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首页> 外文学位 >Development of a mariner based transposon and identification of Listeria monocytogenes determinants, including the peptidyl-prolyl isomerase PrsA2, that contribute to its hemolytic phenotype.
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Development of a mariner based transposon and identification of Listeria monocytogenes determinants, including the peptidyl-prolyl isomerase PrsA2, that contribute to its hemolytic phenotype.

机译：基于水手的转座子的开发和李斯特菌单核细胞增生因子决定簇的鉴定，包括有助于其溶血表型的肽基-脯氨酰异构酶PrsA2。

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摘要

Listeria monocytogenes is a Gram-positive, food-borne, facultative intracellular pathogen of animals and humans. Although initially confined to phagosomal compartment after phagocytosis or induced uptake, L. monocytogenes is adapted for growth and replication within the cytosol. Central to the virulence of L. monocytogenes is the cholesterol-dependent pore-forming cytolysin listeriolysin O (LLO), which facilitates the escape of the pathogen from the phagosome into the cytoplasm. However, the activity of this toxin must be compartmentalized to avoid lysing the host cell membrane. Although considerable work has focused on intragenic mechanisms of LLO regulation, little is known about the extragenic factors that regulate LLO.;Here we undertook a broad, genetic screen for factors that mediate the production or activity of LLO. We describe the design and construction of a new Himar1 mariner transposon system for use in L. monocytogenes. We observed that libraries generated by this new system are more complex than those generated by the previous standard transposon system, therefore increasing the resolution of forward genetic screens. Approximately 50,000 insertion mutants were then analyzed on blood agar plates for either a hypohemolytic or hyperhemolytic phenotype. Nearly 450 mutants were identified: transposon insertions in 63 unique features resulted in a hypohemolytic phenotype and insertions into 82 unique regions resulted in a hyperhemolytic phenotype.;The hypohemolytic mutants were analyzed further, and six were found to have a plaque defect, a hemolytic defect, and an in vivo defect: lmo0964 (similar to yjbH); lmo1268 (clpX); lmo1401 (similar to ymdB); lmo1575 (similar to ytq1 ); lmo1821 (similar to prpC); and lmo2219 (prsA2). One gene in particular, prsA2, had a profound influence on virulence, and mutants were essentially avirulent. The homolog in Bacillus subtilis is responsible for folding exported substrates, and consistent with a similar role for PrsA2 in L. monocytogenes, multiple virulence factors were affected upon its deletion. In addition to LLO, a broad range phospholipase C (PC-PLC) was less active in mutant strains. Our results suggest that PrsA2 may have evolved to alleviate the increase in export stress that occurs during an infection.;In summary, we updated the genetic tools available for use in L. monocytogenes, and have begun to explore the factors involved in regulating the mechanisms necessary for virulence.
机译：单核细胞增生李斯特菌是动物和人类的革兰氏阳性，食源性兼性细胞内病原体。尽管最初在吞噬作用或诱导摄取后局限于吞噬细胞区室，但单核细胞增生李斯特氏菌适于在细胞质内生长和复制。单核细胞增生李斯特氏菌毒力的核心是胆固醇依赖性孔形成性溶细胞素李斯特菌溶血素O（LLO），它有助于病原体从吞噬体逸出进入细胞质。但是，该毒素的活性必须区分开，以避免裂解宿主细胞膜。尽管大量工作集中在调节LLO的基因内机制上，但对调节LLO的外源因子知之甚少。在这里，我们对介导LLO产生或活性的因子进行了广泛的遗传筛选。我们描述了用于单核细胞增生李斯特菌的新型Himar1 Mariner转座子系统的设计和构建。我们观察到，由这个新系统生成的文库比由以前的标准转座子系统生成的文库更为复杂，因此提高了正向遗传筛选的分辨率。然后在血琼脂平板上分析大约50,000个插入突变体的低溶血或超溶血表型。鉴定了近450个突变体：转座子插入63个独特特征导致低溶血性表型，插入82个独特区域导致高溶血性表型。;进一步分析了低溶血突变体，发现6个具有噬斑缺陷，溶血缺陷。 ，以及体内缺陷：lmo0964（类似于yjbH）； lmo1268（clpX）; lmo1401（类似于ymdB）； lmo1575（类似于ytq1）； lmo1821（类似于prpC）；和lmo2219（prsA2）。特别是一个基因prsA2，对毒力具有深远的影响，而突变体基本上是无毒的。枯草芽孢杆菌中的同源物负责折叠输出的底物，并与单核细胞增生李斯特氏菌中PrsA2的相似作用一致，多种毒力因子在其缺失后受到影响。除LLO外，大范围的磷脂酶C（PC-PLC）在突变菌株中的活性也较低。我们的结果表明，PrsA2可能已经进化为减轻感染过程中出口压力的增加。;总而言之，我们更新了可用于单核细胞增生李斯特氏菌的遗传工具，并已开始探索涉及调节机制的因素毒力所必需。

著录项

  • 作者

    Zemansky, Jason Eron.;

  • 作者单位

    University of California, Berkeley.;

  • 授予单位 University of California, Berkeley.;
  • 学科 Biology Molecular.;Biology Microbiology.;Biology Bioinformatics.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 129 p.
  • 总页数 129
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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