Identification of the chlamydial adhesin and receptor

描述(申请人提供)：衣原体是一种专性细胞内细菌，可引起人类多种疾病，如沙眼、不孕症、肺炎和可能的动脉粥样硬化。衣原体进入有利的细胞内生态位是其生存的绝对要求，因此，人们推测这种生物已经开发出一种非常有效的进入宿主细胞的方法。衣原体是如何进入宿主细胞的尚不清楚，这种对衣原体入侵机制的明显缺乏源于其未开发的遗传系统。最近分离的中国仓鼠卵巢(CHO)突变克隆，由CHO-K1细胞的随机化学诱变和对L2裂解血清型感染的抗性筛选而来，表明需要一个二级受体。衣原体基本体与该受体的结合位于硫酸乙酰肝素结合的下游。这项拨款提案将侧重于两个领域的研究，这两个领域利用了这种新的细胞系，并依赖于旨在保护病原体和宿主的表面结构的实验方法。首先，该细胞系将用于通过cDNA互补、功能结合分析和质谱学鉴定衣原体受体。对候选基因的验证将包括引入该基因的野生型副本，以恢复易感表型。此外，该基因的敲除等位基因将在敏感细胞中产生，以概括抗性表型。第二个研究目标将集中在利用质谱学鉴定衣原体配体。检测将利用衣原体粘附素受体的天然亲和力。将确定在其他血清型中是否存在性性淋巴肉芽肿(LGV)血清L2粘附素的同源物，并将调查可能导致生物学差异的遗传多态的存在，如组织嗜性。这两个研究目的的结果将与LGV和非LGV血清型之间的生物学差异相关。从这些研究领域获得的知识应该有助于我们理解衣原体的发病机制，并导致合理合理的策略来对抗这种具有临床相关性的微生物的感染。

DESCRIPTION (provided by applicant): Chlamydiae are obligate intracellular bacteria that cause a wide spectrum of disease in humans, such as trachoma, infertility, pneumonia, and possibly atherosclerosis. Entry of chlamydia into a favorable intracellular niche is an absolute requirement for its survival, and thus, it has been speculated that this organism has developed a very efficient means of entering a host cell. How chlamydia enters the host cell is unknown, and this glaring lack of knowledge of the mechanisms involved in chlamydial invasion stems from its undeveloped genetic system. A recently isolated Chinese hamster ovary (CHO) mutant clone that arose from random chemical mutagenesis of the CHO-K1 cell line and selection of resistance to infection by the lyric serovar L2, indicate the requirement for a secondary receptor. Engagement of this receptor by chlamydial elementary bodies is downstream of binding of heparan sulfate. This grant proposal will focus on two areas of research that takes advantage of this novel cell line and relies on experimental approaches that are designed to preserve the surface structures of both pathogen and host. Firstly, this cell line will be used to identify the chlamydial receptor using cDNA complementation, functional binding assays, and mass spectrometry. Verification of candidates will involve the introduction of the wild type copy of the gene to restore the susceptibility phenotype. In addition, knockout alleles of the gene will be created in susceptible cells to recapitulate the resistance phenotype. The second research aim will focus on the identification of chlamydial ligands by mass spectrometry. Assays will take advantage of the natural affinity of the receptor for the chlamydial adhesin. Existence of homologs of the lympogranuloma venereum (LGV) serovar L2 adhesin in other serovars will be determined, and the presence of genetic polymorphisms that may contribute to the biological differences, such as tissue tropism, will be investigated. Findings from the two research aims will be correlated with the biological differences between LGV and non-LGV serovars. Knowledge gained from these research areas should contribute to our understanding of chlamydial pathogenesis and lead to rationally sound strategies in combating infection by this clinically relevant organism.