Tryptophan derivatives in EHEC pathogenesis

描述(由申请人提供)：人类胃肠道(GI)中居住着数以万亿计的共生细菌，它们在人类生理中扮演着至关重要的角色。微生物与宿主之间的化学交流是它们联系的基础，引起全世界血性腹泻爆发的胃肠道病原体肠出血性大肠杆菌(EHEC)利用胃肠道微生物区系和宿主之间的细胞对细胞信号作为测量和识别宿主环境的一种手段。这种信号是基于结肠中的色氨酸衍生物的信号分子，如宿主神经递质5-羟色胺和产生吲哚的微生物群。5-羟色胺是一种神经递质，主要在胃肠道合成，5-羟色胺被释放到固有层以激活蠕动、分泌和血管扩张。然而，最近有报道称，5-羟色胺也分泌到管腔内，但它在这个腔内的功能尚不清楚。许多功能性胃肠道疾病与5-羟色胺信号的改变有关，但这种影响 5-羟色胺信号在细菌介导的胃肠道疾病中的作用尚不清楚。虽然许多细菌和病毒已被证明影响5-羟色胺信号转导，但尚未有研究调查5-羟色胺在细菌发病机制中的作用。细菌产生的色氨酸衍生物吲哚已知可以影响肠道屏障功能，也被证明可以调节EHEC的毒力基因表达。我们鉴定了细菌膜结合的组氨酸感受器激酶(HK)CpxA是5-羟色胺和吲哚的感受器。CpxA是在自动磷酸化后，磷酸转移到反应调节因子(RR)CpxR的HK，这是一种转录因子。CpxA也使这个RR去磷酸化，CpxR的磷酸化状态定义了它与DNA靶标上的哪些位点结合。通过CpxA，5-羟色胺和吲哚均可降低EHEC中肠上皮细胞消失点(Lee)的表达，从而编码EHEC所需的三型分泌系统(T3SS)、效应物和粘附素，从而在EHEC上形成病变，导致肠道疾病。5-羟色胺和吲哚都能抑制CpxA的自磷酸化，从而减少其靶标的表达。综上所述，这些结果表明CpxA是一种重要的小分子受体，对细胞间信号转导和抑制胃肠道病原体的毒力至关重要。因此，这一应用的具体目的是：1)色氨酸衍生物对毒力基因调控的体外机制研究。2)微生物区系、病原菌和寄主细胞界面的5-羟色胺和吲哚信号转导。3)5-羟色胺对小鼠胃肠道细菌毒力的调节。

DESCRIPTION (provided by applicant): The human gastrointestinal (GI) tract is inhabited by trillions of commensal bacteria that play crucial roles in human physiology. Chemical communication between microbes and their hosts underlies the basis of their associations The GI pathogen enterohemorrhagic E. coli (EHEC), responsible for outbreaks of bloody diarrhea worldwide, exploits cell-to-cell signaling between the gastrointestinal microbial flora and the hos as a means to gage and recognize the host environment. This signaling is predicated upon signaling molecules that are tryptophan derivatives in the colon such as the host neurotransmitter serotonin and the microbiota produced indole. Serotonin is a neurotransmitter that is primarily synthesized in the GI tract Serotonin is released into the lamina propria to activate peristalsis, secretion, and vasodilation. However, it has been recently reported that serotonin is also secreted into the lumen, but its function in this compartment remains unclear. Many functional GI disorders are associated with alterations in serotonin signaling, but the effect of serotonin signaling on bacterial-mediated GI disorders remains unknown. Although a number of bacteria and viruses have been show to influence serotonin signaling, no study has investigated the effect that serotonin has on bacterial pathogenesis. The bacterially produced tryptophan derivative indole is known to influence intestinal barrier function, and has also been shown to regulate virulence gene expression in EHEC. We identified the bacterial membrane bound histidine sensor kinase (HK) CpxA as a sensor of serotonin and indole. CpxA is the HK that upon autophosphorylation, phosphotranfers to the response regulator (RR) CpxR, which is a transcriptional factor. CpxA also dephosphorylates this RR, and the phosphorylation state of CpxR defines which sites it binds to on its DNA targets. Through CpxA, both serotonin and indole decrease expression of the locus of enterocyte effacement (LEE) in EHEC, which encodes a type three secretion system (T3SS), effectors and an adhesin necessary for EHEC to form lesions on enterocytes leading to intestinal disease. Both serotonin and indole inhibit CpxA's autophosphorylation, consequently decreasing the expression of its targets. Altogether, these results suggest that CpxA is an important small molecule receptor crucial for cell-to-cell signaling and inhibition of virulence of GI pathogens. Accordingly, the specific aims of this application are: 1) In vitro mechanistic studies on virulence gene regulation by tryptophan derivatives. 2) Serotonin and indole signaling at the microbiota, pathogen and host cell interface. 3) Serotonin regulation of bacterial virulence in the murine GI tract.