Genital Chlamydia trachomatis serovars D-K are responsible for epidemic sexually transmitted infections in the USA, affecting over 4 million men, women and infants per year. In women, without treatment, chlamydiae may spread canalicularly from the endocervical canal to the endometrium (endometritis), and the fallopian tubes (salpingitis) - the constellation of which is defined as pelvic inflammatory disease. As a result of tubal scarring and/or impaired ovum transportation, ectopic pregnancy and infertility can occur. Clearly, chlamydial diseases constitute significant primary, secondary and tertiary health care concerns in which women bear a special burden because of their increased risk of adverse reproductive consequences. Our ultimate goal is to understand the basic biology of chlamydial growth in a polarized, hormone-responsive human genital epithelial cell model system in order to learn more about the mechanism of primary and persistent infection.
In Specific Aim 1, we shall analyze the function of 3 chlamydial gene products - OrfA, GrpE, and DnaK - in C. trachomatis adherence by (i) assessment of attachment of engineered subclones to determine which ORF(s) are responsible for the attachment phenotype, and (ii) binding and chlamydial competition/inhibition analyses using the recombinant proteins, purified by non-denaturing methods, and monospecific antibodies against these proteins. To test the authenticity of gene expression and its relationship to functional adherence will require reintroduction of the cloned chlamydial DNA into C. trachomatis.
In Specific Aim 2, the promoter region(s) of orfA-grpE-dnaK will be evaluated for use in our tested shuttle vector. In addition, we shall investigate a derivative of the OMEGON transposon, containing a promoterless CAT::luciferase reporter gene fusion, for promoter detection and transposition events in chlamydiae following electroporation.
In Specific Aim 3, infectious chlamydiae and recombinant proteins, reconstituted into liposomes, will be bound to isolated, radiolabeled """"""""right-side-out"""""""" apical membrane vesicles, generated by vinblastin/cytochalasin D or phenylarsine oxide; the complex will be immunoprecipitated, subjected to SDS-PAGE, autoradiography and electroblot for preliminary identification of epithelial receptors. Finally, the pH of SNAFL-conjugated chlamydiae in early endosomes - in unexposed versus ouabain- and bafilomycin-exposed infected host cells - will be determined with dual channel scanning confocal fluorescence microscopy.
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