VIRULENCE FACTORS ASSOCIATED WITH H INFLUENZA HIT B

与 H 型流感 HIT B 相关的毒力因子

• 批准号: 2057606
• 负责人: WENDY J WATSON
• 金额: $ 9.08万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-01 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2057606
• 关键词: Haemophilus influenzae; SDS polyacrylamide gel electrophoresis; adhesin; bacterial genetics; bacterial proteins; bacterial toxins; cytotoxicity; epidemiology; gene expression; genetic strain; membrane transport proteins; molecular cloning; mutant; oligonucleotides; open reading frames; phenotype; pilin; protein biosynthesis; protein sequence; protein structure function; protein transport; southern blotting; tissue /cell culture; virulence; western blottings

This proposal is to study a candidate virulence gene and its role in H influenzae disease. The specific aims of this project are: 1) define the complete nucleotide sequence of hitB, a homolog of the E coli hemolysin transport gene hlyB, in Haemophilus influenzae type b (Hib) and nontypable strains (NTHi); 2) use a genetic approach and protein analysis strategy to identify the putative protein(s) transported by HitB; 3) characterize hit B function in H. influenzae using hitB mutants and screening of clinical isolated. Preliminary nucleotide sequence data has identified a region homologous to hlyB on subclones containing Hib chromosomal DNA. In addition, Southern analysis suggests that this region is also present in NTHi. HlyB is associated with hemolysin transport in E coli, and analogous proteins in the Pasteurellaceae family transport various cytotoxins of the RTX family. The genes involved in encoding the RTX toxin, RTX activators, and transporters of the toxin are classically organized in a cluster on the chromosome. This proposal will investigate the relationship of hitB with virulence factor transport in H. influenzae. Nucleotide sequence analysis will be performed to define the boundaries of hitB in Hib. The homologous region will be cloned from NTHi and the sequence of the hitB gene in NTHi will be determined. Identification of the protein(s) transported by HitB will use several approaches. The region flanking hitB will undergo sequence analysis to determine the presence of genes encoding transported protein candidates. Protein analysis of various cell fractions of wild type Hib in comparison with hitB mutants will be used to identify differential protein transport. In addition, the HitB protein will be utilized in binding assays to identify and purify a candidate protein transported by HitB. An RTX cytotoxin gene or protein will be screened for using Southern blots or immunoblots. Finally hitB function will be characterized by examining the phenotypic effects produced by mutations in hitB on growth, piliation, hemagglutination and cytotoxic activity, and by screening for HitB expression in H. influenzae associated with disease and with normal flora.

本研究旨在研究一个候选毒力基因及其在H 流行性感冒 该项目的具体目标是：1）确定 hitB的完整核苷酸序列， 溶血素转运基因hly B，在流感嗜血杆菌B型（Hi B）中，和 不可分型菌株（NTHi）; 2）使用遗传方法和蛋白质分析 鉴定HitB转运的推定蛋白的策略; 3） 在H中表征命中B函数。使用hitB突变体的流感病毒， 临床分离株的筛选。 初步的核苷酸序列数据已经确定了一个区域同源 在含有Hib染色体DNA的亚克隆上转化为hlyB。此外，本发明还提供了一种方法， 南方分析表明，这一地区也存在于NTHi。 HlyB与大肠杆菌中的溶血素转运相关， 巴氏杆菌科中的蛋白质转运多种细胞毒素， RTX家族 参与编码RTX毒素的基因， 毒素的活化剂和转运体典型地组织在 染色体上的一簇 本研究将探讨hitB与毒力的关系 H.流感。 核苷酸序列分析将是 定义Hib中hitB的边界。 同源区域 将从NTHi中克隆，NTHi中hitB基因的序列将 被确定。 鉴定HitB转运的蛋白质将 使用几种方法。 hitB侧翼的区域将经历序列 分析以确定编码转运蛋白的基因的存在 候选人 野生型Hib的各种细胞级分的蛋白质分析 与hitB突变体相比， 蛋白质转运 此外，HitB蛋白将用于 结合试验，以鉴定和纯化通过 HitB. RTX细胞毒素基因或蛋白将被筛选用于使用 Southern印迹或免疫印迹。 最后，hitB函数将是 通过检查突变产生的表型效应来表征 在hitB中对生长、毛化、血凝和细胞毒活性， 并通过筛选HitB在H.流感相关 疾病和正常植物群。