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.
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