Identification of the chlamydial adhesin and receptor

描述（由申请人提供）：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。衣原体进入一个有利的细胞内生态位是其生存的绝对要求，因此，据推测，这种生物体已经开发出一种非常有效的进入宿主细胞的手段。衣原体是如何进入宿主细胞的尚不清楚，而对衣原体入侵机制的认识明显缺乏，这是因为衣原体的遗传系统不发达。最近分离的中国仓鼠卵巢（CHO）突变体克隆，从随机化学诱变的CHO-K1细胞系和选择的感染抗性的抒情血清型L2，表明二级受体的要求。该受体与衣原体原体的结合是硫酸乙酰肝素结合的下游。这项拨款提案将集中在两个研究领域，利用这种新的细胞系，并依赖于旨在保护病原体和宿主表面结构的实验方法。首先，该细胞系将用于使用cDNA互补、功能结合测定和质谱法鉴定衣原体受体。候选物的验证将涉及引入基因的野生型拷贝以恢复易感性表型。此外，将在易感细胞中产生基因的敲除等位基因以重现抗性表型。第二个研究目标将集中在衣原体配体的质谱鉴定。试验将利用受体对衣原体粘附素的天然亲和力。将确定其他血清型中是否存在性病性肉芽肿（LGV）血清型L2粘附素的同源物，并研究是否存在可能导致生物学差异（如组织嗜性）的遗传多态性。这两个研究目标的结果将与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有助于我们了解衣原体的发病机制，并导致合理的合理的战略，在打击感染，这种临床相关的有机体。

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.