Over four million men, women and infants suffer from chlamydial genital infection annually. Women bear a special burden because of their increased risk of adverse reproductive consequences. C. trachomatis is responsible for 25-50% of the estimated one million cases of pelvic inflammatory disease/year. The majority of cases of tubal disease associated with chlamydial salpingitis appear to result from chronic, subclinical infection. Since the disease process is not understood, our ultimate goal is to elucidate the basic biology of chlamydial growth in human epithelial cells in order to learn more about the mechanism of persistent infection. The in vitro model system we are using consists of estrogen-exposed, polarized human endometrial gland epithelial cells (HEGEC). The four Specific Aims are to: identify the chlamydial adhesin(s) and the human epithelial receptor(s), and to devise a system to reintroduce into chlamydiae altered genetic material for substantiating the identity of the proposed adhesin genes as well as for opening up exciting possibilities for exploring gene regulation. Two functional methods have been used to screen the C. trachomatis genomic library, generated with pUC19. The whole cell assay detects E. coli recombinants bound to the apical surfaces of HEGEC. On the assumption that a chlamydial adhesin may not properly fold and be expressed on the surface of E. coli, the second screening method tests lysates of recombinants for binding to 35S met/cys-labeled (i) CHAPS- solubilized epithelial plasma membrane and (ii) """"""""right-side out"""""""" membrane vesicles generated by vinblastin/cytochalasin or phenylarsine oxide treatment. Several recombinants, positive in both screening methods, show promise in that they express chlamydia-specific proteins in their outer membranes and their binding characteristics parallel those of chlamydiae.
In Specific Aim 4, the same radiolabeled apical membranes will be bound to infectious chlamydiae, the complex immuno-precipitated with anti-chlamydial antibody, subjected to SDS-PAGE, autoradiography and electroblot for preliminary identification of epithelial receptors. Chlamydial promoters from the 7.5kb plasmid, identified via use of promoterless vectors and active in both E. coli and replicating C. trachomatis (RB) in vitro, will be cloned for construction of shuttle vectors. These vectors will serve to reintroduce genetic material into infectious as well as actively growing chlamydiae in infected host cells by electroporation or microinjection.
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