Mechanisms of Inhibition of Intestinal SERT by EPEC Infection.

描述（由申请人提供）：由传染性病原体引起的腹泻疾病继续造成严重的健康问题，特别是在婴儿中。因此，有必要开发新的有效的药物来治疗腹泻。肠5-羟色胺转运蛋白（SERT）参与调节管腔5-HT的可用性，是一个重要的候选目标，因为 它在几种神经系统疾病中的意义。本研究主要探讨血清素转运蛋白（SERT）在重要食源性致病菌肠致病性大肠杆菌感染相关腹泻的病理生理学中的作用。coli（EPEC）。由于缺乏已知的毒素，EPEC诱导的早期腹泻的病理生理学多年来仍然难以捉摸。新兴的研究表明，EPEC相关腹泻的潜在机制是多因素的。我们最近的研究表明，EPEC通过激活蛋白酪氨酸磷酸酶抑制肠上皮细胞的SERT。然而，EPEC诱导的SERT抑制的详细机制尚不清楚。我们假设EPEC诱导的信号传导事件改变SERT磷酸化水平，导致其抑制，这可能有助于相关的腹泻。本研究将系统地研究EPEC感染对SERT的影响，利用体外和体内模型。具体目标1中提出的研究将确定感染后SERT调节中涉及的特定丝氨酸/苏氨酸/酪氨酸磷酸酶，并将确定含有SERT磷酸化位点的结构域。具体目标2将通过评估天然小鼠回肠中细胞磷酸酶的作用，严格阐明SERT对EPEC感染的急性抑制机制。此外，将检查磷酸酶对EPEC能机制（5-HT合成和释放）的影响，以模拟EPEC感染在复杂的天然肠道中的净影响。总体而言，拟议的研究将有助于提供有价值的见解宿主-微生物上皮相互作用的基础EPEC相关腹泻的病理生理学，以及将增加我们的理解磷酸化/去磷酸化机制，在生理和病理生理条件下的SERT活性。 公共卫生相关性：腹泻病是五岁以下儿童死亡的第二大原因，每年造成150万儿童死亡。因此，重要的是要发现新的药物，可以有效地治疗腹泻。我们的研究旨在探讨食源性细菌EPEC如何调节肠道5-羟色胺转运蛋白（SERT）引起腹泻。由于SERT在肠道疾病中的意义，这些研究的结果可能为肠道感染或肠道疾病的治疗方式提供新的见解。

DESCRIPTION (provided by applicant): Diarrheal diseases caused by infectious agents continue to pose a serious health problem, especially in infants. It is, therefore, necessary to develop novel efficacious drugs to treat diarrhea. Intestinal serotonin transporter (SERT), involved in regulation of luminal 5-HT availability, is an important candidate to target because of its implications in several diarrheal disorders. The proposed studies are focused at examining the role of serotonin transporter (SERT) in the pathophysiology of diarrhea associated with infection by an important food-borne pathogen, Enteropathogenic E. coli (EPEC). Because of the lack of a known toxin, pathophysiology of EPEC-induced early diarrhea remained elusive for many years. Emerging studies have revealed that mechanism(s) underlying EPEC associated diarrhea are multi-factorial. Our recent studies showed that EPEC inhibits SERT in the intestinal epithelial cells via activation of protein tyrosine phosphatases. However, the detailed mechanisms underlying EPEC induced inhibition of SERT are not known. We hypothesize that EPEC induced signaling events alter SERT phosphorylation levels to cause its inhibition that may contribute to associated diarrhea. The present studies will systematically examine the effects of EPEC infection on SERT utilizing both in vitro and in vivo models. Studies proposed in Specific Aim 1 will identify the specific serine/threonine/tyrosine phosphatases involved in SERT modulation post-infection and will identify the structural domains that harbor SERT phosphorylation site(s). Specific Aim 2 will critically elucidate the mechanisms of acute inhibitio of SERT in response to EPEC infection by assessing the role of cellular phosphatases in the native mouse ileum. In addition, the effect of phosphatases on serotonergic machinery (5-HT synthesis and release) will be examined to model the net impact of EPEC infection in complex native intestine. Overall, the proposed studies will help provide valuable insights into the host-microbial epithelial interaction underlying pathophysiology of EPEC- associated diarrhea as well as will increase our understanding of phosphorylation/dephosphorylation mechanisms governing SERT activity under physiological and pathophysiological conditions. PUBLIC HEALTH RELEVANCE: Diarrheal disease is the second leading cause of death in children under five years old, and is responsible for killing 1.5 million children every year. Therefore, it is important to discover new drugs that can effectively treat diarrhea. Our studies are aiming to investigate how a food-borne bacterium, EPEC modulates intestinal serotonin transporter (SERT) to cause diarrhea. Because of implication of SERT in diarrheal disorders, the outcome of these studies may provide new insights into therapeutic modalities for intestinal infectious or diarrheal disorders.