Identification of the chlamydial adhesin and receptor

描述（由申请人提供）：衣原体是专性细胞内细菌，可引起人类广泛的疾病，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需要，因此，人们推测这种生物已经发展出一种非常有效的进入宿主细胞的方法。衣原体如何进入宿主细胞尚不清楚，对衣原体入侵机制的明显缺乏源于其不发达的遗传系统。最近分离的中国仓鼠卵巢（CHO）突变克隆是由CHO- k1细胞系随机化学诱变和lyric血清型L2对感染的抗性选择产生的，表明需要一个次级受体。衣原体初级体与该受体的结合在硫酸肝素结合的下游。这项拨款提案将集中在两个研究领域，利用这种新型细胞系的优势，并依赖于旨在保存病原体和宿主表面结构的实验方法。首先，该细胞系将使用cDNA互补、功能结合测定和质谱法鉴定衣原体受体。候选者的验证将涉及引入基因的野生型拷贝以恢复易感表型。此外，基因的敲除等位基因将在易感细胞中产生，以再现抗性表型。第二个研究目标将集中在衣原体配体的质谱鉴定。检测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有性肉芽肿（LGV）血清型L2粘附素的同源物，并研究可能导致组织趋向性等生物学差异的遗传多态性的存在。这两个研究目标的发现将与LGV和非LGV血清型之间的生物学差异相关。从这些研究领域中获得的知识应该有助于我们对衣原体发病机制的理解，并为对抗这种临床相关生物的感染提供合理的策略。

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.