NOVEL CELLULAR & GENETIC ANALYSES USING A NEW CHLAMYDIA PENETRANT PEPTIDE

描述（由申请方提供）：本提案的目的是阐明沙眼衣原体通过减弱IFN？-γ逃避免疫应答的机制。依赖的信号转导，并开发一种新的反向遗传策略来研究C.沙眼的发病机制和生物学。 性传播C。主要由血清型D-F引起的沙眼感染是一个主要的公共卫生问题，对女性生育力和新生儿健康有不利影响。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，并继续上升，导致每年的医疗保健费用> 24亿美元。在>70%的女性和>30%的男性中，无症状感染可能需要数月至数年才能解决，从而加剧了C。 沙眼对再次感染的保护期短，会加剧传播。在人类上皮细胞中的体外研究和使用C. muridarum，已划定干扰素？作为主要的抗衣原体介质。在体内，虽然IFN？临床资料表明，C.沙眼可以逃避免疫反应。 我们已经开发了一种创新的方法，使用一种新的渗透肽（CPP）标记C。 沙眼原体（EB）。我们的方法可以从暴露于C.沙眼纯种群分析表明，C.沙眼使IFN？减弱吲哚胺-2，3-双加氧酶（IDO 1）的依赖性表达。衰减允许C。沙眼衣原体，色氨酸营养缺陷型，以改善IDO 1对细胞内色氨酸消耗的影响。本申请的第一个目标是了解衣原体如何阻断宿主细胞IFN？-依赖信号了解这一机制将有助于开发针对C.沙眼 缺乏易于应用的遗传工具来操纵C。沙眼衣原体的研究限制了研究沙眼衣原体机制的能力。导致免疫逃避和发病的沙眼。本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来运载抑制特定C的表达的货物。沙眼相关基因当开发和验证，我们的策略将允许强大的反向遗传方法，从分子上剖析C。 沙眼 这些研究与我们的新的CPP将显着改变当前的范式和革命性的方法在衣原体研究：1）建立一个强大的方法来阐明以前未确定的免疫逃避策略使用的衣原体。沙眼;和2）首次提供了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，因为慢性疾病的破坏性生殖后果。关于这种生物如何生长或适应其在人类女性生殖道中的自然栖息地，我们知之甚少，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需要这么长的时间来清除一些妇女，为什么它可能成为慢性在其他人。

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.