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首页> 外文学位 >Listeria adhesion protein and heat shock protein 60: Application in pathogenic Listeria detection and implication in Listeriosis prevention.
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Listeria adhesion protein and heat shock protein 60: Application in pathogenic Listeria detection and implication in Listeriosis prevention.

机译：李斯特菌粘附蛋白和热休克蛋白60：在李斯特菌病原体检测中的应用及其在李斯特菌病预防中的意义。

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摘要

Listeria adhesion protein (LAP, lmo1634), also known as a housekeeping enzyme alcohol acetaldehyde dehydrogenase, is a cell surface protein involved in adhesion and translocation of L. monocytogenes to human intestinal cells. The pathogen crosses the intestinal barrier following interaction with the receptor present on intestinal epithelial cells. Heat shock protein 60 (Hsp60), a eukaryotic mitochondrial chaperon protein is the receptor for LAP. LAP is present in all Listeria species except L. grayi, and the purified LAP from all Listeria species had similar molecular characteristics such as high sequence similarity, strong binding to Hsp60 and enzyme activity. Interestingly, secretion of LAP was only observed in pathogenic L. monocytogenes and one of our previous study suggested that LAP secretion is SecA2-dependent.Because of the specific interaction between surface exposed LAP and Hsp60, strategies could be employed to potentially protect the host from the infection by blocking the attachment. The overall goals of this project were to enhance our understanding of the adhesion mechanism and to apply this knowledge in developing a recombinant probiotic bacterial strain to prevent infection in a cell culture model and to develop method(s) to improve L. monocytogenes detection from food matrices.;Antibodies have been widely used for detection of specific antigens; however, earlier studies have shown that non-specific interactions may reduce specificity of antibody detection. As an alternate method, Hsp60 was used on microfluidic biochip and paramagnetic beads to capture and concentrate L. monocytogenes from contaminated foods. The efficiency of Hsp60 to capture L. monocytogenes was 83 times greater than monoclonal antibody MAb-C11E9, on the biochip. Hsp60 also showed specific interaction with L. monocytogenes compared to other Listeria spp. and other food-borne bacteria on both detection platforms. On paramagnetic beads, Hsp60 was able to capture 2.1- to 6.2-fold more of L. monocytogenes than L. innocua in hotdog samples under a coculture environment.;We also examined if LAP can be expressed in a probiotic bacterium to prevent Listeria attachment and colonization in an in vitro Caco-2 cell culture model. Wild type probiotic Lactobacillus species were unable to reduce the adhesion of L. monocytogenes; however, Lb. paracasei expressing LAP caused significant reduction in L. monocytogenes adhesion (25-31%) and transepithelial translocation (40%) in Caco-2 cells. Furthermore, re-association of secreted LAP to the surface of Listeria strain and recombinant Lactobacillus was found to be essential to promote interaction with host cells. Non-pathogenic Listeria lacks surface molecule(s) that aid in LAP re-association, hence it unables to interact with host cells. Collectively, our data show that Hsp60 interacts strongly with only pathogenic Listeria, including L. monocytogenes, and can be used to capture and detect the pathogen on microfluidic biochip or on magnetic beads. Furthermore, LAP expressing probiotic Lactobacillus has the potential to protect host against infection by specific blocking of Listeria interaction with the host receptor.
机译：李斯特菌粘附蛋白（LAP，lmo1634），也称为管家酶乙醇乙醛脱氢酶，是一种细胞表面蛋白，参与单核细胞增生李斯特氏菌向人肠道细胞的粘附和转运。在与肠道上皮细胞上存在的受体相互作用后，病原体穿过肠道屏障。热休克蛋白60（Hsp60）是一种真核线粒体伴侣蛋白，是LAP的受体。 LAP存在于除李斯特菌之外的所有李斯特菌属物种中，并且从所有李斯特菌属物种中纯化得到的LAP具有相似的分子特征，例如高序列相似性，与Hsp60的强结合以及酶活性。有趣的是，仅在致病性单核细胞增生李斯特菌中观察到LAP的分泌，而我们之前的一项研究表明LAP的分泌是SecA2依赖性的。由于表面暴露的LAP与Hsp60之间存在特异性相互作用，因此可以采取策略来潜在地保护宿主免受宿主通过阻止附件感染。该项目的总体目标是加深我们对黏附机制的了解，并将此知识应用于开发重组益生菌菌株以预防细胞培养模型中的感染，并开发改进食品中单核细胞增生李斯特氏菌检测的方法。抗体已被广泛用于检测特定抗原。但是，较早的研究表明，非特异性相互作用可能会降低抗体检测的特异性。作为一种替代方法，将Hsp60用于微流控生物芯片和顺磁珠，以捕获和浓缩受污染食品中的单核细胞增生李斯特菌。在生物芯片上，Hsp60捕获单核细胞增生李斯特菌的效率比单克隆抗体MAb-C11E9高83倍。与其他李斯特菌属菌种相比，Hsp60还显示出与单核细胞增生李斯特菌的特异性相互作用。以及两个检测平台上的其他食源性细菌。在共培养环境下，在顺磁珠上，Hsp60能够捕获热狗样品中单核细胞增生李斯特氏菌多2.1到6.2倍;我们还检查了LAP是否可以在益生菌中表达以防止李斯特菌附着和在体外Caco-2细胞培养模型中的定植野生型益生菌乳酸菌无法减少单核细胞增生李斯特菌的粘附。但是，磅。在Caco-2细胞中，表达LAP的paracasei引起单核细胞增生李斯特氏菌粘附（25-31％）和上皮易位（40％）的显着降低。此外，发现分泌的LAP与李斯特菌菌株和重组乳杆菌的表面再结合对于促进与宿主细胞的相互作用至关重要。非致病性李斯特菌缺乏帮助LAP重新结合的表面分子，因此它无法与宿主细胞相互作用。总体而言，我们的数据表明，Hsp60仅与致病性李斯特菌（包括单核细胞增生李斯特菌）发生强烈相互作用，可用于捕获和检测微流生物芯片或磁珠上的病原体。此外，表达LAP的益生菌乳杆菌具有通过特异性阻断李斯特菌与宿主受体相互作用而保护宿主免受感染的潜力。

著录项

  • 作者

    Koo, Ok Kyung.;

  • 作者单位

    Purdue University.;

  • 授予单位 Purdue University.;
  • 学科 Agriculture Food Science and Technology.;Biology Microbiology.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 184 p.
  • 总页数 184
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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