GENETIC SYSTEMS TO STUDY VIRULENCE IN BACTEROIDES

研究拟杆菌毒力的遗传系统

• 批准号: 3128853
• 负责人: MICHAEL H MALAMY
• 金额: $ 19.73万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-01-01 至 1989-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3128853
• 关键词: Bacteroides; Escherichia coli; R factors; antibacterial agents; bacterial DNA; bacterial genetics; beta lactamase; catalase; chromosome translocation; exo alpha sialidase; gene expression; genetic manipulation; molecular cloning; mutant; tetracyclines; transposon /insertion element; virulence

Bacteroids fragilis is an important human pathogen. Attempts to evaluate and investigate determinants that may contribute to the virulence of this organism have been hampered by the absence of suitable systems of genetic analysis. We propose to continue the development of genetic systems in this organism with two specific aims. To investigate the determinants of antibiotic resistance and their transfer within B. fragilis and from B. fragilis to E. coli. We will characterize pBFTM10, a 15 kb transfer factor that encodes Clnr in B. fragilis and Tetr in E. coli. These resistances are carried on a transposon, Tn4400, that functions in E. coli. Studies are described to determine the transposition properties of this element and the IS elements which are present as direct repeats at each end. Studies will be directed towards understanding the "tetracycline transfer factor" that promotes plasmid and transposon transfer between B. fragilis strains and into E. coli. The second aspect of our proposal is to develop a genetic system to analyze virulence factors in B. fragilis. This will involve isolating mutants of selected phenotypic characteristics and then isolating the gene(s) encoding the determinants by making use of a chromosome library of B. fragilis cloned in E. coli. Tn4400 will be employed as a mutagen in B. fragilis to help locate chromosomal genes suspected to be virulence factors. The determinants to be investigated are imipenem and tetracycline resistance, catalase production and neuraminidase production. The beta-lactamase that inactivates imipenem will be studied first. The genes encoding tetracycline resistance in B. fragilis will be studied and we will attempt to develop a probe to study the mechanism of Tetr transfer. The role of catalase in the ability of B. fragilis to survive in oxygenated tissue will be assessed. Finally, the role that neuraminidase plays in tissue destruction during B. fragilis infections will be investigated. Once the mutants have been isolated and the gene(s) encoding the determinant are located then important questions related to expression can be addressed. The results of these studies should give a clearer picture of how DNA is transferred in B. fragilis and help to provide a genetic system to study virulence factors in this important pathogen.

细菌脆弱是一种重要的人类病原体。 尝试评估 并调查可能有助于这种毒力的决定因素 没有合适的遗传系统阻碍了生物体 分析。 我们建议继续在 这个有两个特定目的的有机体。 调查决定因素 抗生素耐药性及其在Fragilis和B.中的转移 脆弱至大肠杆菌。 我们将表征PBFTM10，一个15 kb的转移因子 这在脆弱的芽孢杆菌中编码CLNR和大肠杆菌中的Tetr。 这些阻力 在转座子TN4400上进行，该转座在大肠杆菌中起作用。 研究 描述以确定此元素的换位属性和 每端直接重复的IS元素。 研究 将针对理解“四环素转移因子” 促进质粒和转座子菌株之间的转移 并进入大肠杆菌。 我们建议的第二个方面是开发 分析脆弱芽孢杆菌中毒力因子的遗传系统。 这会 涉及隔离选定表型特征的突变体，然后 通过使用A隔离编码决定因素的基因 克隆在大肠杆菌中克隆的脆弱芽孢杆菌的染色体文库。 TN4400将是 在Fragilis中用作诱变剂，以帮助定位染色体基因 怀疑是毒力因素。 要研究的决定因素是 亚替林和四环素抗性，过氧化氢酶产生和神经氨酸酶 生产。 将研究失活的β-内酰胺酶 第一的。 编码脆弱芽孢杆菌中四环素抗性的基因将是 研究了，我们将尝试开发一个探针来研究 Tetr转移。 过氧化氢酶在脆弱的芽孢杆菌的能力中的作用 将评估在氧化组织中存活。 最后，角色 神经氨酸酶在脆弱芽孢杆菌感染期间在组织破坏中发挥作用 将被调查。 一旦分离出突变体，并且基因（s） 编码决定因素的位置，然后与 表达可以解决。 这些研究的结果应给出 更清晰的DNA如何在B. fragilis中转移并有助于 提供一个遗传系统来研究这一重要的毒力因子 病原。