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: 2057604
• 负责人: WENDY J WATSON
• 金额: $ 8.96万
• 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 1997-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2057604
• 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赤霉病中的作用。 流感病毒病。该项目的具体目标是：1)确定 大肠杆菌同源基因HITB的全序列测定 B型流感嗜血杆菌溶血素转运基因hlyB和 非分型菌株(NTHi)；2)使用遗传方法和蛋白质分析 HITB运输的推定蛋白(S)的鉴定策略；3) 用HITB突变体和HITB研究流感嗜血杆菌的HIT B功能 临床分离菌株的筛选。 初步的核苷酸序列数据已经确定了一个同源区域 在含有Hib染色体DNA的亚克隆上插入hlyB。此外, 南方分析表明，这一地区也存在于NTHi。 HlyB与溶血素在大肠杆菌中的转运有关，类似于 巴氏杆菌科家族的蛋白质转运多种细胞毒素 腾讯通家族。编码RTX毒素RTX的基因 毒素的激活剂和转运体通常在 染色体上的一簇。 这项建议将调查HITB与毒力的关系 流感嗜血杆菌的因子转运。核苷酸序列分析将是 执行以确定HITB在HIB中的边界。同源区域 将从NTHi中克隆HITB基因，并将其序列 要下定决心。HITB Will转运蛋白(S)的鉴定 使用几种方法。HITB侧翼区域将经历序列 分析以确定编码运输蛋白的基因的存在 候选人。野生型Hib不同细胞组分的蛋白质分析 与HITB相比，突变体将用于鉴定差异 蛋白质运输。此外，HITB蛋白将用于 结合实验鉴定和纯化一种候选蛋白 HITB。RTX细胞毒素基因或蛋白将被筛选用于 Southern印迹或免疫印迹。最后，HITB功能将被 以检测突变产生的表型效应为特征的 在HITB中对生长、皮毛、血凝和细胞毒活性的影响， 通过筛选与HITB相关的HITB在流感嗜血杆菌中的表达 疾病和正常的菌群。