Our laboratory uses molecular methods to study the genetics and regulation of bacterial protein toxins, their roles in pathogenesis of infectious diseases, and their uses for vaccine development. The toxins and regulatory proteins currently being studied include diphtheria toxin from Corynebacterium diphtheriae, cholera toxin from Vibrio cholerae, type I and type II heat-labile enterotoxins from Escherichia coli, the diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae, and the homologous iron-dependent regulator (IdeR) from Mycobacterium tuberculosis.
Diphtheria toxin causes the serious manifestations of diphtheria. DtxR is an iron-activated regulatory protein that negatively controls synthesis of diphtheria toxin and other putative virulence factors of C. diphtheriae. We cloned the dtxR gene from C. diphtheriae and the ideR gene from M. tuberculosis. We purified and characterized DtxR and IdeR in vitro, identified the consensus sequence for DtxR-regulated operators, and determined the crystal structures of DtxR as an apo-repressor, a holo-repressor and a holo-repressor in complex with DNA. Current studies in our laboratory focus on identifying the molecular basis for interactions of DtxR and IdeR with DNA, characterizing the DtxR-specific regulon in C. diphtheriae by genetic, proteomic and microarray-based transcriptional analyses, identifying the importance of DtxR-regulated genes in the pathogenesis of diphtheria, and developing compounds that modulate the biological activities of IdeR as potentially novel therapeutic agents against tuberculosis.
Cholera toxin and the closely related E. coli heat-labile enterotoxins cause the secretory diarrhea of cholera and traveler’s diarrhea, respectively. These toxins consist of a pentameric B subunit that binds to ganglioside receptors on target cells and a monomeric A subunit that activates adenylate cyclase by ADP-ribosylation of the regulatory protein Gs. Current studies in our laboratory are analyzing the structure and function of these enterotoxins in vitro and the mechanisms for their uptake and trafficking in target cells. CT is also a potent mucosal antigen and immunomodulatory agent. We have developed methods to make cholera toxin-like chimeric proteins in which the enzymatic domain of the native A polypeptide is replaced by various other microbial protein antigens. We are testing them as experimental vaccines to determine whether they can present the foreign antigenic domains to the mucosal immune system in a highly immunogenic manner.