TOXIN A SYNTHESIS IN P AERUGINOSA

描述(改编自申请人的摘要)：假单胞菌 铜绿假单胞菌是一种革兰氏阴性条件致病菌，可引起 严重烧伤患者的严重感染，免疫功能受损 宿主和囊性纤维化患者。A毒素是毒性最强的毒素之一 铜绿假单胞菌产生的毒力因子。毒素A的合成 在铜绿假单胞菌中，受不同环境因素的高度调控 尤其是铁。尽管有几项研究，但这种情况的确切机制 监管还没有完全被理解。这样做的长期目标是 建议确定毒素A合成的调控机制 在铜绿假单胞菌和这一调控中涉及的因素。最近， 克隆了一个新的ToxA阳性调控基因ptxR。分析 PtxR表明，它增强了Toxa和Toxa基因的转录 此前已描述了弓形虫调控基因Regab。核苷酸序列 分析揭示了开放阅读框架的存在，该框架编码 一个34 kDa的预测蛋白质。计算机分析揭示了 干扰ptxR功能的相邻基因。公开的读物 从ptxR差异转录的框架(ORF2)(从减号 链)和编码一个30 kDa的蛋白。这预示着 蛋白质与GalR的几种蛋白质有显著的同源性 压迫者家族。这项建议的具体目的是：1) 研究ptxR功能的机制；2)确定 PtxR的转录调控；3)检测ORF2的作用 关于ptxR函数。研究ptxR功能的机制将包括 PtxR产物的纯化、DNA/蛋白质结合实验 (凝胶延迟实验)，磷酸化实验，两个 空间凝胶实验及PAO1同源基因的构建 PtxR中的突变。监管研究将包括转录分析 包括ptxR、Rega和Toxa。分析ORF2的功能将涉及到 体内和体外转录实验、免疫共沉淀 实验，并分离到一株ORF2等基因突变体。

DESCRIPTION (adapted from the applicant's abstract): Pseudomonas aeruginosa is a gram negative opportunistic pathogen which causes serious infections in severely burned patients, immuno-compromised hosts, and Cystic Fibrosis patients. Toxin A is one of the most toxic virulence factors produced by P. aeruginosa. The synthesis of toxin A in P. aeruginosa is highly regulated by different environmental factors especially iron. Despite several studies, the exact mechanism of this regulation is not completely understood. The long term goal of this proposal is to determine the mechanisms of regulating toxin A synthesis in P. aeruginosa and the factors involved in this regulation. Recently, a new toxA positive regulatory gene, ptxR, was isolated. Analysis of ptxR showed that it enhances transcription of both toxA and the previously described toxA regulatory gene, regAB. Nucleotide sequence analysis revealed the presence of an open reading frame which codes for a 34 kDa predicted protein. Computer analysis revealed the presence of an adjacent gene which interferes with ptxR function. An open reading frame (ORF2) which is divergently transcribed from ptxR (from the minus strand) and codes for a 30 kDa protein was identified. This predicted protein has a significant homology to several proteins of the GalR family of repressors. The specific aims of this proposal are: 1) to examine the mechanism of ptxR function; 2) to determine the transcriptional regulation of ptxR; and 3) to examine the effect of ORF2 on ptxR function. Examining the mechanism of ptxR function will include the purification of ptxR production, DNA/protein binding experiments (gel retardation experiments), phosphorylation experiments, two dimensional gel experiments, and the construction of a PAO1 isogenic mutant in ptxR. Regulatory studies will include transcriptional analysis of ptxR, regA, and toxA. Analysis of ORF2 function will involved in vivo and in vitro transcription experiments, co-immunoprecipitation experiments, and the isolation of an ORF2 isogenic mutant.