Neisseria gonorrhoeae produces conserved and unique heat shock proteins, or stress proteins, in response to heat, nutrient deprivation, and anaerobiasis. As the gonococcus is exposed to wide variations in pH in the various niches it infects in vivo, it may produce pH-regulated stress proteins as well. The objective of this proposal is to identify proteins synthesized by N. gonorrhoeae in response to pH stress that are recognized by convalescent serum from patients with gonorrhea. Experimental protocols were chosen as they have been used successfully to describe stress proteins in other pathogenic bacteria. Furthermore, these protocols are quite straightforward, enabling undergraduate participation. Standard one- and two-dimensional electrophoretic analyses will be used to detect induction and repression of cytoplasmic and outer membrane proteins in response to pH changes in vitro. Then, immunoblots of separated outer membrane proteins from organisms grown at different pHs will be probed with immune serum from patients with gonorrhea. Results of immunoblotting experiments will indicate whether pH-induced outer membrane proteins are expressed during gonococcal infection, and if they are immunodominant. Finally, gonococcal pH-regulated proteins recognized by patient immune serum will be purified, blotted onto polyvinylidene difluoride membranes, and the N-terminal 20-30 amino acids sequenced. These sequences will be compared to stress protein amino acid sequences from other organisms. These data will enable classification of gonococcal pH-regulated proteins as members of highly conserved stress protein families, or as unique to the gonococcus. Unique surface-exposed stress proteins would be excellent candidates for a gonococcal vaccine. This research will contribute toward an understanding of the role of gonococcal stress proteins in pathogenesis and the host immune response.
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