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Pathogenomics of Chlamydial Infection

衣原体感染的病理基因组学

基本信息

批准号：
7732543
负责人：
HARLAN D CALDWELL
金额：
$ 76万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7732543
关键词：
Actins Biologic Characteristic Biological Models Blindness Cell Culture System Cells Characteristics Chlamydia Infections Chlamydia trachomatis Clinical Confocal Microscopy Cytoskeleton Disease Disease Outcome Dominant-Negative Mutation Exhibits Eye Infections Genes Genetic Genetic Variation Genome Genomics Goals Growth Human In Vitro Infection Inflammatory Interferon Type II Ligands Link Mammalian Cell Measures Modeling Morphology Outcome Pathogenesis Pathogenicity Phenotype Protein Family Proteins Range Rate Receptor Cell Role Serotyping Site Small Interfering RNA Staging Syndrome Trachoma Tyrosine Tyrosine Phosphorylation Variant Virulence Work base cellular microvillus comparative ezrin gastrointestinal microvillus genome sequencing in vivo Model member moesin nonhuman primate pathogen polypeptide radixin protein receptor size

项目摘要

We have used both in vitro cell culture systems and non-human primate ocular infection models to better understand chlamydial pathogenesis. These studies have identified chlamydial induced host proteins that are tyrosine phosphorylated in a strain specific manner that implicate differences in chlamydial co-receptor usage during pathogen entry. We also have identified by functional comparative genomic studies of trachoma strains a small subset of genes that correlate with chlamydial strain pathogenic diversity. Chlamydia trachomatis is an obligate intracellular pathogen of humans that exhibits species-specific biological characteristics in its early interactions with host cells that are likely important to pathogenesis. One such characteristic is the tyrosine phosphorylation (Tyr-P) of a circa 70 kDa polypeptide that occurs only following infection of mammalian cells by human isolates/strains. We sought to identify this protein because of its potential significance to the pathogenesis of human chlamydial infections. Using an immunoproteomic approach we identified the host protein ezrin, a member of the ezrin/radixin/moesin (ERM) protein family that serves as a physical link between host cell receptors and the actin cytoskeleton. Confocal microscopy studies showed colocalization of ezrin and actin at the tips and crypts of microvilli, the site of chlamydial attachment and entry, respectively. To demonstrate a functional role for ezrin, we infected cells with a dominant negative (DN) ezrin phenotype, or treated cells with ezrin-specific siRNA. We found that both DN and siRNA-treated cells were significantly less susceptible to infection by human chlamydial strains. Moreover, we demonstrated that inhibition of infection in ezrin DN cells occurred at the stage of chlamydial entry. We hypothesize that the C. trachomatis-specific Tyr-P of ezrin might relate to an undefined species-specific mechanism of pathogen entry that involves chlamydial specific ligand(s) and host cell co-receptor usage. Chlamydia trachomatis is the etiological agent of trachoma, the leading cause of preventable blindness. Trachoma presents distinct clinical syndromes ranging from mild and self-limiting to severe inflammatory disease. The underlying host and pathogen factors responsible for these diverse clinical outcomes are unclear. To assess the role of pathogen variation in disease outcome we analyzed the genomes of four trachoma strains representative of the three major trachoma serotypes using microarray based comparative genome sequencing. Outside of ompA trachoma strains differed primarily in a very small subset (twenty-two) of genes. These subtle genetic variations were manifested in profound differences in virulence as measured by in vitro growth rate, burst size, plaque morphology, and interferon-gamma sensitivity but most importantly in virulence as shown by ocular infection of non-human primates. These findings are the first to identify genes that correlate with different trachoma strain pathogenicities.

我们使用体外细胞培养系统和非人灵长类动物眼部感染模型来更好地了解衣原体的发病机制。这些研究已经鉴定了衣原体诱导的宿主蛋白，其以菌株特异性方式被酪氨酸磷酸化，这暗示了病原体进入期间衣原体共受体使用的差异。我们还通过沙眼菌株的功能比较基因组研究，确定了与衣原体菌株致病多样性相关的一小部分基因。沙眼衣原体是一种专性细胞内病原体，在其与宿主细胞的早期相互作用中表现出种特异性生物学特征，这些生物学特征可能对发病机制很重要。一个这样的特征是大约70 kDa多肽的酪氨酸磷酸化（Tyr-P），其仅在哺乳动物细胞被人分离株/菌株感染后发生。我们试图确定这种蛋白质，因为它的潜在意义，人类衣原体感染的发病机制。使用免疫蛋白质组学方法，我们确定了宿主蛋白埃兹蛋白，埃兹蛋白/radixin/膜突蛋白（ERM）蛋白家族的成员，作为宿主细胞受体和肌动蛋白细胞骨架之间的物理联系。共聚焦显微镜研究表明，埃兹蛋白和肌动蛋白在微绒毛的尖端和隐窝，衣原体附着和进入的网站，分别共定位。为了证明ezrin的功能作用，我们用显性阴性（DN）ezrin表型感染细胞，或用ezrin特异性siRNA处理细胞。我们发现DN和siRNA处理的细胞对人衣原体菌株感染的敏感性显著降低。此外，我们证明，在埃兹林DN细胞感染的抑制发生在衣原体进入的阶段。我们假设C.埃兹蛋白的沙眼特异性Tyr-P可能涉及病原体进入的未定义的物种特异性机制，其涉及衣原体特异性配体和宿主细胞共受体的使用。沙眼衣原体是沙眼的病原体，是可预防失明的主要原因。沙眼呈现不同的临床综合征，从轻度和自限性到严重的炎性疾病。导致这些不同临床结果的潜在宿主和病原体因素尚不清楚。为了评估病原体变异在疾病转归中的作用，我们使用基于微阵列的比较基因组测序分析了代表三种主要沙眼血清型的四种沙眼菌株的基因组。在ompA之外，沙眼菌株主要在一个非常小的基因子集（22个）上不同。这些细微的遗传变异表现在毒力的显著差异中，如通过体外生长速率、爆发大小、斑块形态和干扰素-γ敏感性测量的，但最重要的是在毒力中，如通过非人灵长类动物的眼部感染所示的。这些发现是第一个确定与不同沙眼菌株致病性相关的基因。