Function of Longus Type IV pili in enterotoxigenic E coli

描述(申请人提供)：产肠毒素大肠杆菌(ETEC)是发展中国家儿童腹泻的主要原因，也是引起旅行者腹泻的最重要原因。目前还没有针对ETEC的有效疫苗。ETEC的肠道定植是感染过程中至关重要的第一步，它被认为是由物种特异性菌毛介导的。Longus(Long Pilus)是一种新近发现的IV型菌毛(T4P)，也是人类ETEC菌株中最常见的菌毛之一，它与重要的人类细菌病原体表达的众所周知的毒力相关T4P具有结构相似性和蛋白质序列同源性。该项目的长期目标是确定Longus在ETEC在人类肠道定植中的作用，并开发候选疫苗以预防ETEC腹泻。我假设Longus通过与细胞或非细胞表面结合而促进人类肠道的定植，而基于Longus的疫苗能够诱导抗Longus抗体，将阻止ETEC的定植并预防疾病。为了验证这一假设，我提出了以下具体目标：1)确定Longus表达所必需的基因并评估Longus基因的变异；2)评估Longus介导的对人类肠道表面的黏附和定植；以及3)在小鼠模型中测试Longus的免疫原性。我将首先克隆Longus表达所必需的基因，评估世界各地分离株之间的基因变异，并构建Longus表型评估所必需的Longus结构亚单位突变体。野生型和突变型将在细胞对人类肠道细胞的粘附性和生物膜形成试验中进行比较。ETEC的定植将通过使用一种新的人类肠道外植体-严重联合免疫缺陷(SCID)小鼠模型来评估。外植体将被ETEC感染，并通过菌落计数和组织学评估其定植情况。将在表达Longus的减毒沙门氏菌免疫小鼠的血清上测试Longus的免疫原性和与Longus变体的交叉反应。证明Longus介导了与细胞的结合和生物膜的形成，这将大大有助于我们对Longus功能的了解。人外植体/SCID小鼠模型的建立将对从分子水平上理解ETEC的定植具有重要意义。Longus在不同变种之间具有免疫原性以及抗Longus抗体可能阻止定植和预防疾病的信息将有助于构建有效的ETEC疫苗。

DESCRIPTION (provided by applicant): Enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood diarrhea in developing countries and the most important cause of traveler's diarrhea. Effective vaccines for ETEC are not currently available. ETEC intestinal colonization is a crucial first step in the infection process, and it is believed to be mediated by species-specific pili. Longus (long pilus), a recently identified type IV pili (T4P) and one of the most prevalent pili described in human ETEC strains, has structural similarity and protein sequence homology to the well known virulence-associated T4Ps expressed by important human bacterial pathogens. The long-term goals of this project are to identify the role of Longus in ETEC colonization of the human gut and to develop vaccine candidates to prevent ETEC diarrhea. I hypothesize that Longus contributes to colonization of human intestine by binding to cells or non-cell surfaces, and that Longus-based vaccines, capable of inducing anti-Longus antibodies, will block ETEC colonization and prevent disease. To test this hypothesis I propose the following specific aims: 1) Identify the genes necessary for Longus expression and evaluate Longus-gene variation, 2) Evaluate Longus-mediated adherence and colonization of to human intestinal surfaces, and 3) Test immunogenicity of Longus in a mouse model. I will first clone the genes necessary for Longus expression, evaluate gene variation among worldwide isolates, and construct a Longus structural subunit mutant essential for the evaluation of Longus phenotypes. Wild type and mutants will be compared for cell adherence to human intestinal cells, and in biofilm formation assays. ETEC colonization will be evaluated by using a novel human intestine explant-Severe combined immunodeficiency (SCID) mouse model. The explant will be infected with ETEC and evaluated for colonization by colony count and histology. Longus immunogenicity and cross-reactivity with longus variants will be tested on sera from immunized mice with Longus-expressing attenuated Salmonella. Demonstration that Longus mediates binding to cells and biofilm formation will greatly contribute to our knowledge on Longus function. The development of a human explant/SCID mouse model will have crucial implications for the understanding ETEC colonization at the molecular level. Information that Longus is immunogenic across variants and that anti-Longus antibodies may block colonization and prevent disease will facilitate the construction of effective ETEC vaccines.