Pathogenomics of Chlamydial Infection

为了更好地了解衣原体的发病机制，我们使用了体外细胞培养系统和非人灵长类动物眼部感染模型。这些研究已经确定了衣原体诱导的宿主蛋白，这些蛋白以一种菌株特有的方式被酪氨酸磷酸化，这意味着在病原体进入期间衣原体共受体的不同使用。我们还通过沙眼衣原体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。 沙眼衣原体是人类的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物种特异性的生物学特征，这可能在发病机制中起重要作用。其中一个特征是大约70 kDa多肽的酪氨酸磷酸化(Tyr-P)，只有在人类分离株/菌株感染哺乳动物细胞后才会发生。我们试图鉴定这种蛋白，因为它对人类衣原体感染的发病机制具有潜在的意义。利用免疫蛋白质组学方法，我们鉴定了宿主蛋白Ezrin，它是Ezrin/Radisin/moesin(ERM)蛋白家族的成员，它是宿主细胞受体和肌动蛋白细胞骨架之间的物理联系。共聚焦显微镜研究显示，Ezrin和肌动蛋白分别位于微绒毛的顶端和隐窝，即衣原体附着和进入的位置。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处理的细胞对人类衣原体菌株感染的敏感性都显著降低。此外，我们还证明在衣原体进入阶段对Ezrin DN细胞的感染有抑制作用。我们推测Ezrin的沙眼衣原体特异性Tyr-P可能与一种未知的病原体进入物种特异性机制有关，该机制涉及衣原体特异性配体(S)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。沙眼表现出不同的临床症状，从轻度和自限性到严重的炎症性疾病。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预后中的作用，我们使用基于微阵列的比较基因组测序方法分析了代表三种主要沙眼血清型的四株沙眼菌株的基因组。除OmpA外，沙眼病毒株主要在一个非常小的基因子集(22个)上存在差异。这些微妙的遗传变异表现在毒力方面的深刻差异，如体外生长速度、爆裂大小、斑块形态和干扰素-伽马敏感性，但最重要的是毒力方面的差异，如非人类灵长类动物的眼部感染。这些发现首次确定了与不同沙眼菌株致病性相关的基因。

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.