At the same time, chlamy dial strains isolates that are either deficient in the plasmid or carry mutated plasmids have been identified, suggesting that there might CHIR99021 molecular weight also be host immune selection pressure against the plasmid encoded antigens and the plasmid encoded function can be compensated by genes proteins encoded elsewhere. To understand the functions of the plasmid encoded proteins, we tested whether the plasmid proteins are expressed and immunogenic during C. trachomatis infection in humans in the current study. Since it is difficult to directly detect chlamydial proteins and evaluate chlamydial protein immunogenicity in humans, we detected the recognition of chlamydial fusion proteins by human antibodies in ELISA as an indirect indicator for both chlamydial protein expression and immunogenicity in individuals with C.
trachomatis infec tion. We found that the plasmid encoded Inhibitors,Modulators,Libraries 8 proteins were recognized by one or more human serum samples, sug gesting that they were all made during human infection. Importantly, we found that pORF5 was the most immunodominant antigen among the 8 plasmid proteins and as dominant as CPAF, a chlamydial genome encoded protease factor known to be immunodominant and secrete into host cell cytosol. Indeed, the pgp3 fusion pro tein purified human IgG detected the endogenous pgp3 in the cytosol of C. trachomatis infected cells in addition to its intra inclusion localization.
Inhibitors,Modulators,Libraries Interestingly, the human antibody recognition of pgp3 but not CPAF as highly con formation dependent since linearizing or denaturing either pgp3 fusion protein or the endogenous protein blocked the human antibody recognition of pgp3 while similar treatments to CPAF still permitted a significant recognition of CPAF by the same human antibodies. These observations Inhibitors,Modulators,Libraries have not only demonstrated that the fusion protein ELISA is a relevant experimental Inhibitors,Modulators,Libraries system for analyzing antibody responses to chlamydial infection in humans, but also more importantly, provided useful information for further developing pgp3 as a diagnostic reagent and or vaccine candidate. Results 1. Human antibody recognition of C. trachomatis plasmid proteins To determine whether the plasmid encoded proteins are expressed and immunogenic during C. trachomatis infec tion in humans, the 8 pORFs were expressed as GST fusion proteins and the fusion proteins were reacted with 15 human antisera in an ELISA.
Each of the 8 plasmid fusion proteins were positively recognized by at least one antiserum, suggesting that the plasmid proteins are all expressed Inhibitors,Modulators,Libraries during human infec tion. However, there is a great variation in both the anti body binding frequency and titer among the plasmid proteins. The plasmid protein pORF5 was recognized by all fifteen human antisera, pORF7 by three, under pORF1, 4 6 by two and pORF2, 3 8 by one only. Five C.