NOVEL CELLULAR & GENETIC ANALYSES USING A NEW CHLAMYDIA PENETRANT PEPTIDE

描述(申请人提供)：这项建议的目的是阐明沙眼衣原体通过减弱感染宿主细胞中依赖干扰素的信号来逃避免疫反应的机制，并开发一种新的反向遗传策略来研究沙眼衣原体的发病机制和生物学。 性传播沙眼衣原体感染，主要由D-F血清型引起，是一个主要的公共卫生问题，对女性生育和新生儿健康产生不利影响。尽管采取了包括教育、筛查和治疗在内的重大公共卫生干预措施，但美国报告的病例在2006年达到100万例，而且还在继续上升，导致每年的医疗保健费用&gt；24亿美元。在70%的女性和30%的男性中，无症状感染可能需要几个月到几年的时间才能解决，从而加剧了沙眼衣原体的传播。对再次感染的短暂保护加剧了传播。对人类上皮细胞的体外研究和对小鼠隐孢子虫的动物研究已经确定了干扰素？作为主要的抗衣原体调停剂。在体内，虽然在感染的人子宫内膜中发现了高水平的干扰素α，但临床数据表明沙眼衣原体可以逃避免疫反应。 我们开发了一种创新的方法，使用一种新的穿透性多肽(CPP)来标记沙眼衣原体基本体(EB)。我们的方法允许从暴露于沙眼衣原体的混合细胞池中分离出受感染的宫颈上皮细胞的纯群体。对纯种群的分析表明，沙眼衣原体可减弱依赖干扰素的吲哚胺-2，3-双加氧酶(IDO1)的表达。衰减允许沙眼衣原体，一种色氨酸营养缺陷型，改善IDO1对细胞内色氨酸耗竭的影响。这个应用程序的第一个目标是了解衣原体如何阻止宿主细胞依赖干扰素？的信号。了解这一机制将有助于开发针对沙眼衣原体免疫逃避的新策略。 缺乏易于应用的基因工具来操纵沙眼衣原体，限制了研究沙眼衣原体驱动免疫逃避和致病机制的能力。这项申请通过提出一种创新的方法来解决这一重大缺陷，该方法使用相同的CPP来运输抑制特定沙眼衣原体基因表达的货物。当开发和验证时，我们的策略将允许强大的反向遗传方法来分子剖析沙眼衣原体的毒力决定因素。 我们的这些新型CPP研究将显著改变目前的研究范式，并通过以下方式彻底改变衣原体研究的方法：1)建立一种强大的方法来阐明沙眼衣原体以前未知的免疫逃避策略；以及2)首次提供一种强大的反向遗传技术来剖析特定细菌基因在发育周期和发病机制中的作用。 公共卫生相关性：性传播沙眼衣原体感染是一个重要的公共卫生问题，因为慢性病会造成毁灭性的生殖后果。人们对这种生物如何在人类女性生殖道中生长或适应其自然栖息地知之甚少，这是我们应用的重点。我们在这里提出的研究可能有助于阐明为什么感染在一些女性身上需要这么长时间才能清除，为什么在其他女性身上它可能会变成慢性感染。

DESCRIPTION (provided by applicant): The objectives of this proposal are to elucidate the mechanism by which Chlamydia trachomatis evades the immune response by attenuating IFN?-dependent signaling in infected host cells, and to develop a new reverse genetic strategy to study C. trachomatis pathogenesis and biology. Sexually transmitted C. trachomatis infections, primarily caused by serovars D-F, are a major public health concern with adverse effects on female fertility and neonatal health. Despite significant public health interventions including education, screening and treatment, reported cases n the US reached 1 million in 2006 and continue to rise, resulting in annual health care costs >$2.4 billion. Asymptomatic infections in >70% of women and >30% of men, can take months to years to resolve, and thereby compounding transmission of C. trachomatis. Transmission is exacerbated by short-lived protection to re-infection. In vitro studies in human epithelial cells, and animal studies using C. muridarum, have demarcated IFN? as a major anti-chlamydial mediator. In vivo, although elevated IFN??is found in the infected human endocervix, clinical data indicate that C. trachomatis can evade immune responses. We have developed an innovative approach using a novel penetrant peptide (CPP) to label C. trachomatis Elementary Bodies (EB). Our approach has permitted the isolation of pure populations of infected endocervical epithelial cells, from mixed pools of cells exposed to C. trachomatis. Analyses of pure populations has revealed that C. trachomatis attenuates IFN?-dependent expression of indoleamine-2,3-dioxygenase (IDO1). Attenuation permits C. trachomatis, a tryptophan auxotroph, to ameliorate the effect of intracellular tryptophan depletion by IDO1. The first goal of this application is to understand how Chlamydia blocks host-cell IFN?-dependent signaling. Understanding this mechanism will permit development of new strategies that target immune evasion by C. trachomatis. The paucity of easily applicable genetic tools to manipulate C. trachomatis has limited the capacity to investigate mechanisms employed by C. trachomatis that drive immune evasion and pathogenesis. This application addresses this significant deficiency by proposing an innovative approach using the same CPP to carry cargoes that repress expression of specific C. trachomatis genes. When developed and validated, our strategy will permit robust reverse genetic approaches to molecularly dissect virulence determinants of C. trachomatis. These studies with our novel CPP will significantly shift current paradigms and revolutionize approaches in Chlamydia research by: 1) Establishing a robust method to elucidate previously unidentified immune evasion strategies used by C. trachomatis; and 2) Providing, for the first time, a robust reverse genetic technique to dissect the role of specific bacterial genes in the developmental cycle and pathogenesis. PUBLIC HEALTH RELEVANCE: Sexually transmitted Chlamydia trachomatis infection is an important public health issue because of the devastating reproductive consequences of chronic disease. Little is known about how this organism grows in, or adapts to its natural habitat in the human female genital tract, and this is the focus of our application. The studies we propose here may help to elucidate why infection takes so long to clear in some women, and why it may become chronic in others.