞吞噬（LEE）致病性岛位点的表达。糖酵解环境抑制LEE基因的表达。相反，在糖异生环境中生长会激活这些基因的表达。这种糖依赖性调节部分是通过两个转录因子：KdpE和Cra来实现的。Cra和KdpE相互作用，以代谢产物依赖的方式直接激活LEE基因的表达。考虑到kdpE突变体在体内被减弱，这种糖依赖性调节在哺乳动物宿主感染期间是关键。此外，一种名为FusKR的新型双组分系统（FusK是一种膜结合组氨酸传感器激酶（HK）， FusR是一种反应调节因子（RR））可以感知病灶，控制LEE基因的表达。这种聚焦感应系统是肠出血性大肠杆菌肠道定植所必需的。在黏液生长过程中，胃肠道菌群中主要的糖噬菌——拟杆菌（Bacteroides thetaiotaomicron）为肠出血性大肠杆菌提供聚焦，调节其毒力基因的表达。然而，仍有几个问题有待解决，例如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？我们也知道FusK能感知焦点，但缺乏对该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是否只磷酸化其同源RR FusR，或者是否也能磷酸化其他非同源RR。最后，在哺乳动物感染过程中，这些复杂的糖传感相互作用的含义，尽管由于体内kdpE和fusR突变体的衰减而明显重要，但仍有待解决。因此，本基金的具体目标是：目标1：揭示KdpE、Cra和FusR对LEE基因表达的机制相互作用。目的2：研究FusK磷接力信号级联。目的3：糖在哺乳动物感染期间的毒力调节。

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.