GENETIC REGULATION OF HEAT LABILE ENTEROTOXIN SYNTHESIS IN E COLI

产肠毒素大肠杆菌(BTEC)产生不耐热的肠毒素(LT)，是引起人和农业动物腹泻疾病的主要原因之一。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导致疾病，以及改进疫苗的开发。LT获得的信息可能为由其他大肠杆菌和志贺氏菌引起的其他肠道疾病提供信息，志贺氏菌的毒力因子也受相同的调节蛋白H-NS的热调节。凝胶迁移率改变分析(EMSA)和体外转录实验将被用来确定H-NS是否通过直接与LT操纵子DNA，特别是下游调节元件(DRE)结合来调节其对LT表达的温度控制。通过进行化学诱变和寡核苷酸定向诱变获得的LT表达改变的突变株的分离，将实现对LToperon DNA的进一步鉴定。H-NS与突变DNA的相互作用将通过EMSA和体外转录进行评估，如果合适的话。一些对H-NS敏感的毒力因子的表达已被证明随着DNA拓扑结构的变化而改变。通过评估旋转酶抑制剂的效果，通过使用编码顶部异构酶L和旋转酶的两个亚单位的基因突变的菌株，以及通过评估温度以外的环境条件来研究超螺旋对LT表达的影响，因为许多H-NS结合位点已经被证明显示出内在的曲率，因此LT操纵子DNA的这一特征也将被研究。将对LT DRE进行研究，以确定该DNA片段在低温下是否会异常地通过琼脂糖凝胶或丙烯酰胺凝胶。如果发现LT DRE是弯曲的，可以使用抗生素地塞霉素来确定这一弯曲是否对H-NS或其他调节蛋白的结合是必要的(尚未确定)。

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).