LISTERIA HEMOLYSIN AND INTRACELLULAR GROWTH

李斯特菌溶血素和细胞内生长

• 批准号: 3455054
• 负责人: DANIEL A PORTNOY
• 金额: $ 11.88万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-06-01 至 1993-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3455054
• 关键词: Enterococcus; Escherichia coli; Listeria; Listeria infections; ampicillin; bacterial antigens; bacterial genetics; electron microscopy; fibroblasts; genetic library; genetic manipulation; hemolysin; host organism interaction; intracellular parasitism; laboratory mouse; macrophage; microorganism genetics; microorganism growth; molecular cloning; mutant; nucleic acid probes; nucleic acid sequence; plasmids; population genetics; protein sequence; tissue /cell culture; transposon /insertion element; virulence

Intracellular pathogens are responsible for an extensive amount of morbidity and mortality world-wide. Our current understanding of host reponse to intracellular pathogens stems mainly from extensive analysis of murine cell-mediated immunity to the facultative intracellular bacterial pathogen Listeria monocytogenes. Athough the immune response to L. monocytogenes has received enormous attention surprising little research has been devoted to understanding the cell biology of intracellular growth or to bacteria determinants of pathogenicity. The overall goal of the proposed research is to define in molecular terms listerial determinants required for intracellular growth. One likely determinant of L. monocytogenes pathogenesis is the elaboration of a sulfhydryl-activated hemolysin. In preliminary studies, conjugative transposons were used to isolate non- hemolytic mutants. The mutants fail to grow in the mouse macrophage like cell line J774. In the proposed study, the mutants will be evaluated with respect to growth in vivo as well as intracellularly, in vitro. Preliminary data support that L. monocytogenes grows freely in the eucaryotic cell cytoplasm. Electron microscopy of thin-sectioned infected cells will be used to evaluated the role of hemolysin for intracellular localization. As an initial step in structure-function analysis of the hemolysin, the hemolysin gene will be cloned from L. monocytogenes cosmid DNA libraries in E. coli. The primary amino acid sequnce will be deduced from the nucleotide sequence and compared with other pore-forming proteins. The cloned gene will also be used as a hybridization probe to examine its conservation among Listeria species. The hemolysin gene will be cloned into a streptococcal shuttle vector, transformed into a streptococcal strain and conjugated to a non-hemolytic strain of L. monocytogenes. This methodology will facilitate future work in which in vitro constructed hemolysin mutations can be studied in their normal background. Genes other than hemolysin are likely to contribute to listerial infectivity. An attempt will be made to isolate tansposon insertions in genes other than hemolysin which are required for intracellular multiplication. Mutants will be selected inside tissue culture cells in the presence of ampicillin, an antibiotic that kills only growing bacteria.

细胞内病原体负责大量的 全世界的发病率和死亡率。 我们目前的理解 宿主对细胞内病原体的反应主要源于 小鼠细胞介导的免疫的广泛分析 兼性细胞内细菌病原体李斯特菌 单核细胞增多症。 尽管对 L 的免疫反应。 单核细胞增多症受到了极大的关注，但令人惊讶的是 研究致力于了解细胞生物学 细胞内生长或细菌致病性的决定因素。 拟议研究的总体目标是在分子中定义 术语“李斯特菌”是细胞内生长所需的决定因素。 单核细胞增生李斯特菌发病机制的一个可能的决定因素是 巯基激活的溶血素的研制。 在初步 研究中，接合转座子被用来分离非 溶血突变体。 突变体无法在小鼠体内生长 巨噬细胞样细胞系J774。 在拟议的研究中， 还将评估突变体的体内生长情况 如细胞内、体外。 初步数据支持 L. 单核细胞增多症在真核细胞的细胞质中自由生长。 将使用受感染细胞薄切片的电子显微镜 评估溶血素在细胞内定位中的作用。 作为溶血素结构功能分析的第一步， 溶血素基因将从单增李斯特菌粘粒中克隆 大肠杆菌中的 DNA 文库。 初级氨基酸序列将是 从核苷酸序列推导出来并与其他序列进行比较 成孔蛋白。 克隆的基因也将被用作 杂交探针检查其在李斯特菌中的保守性 物种。 溶血素基因将被克隆到链球菌中 穿梭载体，转化为链球菌菌株并 与单核细胞增生李斯特氏菌的非溶血菌株缀合。 这 方法将促进未来的工作，其中体外 构建的溶血素突变可以在正常情况下进行研究 背景。 溶血素以外的基因可能与李斯特菌有关 传染性。 将尝试分离转座子 插入溶血素以外的基因，这是 细胞内增殖。 突变体将在组织内被选择 在氨苄西林（一种杀死细胞的抗生素）存在的情况下培养细胞 只生长细菌。