LISTERIA HEMOLYSIN AND INTRACELLULAR GROWTH

细胞内的病原体是造成大量 发病率和死亡率。 我们目前的理解 宿主对胞内病原体的反应主要来自于 广泛分析小鼠细胞介导的免疫力， 兼性胞内细菌病原体李斯特菌 单核细胞增多症 尽管对L. 单核细胞增多症受到了极大的关注， 研究一直致力于了解细胞生物学的 细胞内生长或致病性的细菌决定因素。 拟议研究的总体目标是在分子水平上定义 术语胞内生长所需的细胞因子。 L的一个可能的决定因素。单核细胞增多症的发病机制是 巯基活化溶血素的制备。 初步 研究中，接合转座子被用来分离非- 溶血突变体 突变体不能在老鼠体内生长 巨噬细胞样细胞系J774。 在拟议的研究中， 突变体也将在体内生长方面进行评价 在细胞内，在体外。 初步数据支持L. 单核细胞增生在真核细胞的细胞质中自由生长。 将使用薄切片感染细胞的电子显微镜检查 评价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L.单核细胞增生粘粒 E.杆菌 一级氨基酸序列将是 从核苷酸序列推导并与其他序列进行比较 成孔蛋白 克隆的基因也将被用作 杂交探针检测李斯特菌保守性 物种 溶血素基因将被克隆到链球菌 穿梭载体，转化到链球菌菌株中， 与L.单核细胞增多症 这 方法将促进今后的工作， 构建的溶血素突变可以在它们的正常 背景 溶血素以外的其他基因可能也会导致 传染性 我们将尝试分离出转座子 在溶血素以外的基因中插入， 细胞内增殖 突变体将在组织内被选择 在氨苄青霉素的存在下培养细胞，氨苄青霉素是一种抗生素， 只会滋生细菌

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.