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首页> 外文学位 >Identification of Leptospira interrogans adhesins and host cell surface receptors.
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Identification of Leptospira interrogans adhesins and host cell surface receptors.

机译：鉴定钩端螺旋体粘附素和宿主细胞表面受体。

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摘要

Leptospirosis, caused by pathogenic species of Leptospira, is the most widespread zoonosis and has emerged as a major public health problem worldwide. The adhesion of pathogenic Leptospira to host cells, and to extracellular matrix (ECM) components, is likely to be necessary for the ability of leptospires to penetrate, disseminate and persist in mammalian host tissues. Previous work demonstrated that pathogenic L. interrogans binds to host cells more efficiently than to ECM. However, the Leptospira adhesion molecules and host cell surface receptors involved in these interactions have not yet been determined.;Using two independent screening approaches, mass spectrometry and protein array, members of the cadherin family were identified as possible surface receptors for L. interrogans. My first aim was to extend and validate the preliminary results of the screens to identify mammalian host cell receptors that facilitate pathogenic L. interrogans attachment. My investigation focused on vascular endothelial (VE)-cadherin, which is widely expressed on endothelia and is primarily responsible for endothelial cell-cell adhesion. Monolayers of EA.hy926 and HMEC-1 endothelial cells produce VE-cadherin, bind L. interrogans in vitro, and are disrupted upon incubation with the bacteria, which may reflect the endothelial damage seen in vivo. Dose-dependent and saturable binding of L. interrogans to the purified VE-cadherin receptor was demonstrated and pretreatment of purified receptor or endothelial cells with function-blocking antibody against VEcadherin significantly inhibited bacterial attachment. The contribution of VE-cadherin to leptospiral adherence to host endothelial cell surfaces is biologically significant because VE-cadherin plays an important role in maintaining the barrier properties of the vasculature. Attachment of L. interrogans to the vasculature via VE-cadherin may result in vascular damage, facilitating the escape of the pathogen from the bloodstream into different tissues during disseminated infection, and may contribute to the hemorrhagic manifestations of leptospirosis. This work is first to describe a mammalian cell surface protein as a receptor for L. interrogans..;My second aim focused on identifying bacterial factors involved in interactions with host cells leading to infection and disease. Cell blot analysis confirmed presence of surface-exposed Leptospira proteins that recognize epithelial and endothelial cells in vitro. Phage display was employed to identify Leptospira outer membrane proteins involved in direct host cell interactions. DNA fragments of L. interrogans sv. Copenhageni st. Fiocruz L1-130 were inserted into a gene encoding a phage coat protein and expressed on the surface of the phage particle. Independent pools of the phage display library were pre-adsorbed on ECM and the unbound phage particles were subjected to three rounds of selection for phage that bind to Ea.hy926 endothelial cells in vitro. The selected phage clones were analyzed to determine L. interrogans genes inserted