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首页> 外文学位 >Listeria adhesion protein mediated Listeria monocytogenes translocation and infection in cell culture model.
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Listeria adhesion protein mediated Listeria monocytogenes translocation and infection in cell culture model.

机译：李斯特菌粘附蛋白介导的单核细胞增生李斯特菌易位和细胞培养模型中的感染。

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摘要

Listeria adhesion protein (LAP), an alcohol acetaldehyde dehydrogenase (lmo1634) in Listeria monocytogenes, has dual function: enzymatic activity, and adhesion capacity. Recently, our lab reported that LAP is involved in transepithelial translocation of L. monocytogenes through a paracellular route in Caco-2 cell model. In addition, LAP secretion is facilitated by SecA2 and secreted LAP is re-associated on bacterial cell wall to enhance LAP-mediated infection. However, the importance of secreted LAP and the cellular/molecular mechanisms of LAP-mediated translocation were unknown. In this study, LAP secretion levels were examined in fifty-two clinical isolates of L. monocytogenes. Experiments with three high and three low secreting isolates indicated that LAP-mediated L. monocytogenes adhesion, invasion and translocation in Caco-2 cells are each directly related to secreted LAP levels. There was minimal (2.0%) sequence heterogeneity in lap genes from high and low LAP secreting isolates and they all expressed similar levels of lap transcripts. Western blot analysis of different cell fractions indicated that low LAP-secreting cells accumulated largely in the intracellular (cytosolic) fractions.;To investigate the mechanism of LAP-mediated transepithelial translocation, we performed human gene chip microarray analysis to determine the proteins that may be involved in tight junction opening and also examined the role of TNF-alpha, NF-kappaB, and myosin light chain kinase (MLCK) in this process. Data showed LAP increased TNF-alpha expression in Caco-2 cells likely through activation of NF-kappaB pathway. Curcumin, a NF-kappaB inhibitor, affected TNF-alpha production and also bacterial translocation. MLCK inhibitor, ML-9, affected DextranFITC permeability and also bacterial translocation. Microarray data indicated that LAP down regulates expression of adherence junction proteins, E-cadherin and beta-catenin, which were further confirmed by qRT-PCR, Western blot and fluorescence microscopy staining. In summary, this study revealed that secreted LAP is a key player in LAP-mediated Listeria infection and LAP activates host signaling pathway to modify tight junction proteins which facilitates L. monocytogenes transepithelial translocation through a paracellular route.
机译：李斯特菌粘附蛋白（LAP）是单核细胞增生李斯特菌中的一种醇乙醛脱氢酶（lmo1634），具有双重功能：酶促活性和粘附能力。最近，我们的实验室报告说，LAP通过Caco-2细胞模型中的细胞旁途径参与了单核细胞增生李斯特氏菌的跨上皮易位。此外，SecA2促进了LAP的分泌，并且分泌的LAP在细菌细胞壁上重新关联，从而增强了LAP介导的感染。但是，未知的LAP的重要性和LAP介导的易位的细胞/分子机制尚不清楚。在这项研究中，检查了单核细胞增生李斯特菌的52种临床分离株中的LAP分泌水平。用三个高分泌和三个低分泌分离株进行的实验表明，LAP介导的单核细胞增生李斯特氏菌在Caco-2细胞中的粘附，侵袭和易位均与分泌的LAP水平直接相关。来自高和低LAP分泌分离株的lap基因中的序列异质性极小（<2.0％），并且它们都表达了相似水平的lap转录物。对不同细胞级分的蛋白质印迹分析表明，低LAP分泌细胞大量积累在细胞内（胞质）级分中。为了研究LAP介导的上皮易位的机制，我们进行了人类基因芯片微阵列分析，以确定可能是参与紧密连接的开放，还检查了TNF-α，NF-κB和肌球蛋白轻链激酶（MLCK）在此过程中的作用。数据显示，LAP可能通过激活NF-κB途径使Caco-2细胞中的TNF-α表达增加。姜黄素是一种NF-κB抑制剂，影响TNF-α的产生以及细菌移位。 MLCK抑制剂ML-9影响DextranFITC的通透性以及细菌移位。微阵列数据表明，LAP下调了粘附连接蛋白，E-钙粘蛋白和β-连环蛋白的表达，qRT-PCR，Western印迹和荧光显微镜染色进一步证实了LAP的表达。总而言之，这项研究表明，分泌的LAP是LAP介导的李斯特菌感染的关键因素，并且LAP激活宿主信号通路以修饰紧密连接蛋白，从而促进单核细胞增生李斯特氏菌通过细胞旁途径跨上皮易位。

著录项

  • 作者

    Kim, Hyochin.;

  • 作者单位

    Purdue University.;

  • 授予单位 Purdue University.;
  • 学科 Biology Molecular.;Agriculture Food Science and Technology.
  • 学位 Ph.D.
  • 年度 2011
  • 页码 213 p.
  • 总页数 213
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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