GENETIC REGULATION OF HEAT LABILE ENTEROTOXIN SYNTHESIS IN E COLI

大肠杆菌中不耐热肠毒素合成的遗传调控

• 批准号: 6246577
• 负责人: JULIE D TRACHMAN
• 金额: $ 10.16万
• 依托单位: LONG ISLAND UNIVERSITY BROOKLYN CAMPUS
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-30 至 2003-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6246577
• 关键词: DNA; DNA binding protein; Escherichia coli; biosynthesis; enterotoxins; gel mobility shift assay; gene expression; genetic regulation; genetic transcription; heat stimulus; nucleic acid sequence; nucleic acid structure; protein binding; protein structure function; transcription factor

Enterotoxinogenic E.coli (BTEC) which produce heat-labile enterotoxin (LT are among the leading causes of diarrheal disease in man and agricultural animals. Investigation of the genetic regulation of LT will lead hopefully lead to a clearer understanding of how ETEC strains interacts with the host and causes disease as well as to the development of improved vaccines. Information gained with LT may provide information that is usefiil for other enteric diseases caused by other E. coli strains and Shigella whose virulence factors are also thermoregulated by the same regulatory protein, H-NS. Electrophoretic mobility shift assays (EMSA) and in vitro transcription experiments will be used to determine if H-NS mediates its temperature control on LT expression by directly binding to LT operon DNA, specifically the downnstream regulatory element (DRE). Further characterization of the LToperon DNA will be achieved by isolating mutants with altered LT expression that have been obtained by carrying out chemical mutagenesis and then oligonucleotide-directed mutagenesis. H- NS interactions with the mutated DNA will be evaluated by EMSA and in vitro transcription if appropriate. Several H-NS-sensitive virulence factors have been shown to have their expression alterated by changes in DNA topolgy. Supercoiling effects on LT expression will be investigated by evaluating the effects of gyrase inhibitors , by using strains that have mutations in the genes encoding top oisomerase l and the two subunits of gyrase, and by evaluating environmental conditions beside temperature which are known to alter supercoiling Since many H-NS binding sites have been shown to display intrinsic curvature, this characteristic of LT operon DNA will also be investigated. The LT DRE will be studied to see if this DNA fragment moves aberrantly through agarose or acrylamide gels at low temperatures. If the LT DRE is found to be curved, the antibiotic distamycin can be used to determine if this curvature is essential for binding of H-NS or other regulatory proteins (yet to be determined).

产生不耐热肠毒素（LT）的产肠毒素大肠杆菌（BTEC）是人类和农业动物腹泻病的主要原因之一。对 LT 基因调控的研究有望使人们更清楚地了解 ETEC 菌株如何与宿主相互作用并引起疾病，并开发改进的疫苗。通过 LT 获得的信息可能为其他肠毒素提供有用的信息。 由其他大肠杆菌菌株和志贺氏菌引起的疾病，其毒力因子也受到相同调节蛋白 H-NS 的温度调节。电泳迁移率变动分析 (EMSA) 和体外转录实验将用于确定 H-NS 是否通过直接结合 LT 操纵子 DNA，特别是下游调节元件 (DRE) 来介导对 LT 表达的温度控制。进一步表征 LToperon DNA 将通过分离 LT 表达改变的突变体来获得，这些突变体是通过化学诱变和寡核苷酸定向诱变获得的。 H-NS 与突变 DNA 的相互作用将通过 EMSA 和体外转录（如果合适）进行评估。几种 H-NS 敏感毒力因子已被证明其表达会因 DNA 拓扑的变化而改变。超螺旋对 LT 的影响 将通过评估促旋酶抑制剂的作用、通过使用在编码顶部异构酶l和促旋酶的两个亚基的基因中具有突变的菌株以及通过评估已知会改变超螺旋的温度以外的环境条件来研究表达。由于许多H-NS结合位点已被证明显示出内在曲率，因此还将研究LT操纵子DNA的这一特征。将研究 LT DRE 看看该 DNA 片段在低温下通过琼脂糖或丙烯酰胺凝胶时是否异常移动。如果发现 LT DRE 弯曲，则可以使用抗生素偏端霉素来确定该弯曲是否对于 H-NS 或其他调节蛋白的结合至关重要（尚未确定）。