in the phage, and genes encoding proteins that are known or predicted to be surface localized were of interest in terms of their potential role in mammalian host cell interaction. The surface proteins selected by in vitro phage display and prioritized for further screening include putative lipoproteins LIC13411 and LIC10508, and conserved hypothetical proteins LIC12341 and LIC11574.;Our findings reveal that the recombinant form of LIC11574, but not its L. biflexa homologue LBF1629, exhibited binding to both endothelial and epithelial cell monolayers. LIC12341 was also observed to bind to epithelial cells in vitro. Both LIC11574 and LIC13411 exhibited highest binding to VE- and E-cadherin, host receptors identified in the first aim. Whether the ability of LIC11574 to bind to host receptors VE-cadherin and E-cadherin confer the bacterial attachment to endothelial and epithelial cells respectively, needs to be verified experimentally. All candidate adhesins were shown to be recognized by sera obtained from leptospirosis patients but not by sera from healthy individuals as assessed by western blot. Future work includes determination of the surface localization of these proteins.;In summary, this work has identified bacterial adhesins and endothelial host receptors that are potentially involved in the early steps of L. interrogans infection of and dissemination to the hosts. Our findings may be of value in leptospirosis control and prevention, with host cell receptors as potential targets of prophylactic drugs, and bacterial adhesins used for diagnostic applications and as potential vaccinogens.
机译：由钩端螺旋体病原体引起的钩端螺旋体病是最广泛的人畜共患病，已成为全球主要的公共卫生问题。致病性钩端螺旋体对宿主细胞和细胞外基质（ECM）成分的粘附可能是钩端螺旋体在哺乳动物宿主组织中渗透，传播和持续生存的能力所必需的。先前的研究表明，致病的问号乳杆菌比宿主细胞更有效地结合宿主细胞。然而，尚未确定参与这些相互作用的钩端螺旋体粘附分子和宿主细胞表面受体。通过使用两种独立的筛选方法，质谱法和蛋白质阵列，钙粘着蛋白家族成员被确定为问号李斯特菌的可能表面受体。我的第一个目标是扩展和验证筛选的初步结果，以鉴定有助于致病性问号乳杆菌附着的哺乳动物宿主细胞受体。我的研究集中在血管内皮（VE）-钙粘着蛋白上，该蛋白在内皮细胞上广泛表达，主要负责内皮细胞之间的粘附。 EA.hy926和HMEC-1内皮细胞的单层细胞产生VE-钙黏着蛋白，在体外结合问号乳杆菌，并在与细菌孵育后被破坏，这可能反映了体内对内皮的损害。证实了问号乳酸杆菌与纯化的VE-钙粘蛋白受体的剂量依赖性和饱和结合，并且用抗VEcadherin的功能阻断抗体对纯化的受体或内皮细胞进行预处理可显着抑制细菌附着。 VE-钙粘着蛋白对钩端螺旋体粘附到宿主内皮细胞表面的贡献在生物学上很重要，因为VE-钙粘着蛋白在维持脉管系统的屏障特性中起着重要的作用。通过VE-钙黏着蛋白将问询乳杆菌附着到脉管系统可能导致血管损伤，在传播感染期间促进病原体从血流中逸出到不同组织中，并可能导致钩端螺旋体病的出血表现。这项工作首先描述了哺乳动物细胞表面蛋白作为询问乳杆菌的受体。.我的第二个目标集中在鉴定与宿主细胞相互作用导致感染和疾病的细菌因子。细胞印迹分析证实存在可在体外识别上皮和内皮细胞的表面暴露的钩端螺旋体蛋白。噬菌体展示被用于鉴定参与直接宿主细胞相互作用的钩端螺旋体外膜蛋白。询问乳杆菌的DNA片段哥本哈根街将Fiocruz L1-130插入编码噬菌体外壳蛋白的基因中，并在噬菌体颗粒的表面表达。将噬菌体展示库的独立库预先吸附在ECM上，并对未结合的噬菌体颗粒进行三轮筛选，筛选出与Ea.hy926内皮细胞体外结合的噬菌体。分析选择的噬菌体克隆以确定插入噬菌体的询问乳杆菌基因，就其在哺乳动物宿主细胞相互作用中的潜在作用而言，编码已知或预测为表面定位的蛋白质的基因是令人感兴趣的。通过体外噬菌体展示选择并优先进行进一步筛选的表面蛋白包括推定的脂蛋白LIC13411和LIC10508，以及保守的假设蛋白LIC12341和LIC11574。与内皮和上皮细胞单层结合。还观察到LIC12341在体外与上皮细胞结合。 LIC11574和LIC13411都显示出与VE-和E-cadherin的最高结合，VE-cadherin是第一个目标中鉴定的宿主受体。 LIC11574与宿主受体VE-钙黏着蛋白和E-钙黏着蛋白结合的能力是否分别使细菌附着于内皮细胞和上皮细胞，需要通过实验验证。 Western blot评估显示，所有候选粘附素均能被钩端螺旋体病患者的血清识别，但不能被健康个体的血清识别。未来的工作包括确定这些蛋白质的表面定位。总之，这项工作已经确定了细菌粘附素和内皮宿主受体，这些细菌粘附素和内皮宿主受体可能与询问猪乳杆菌感染宿主和传播到宿主的早期步骤有关。我们的发现可能在钩端螺旋体病的控制和预防中具有价值，宿主细胞受体可作为预防药物的潜在靶标，细菌粘附素可用于诊断应用和潜在的疫苗原。

著录项

  • 作者

    Evangelista, Karen Villa.;

  • 作者单位

    The Medical College of Wisconsin.;

  • 授予单位 The Medical College of Wisconsin.;
  • 学科 Biology Microbiology.;Biology Cell.;Health Sciences Pathology.
  • 学位 Ph.D.
  • 年度 2014
  • 页码 218 p.
  • 总页数 218
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 高分子化学（高聚物）;
  • 关键词

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