Heat-Labile Enterotoxin Secretion

描述（由申请人提供）： 本文对产肠毒素大肠杆菌产生的不耐热肠毒素（LT）的分泌和细菌结合进行了探索性研究。大肠杆菌（ETEC）。 ETEC在第三世界国家是一种重要的病原体，是一种生物防御威胁，因为军事人员很可能从野外的非无菌水和食物来源获得这种病原体。该研究实验室已经发现，LT通过LT（LTB）的B亚基和LPS的Kdo核心之间的相互作用结合细菌表面和分泌的外膜囊泡表面上的脂多糖（LPS）。所描述的研究将使用基于寡糖的可能结合位点的定点诱变来阐明LPS在LTB上的结合位点。此外，结合研究将揭示LTB与LPS组分之间的亲和力。将研究LT与LPS结合和毒性的关系。毒力因子的细菌定位可能影响其在致病中的作用。由于霍乱弧菌分泌的霍乱毒素（CT）与LT具有高度同源性，并与大肠杆菌（E. coli LPS、LT和CT在结合和定位研究中进行比较。从这些目标产生的数据将开辟新的研究途径，有关凝集素结合位点，LT的定位，以及ETEC和霍乱弧菌引起的肠道毒性的差异。

DESCRIPTION (provided by applicant): This proposal describes exploratory research into the secretion and bacterial association of heat-labile enterotoxin (LT) produced by enterotoxigenic E. coli (ETEC). ETEC is an important diarrheagenic pathogen in third world countries and is a biodefense threat since military personel are likely to acquire this pathogen from non-sterile water and food sources in the field. This research laboratory has discovered that LT binds lipopolysaccharide (LPS) on the bacterial surface and on the surface of secreted outer membrane vesicles via an interaction between the B subunit of LT (LTB) and the Kdo core of LPS. The described study will elucidate the site for LPS binding on LTB using site-directed mutagenesis based on the likely binding site for the oligosaccharide. In addition, binding studies will reveal the affinity between LTB and components of LPS. The relationship of LT binding to LPS and toxicity will be investigated. The bacterial localization of virulence factors may influence their role in pathogenesis. Therefore, fluorescence microscopy and tagging strategies will be used to investigate the bacterial surface localization of secreted, bound LT. Since cholera toxin (CT) secreted by Vibrio cholera is highly homologous to LT and was also determined to bind to E. coli LPS, LT and CT will be compared in binding and localization studies. The data resulting from these aims will open new avenues of research regarding lectin binding sites, the localization of LT, and the difference in the enteric toxicity caused by ETEC and V. cholerae.