Pathogenomics of Chlamydial Infection

衣原体感染的病理基因组学

基本信息

批准号：
7592240
负责人：
HARLAN D CALDWELL
金额：
$ 86.23万
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/7592240
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.

我们已经使用了体外细胞培养系统和非人类灵长类动物眼感染模型来更好地了解衣原体发病机理。这些研究已经确定了衣原体诱导的宿主蛋白，这些宿主蛋白是以特异性方式磷酸化的酪氨酸，这暗示了病原体进入过程中衣原体共受体使用的差异。我们还通过对沙眼菌株的功能比较基因组研究鉴定了与衣原体菌株致病多样性相关的一小部分基因。沙眼衣原体是人类的强制性细胞内病原体，在与可能对发病机理很重要的宿主细胞相互作用中表现出物种特异性的生物学特征。这样的特征之一是大约70 kDa多肽的酪氨酸磷酸化（Tyr-P），该酪氨酸仅在人类分离株/菌株感染哺乳动物细胞后才发生。我们试图确定该蛋白质，因为它对人类衣原体感染的发病机理具有潜在的意义。使用免疫蛋白质组学方法，我们鉴定了宿主蛋白Ezrin，Ezrin，Ezrin/radixin/Moesin（ERM）蛋白家族的成员，该蛋白是宿主细胞受体与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，在微绒毛的尖端和地下室（分别是衣原体附着和进入的位置）的尖端和隐窝。为了证明ezrin的功能作用，我们感染具有显性阴性（DN）Ezrin表型或Ezrin特异性siRNA的细胞的细胞。我们发现，DN和siRNA处理的细胞均明显不易被人衣原体菌株感染。此外，我们证明了埃兹林DN细胞中感染的抑制作用发生在衣原体进入阶段。我们假设Ezrin的沙眼特异性Tyr-P特异性的Tyr可能与涉及衣原体特异性配体和宿主细胞共受体使用的病原体进入的不确定的物种特异性机理有关。沙眼衣原体是沙丘瘤的病因，这是可预防失明的主要原因。沙眼瘤表现出不同的临床综合征，范围从轻度和自我限制到严重的炎症性疾病。负责这些多种临床结果的基本宿主和病原体因素尚不清楚。为了评估病原体变异在疾病结局中的作用，我们分析了使用基于微阵列的比较基因组测序的四种主要三脊髓瘤血清型的四种沙眼菌株的基因组。在OMPA沙眼菌株之外，主要在很小的子集（22个）基因的基因中有所不同。这些微妙的遗传变异表现为毒力的深刻差异，如通过体外生长速率，爆发尺寸，斑块形态和干扰素 - γ敏感性所测量，但最重要的是毒力，如非人类灵长类动物的眼部感染所示。这些发现是第一个鉴定与不同沙眼菌株致病性相关的基因。