Immunity to Chlamydial Infection

这项工作的目的是了解沙眼衣原体感染如何在人类中持续存在，以及持续感染如何导致严重的感染后后遗症，如失明和不孕。为了实现这一目标，我们对参考实验室菌株和临床分离株进行了功能基因组研究，重点放在病原体的可塑性区域(PZ)中的基因；包含细胞毒素基因、磷脂酶D基因的一个小的多态区域，以及编码色氨酸生物合成途径的最后两个酶(色氨酸B和色氨酸A)的色氨酸操纵子(TrpRBA)。我们发现，感染器官趋向性不同的实验室参考沙眼衣原体菌株与病原体TrpA合成酶的失活突变有关。我们的功能基因组研究发现，为参考血清型建立的范例也适用于临床分离株；具体地说，所有测试的眼沙眼沙眼病毒分离株都有合成酶失活突变，而所有生殖器分离株都编码一种功能性酶；这表明合成酶在衣原体性病的发病机制中具有功能作用。此外，功能性酶活性通过吲哚拯救机制与干扰素-γ抗性直接相关。因此，生殖器菌株存在很大的选择压力，以维持能够利用吲哚进行色氨酸生物合成的功能合酶。从生殖道分离的眼部血清型(B血清型)被发现具有功能性合成酶，这一事实为这种联系提供了更有说服力的证据。这些结果表明，衣原体生殖器株和人类宿主之间存在着重要的宿主-寄生虫关系，这种关系决定了感染的器官嗜性和疾病的病理生理学。我们认为，这种关系涉及阴道微生物菌群的组成部分产生吲哚，使衣原体逃脱干扰素-伽马介导的根除，从而建立持续感染。我们已经证明，衣原体使用色氨酸A和B来避免干扰素-γ对宿主上皮细胞的影响，这是一种允许病原体避开宿主防御并建立持久性的机制。到目前为止，使用传统方法的疫苗接种，如靶向衣原体重组蛋白或DNA编码蛋白，未能在生殖器粘膜诱导保护性抗衣原体免疫。我们的结论是，衣原体抗原的复杂性、其复杂的生命周期以及对粘膜上皮细胞的趋向性对传统疫苗的产生构成了巨大的挑战。因此，我们目前的重点是生产减毒活疫苗株。这是通过克隆选择不能合成色氨酸合成酶的变体来实现的，色氨酸合成酶是病原体在存在宿主防御的情况下持续存在于上皮细胞的能力的关键酶。在体外生长对干扰素-伽马高度敏感的克隆和具有trpRBA基因突变的克隆将在体内进行临床前灵长类感染模型的体内测试，以了解生长和病理特性的减弱。这些研究将产生对开发能够预防或控制衣原体引起的人类性传播疾病的高效和安全疫苗的重要信息。

The purpose of this work is to understand how C. trachomatis infection persists in humans and how persistence contributes to serious post-infection sequelae such as blindness and infertility. To accomplish this goal we have performed functional genomic studies on both reference laboratory strains and clinical isolates focusing on the genes that reside in the pathogen's plasticity zone (PZ); a small polymorphic region that contains cytotoxin genes, phospholipase D genes, and the tryptophan operon (trpRBA) that encodes the last two enzymes (Trp B and Trp A) of the tryptophan biosynthetic pathway. We found that laboratory reference strains of Chlamydia trachomatis differing in infection organotropism correlated with inactivating mutations in the pathogen's Trp A synthase. Our functional genomic studies find that the paradigm established for reference serovars also applies to clinical isolates; specifically, all ocular trachoma isolates tested have inactivating mutations in the synthase, whereas all genital isolates encode a functional enzyme; implicating a functional role for the synthase in the pathogenesis of chlamydial STDs. Moreover, functional enzyme activity was directly correlated to IFN-gamma resistance through an indole rescue mechanism. Hence, a strong selective pressure exists for genital strains to maintain a functional synthase capable of using indole for tryptophan biosynthesis. The fact that ocular serovars (serovar B) isolated from the genital tract were found to possess a functional synthase provided further persuasive evidence of this association. These results argue that there is an important host-parasite relationship between chlamydial genital strains and the human host that determines organotropism of infection and the pathophysiology of disease. We believe that this relationship involves the production of indole by components of the vaginal microbial flora, allowing chlamydiae to escape IFN-gamma mediated eradication and thus establish persistent infections. We have shown that Chlamydiae use Trp A and B to avoid the effect of IFN-gamma on host epithelial cells, a mechanism that allows the pathogen to avoid host defense and establish persistent. To date, vaccination using conventional approaches such as targeted chlamydial recombinant proteins or DNA encoding proteins have failed in eliciting a protective anti-chlamydial immunity at the genital mucosae. Our conclusions are that the antigenic complexity of the chlamydiae, its complex life cycle, and tropism for mucosal epithelial cells constitute overwhelming challenges for the generation of a conventional vaccine. Consequently, we are currently focusing on the generation of live-attenuated vaccine strains. This is being accomplished by the clonal selection of variants that are incapable of synthesizing tryptophan synthase, a key enzyme in the ability of the pathogen to persist in epithelial cells in the presence host defense. Clones whose growth is highly sensitive to IFN-gamma in vitro and that have mutations in the trpRBA genes will be tested in in vivo in pre-clinical primate models of infection for attenuated growth and pathological properties. These studies should yield information important to the development of a highly efficacious and safe vaccine capable of preventing or controlling chlamydial caused STDs in humans.