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- and IdeR-specific regulons in C. diphtheriae by genetic, proteomic and microarray-based transcriptional analyses, identifying the importance of DtxR- and IdeR regulated genes in the pathogenesis of diphtheria and tuberculosis, 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 a . 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 antigens to the mucosal immune system in a highly immunogenic manner.
Selected Publications
Jobling, M.G., and R.K. Holmes. 2002. Mutational
analysis of ganglioside GM1-binding ability, pentamer
formation, and epitopes of cholera toxin B (CTB)
subunits and CTB/heat-labile enterotoxin B subunit
chimeras. Infect. Immun. 70:1260-1271.
Qian, Y, J.H. Lee, and R.K. Holmes. 2002.
Identification of a DtxR-regulated operon that is
essential for siderophore-dependent iron uptake in
Corynebacterium diphtheriae. J. Bacteriol. 184:4846-4856.
Oram, D.M., A. Avdalovic, and R.K. Holmes. 2002.
Construction and characterization of transposon
insertion mutations in Corynebacterium diphtheriae
that affect expression of the diphtheria toxin repressor
(DtxR). J. Bacteriol. 184:5723-5732.
Oram, D.M., A. Avdalovic, and R.K. Holmes. 2004. Analysis of genes that encode DtxR-like transcriptional regulators in pathogenic and saprophytic corynebacterial species. Infect. Immun. 72 :1885-1895.
O'Neal, C.J., E.I. Amaya, M.G. Jobling, R.K. Holmes, and W.G.J Hol. 2004. Crystal structures of an intrinsically active cholera toxin mutant yield insight into the toxin activation mechanism. Biochemistry 43 :3772-3782.
Tripet, B., M.W. Howard, M.G. Jobling, R.K. Holmes, K.V. Holmes and R.S. Hodges. 2004. Structural characterization of the SARS-coronavirus spike S fusion protein core. J. Biol. Chem. 279 :20836-20849.
Wisedchaisri, G., R.K. Holmes, and W.G.J. Hol. 2004. Crystal structure of an IdeR-DNA complex reveals a conformational change in activated IdeR for base-specific interactions. J. Mol. Biol. 342 :1155-1169.
Teter, K., M.G. Jobling, and R.K. Holmes. 2004. Vesicular transport is not required for the cytoplasmic pool of cholera toxin to interact with the stimulatory alpha subunit of the heterotrimeric G protein. Infect. Immun.: (In press).
Li, X., J.L. Erbe, C.V. Lockatell, D.E. Johnson, M.G. Jobling, R.K. Holmes, and H.L.T. Mobley. 2004. Use of a translationsal fusion of the mrpH fimbrial adhesin binding domain with cholera toxin A2 domain, coexpressed with cholera toxin B subunit, as an intranasal vaccine to prevent experimental urinary tract infection by Proteus mirabilis . Infect. Immun.: (In press).
Holmes, R.K. 2005. Diphtheria and other corynebacterial infections, p. 832-837. In D.L. Kasper et al ., Harrison's Principles of Internal Medicine , 16th Edition. McGraw-Hill , New York .
Gilbert, J., J. Dahl, C. Riney, C. Cui, R. Holmes, W. Lencer, and T. Benjamin. Ganglioside GD1a restores infectability to mouse cells lacking functional receptors for polyoma virus. J. Virol.: (In press).
Chou, C.J., G. Wisedchaisri, R.K. Holmes, W.G.J. Hol, and C.C. Beeson. Functional studies of the Mycobacterium tuberculosis iron-dependent regulator. J. Biol. Chem: (In press).
Gorbea, C., G.M. Goellner, K. Teter, R.K. Holmes, and M. Rechsteiner. Characterization of mammalian Ecm29, a 26S proteasome-associated protein that localizes to the nucleus and membrane vesicles. J. Biol. Chem.: (In press).
Oram, D.M., L.M. Must, J.K. Spinler, E.M. Twiddy and R.K. Holmes. Repressor activity and antigenicity of the iron dependent transcriptional regulators from Corynebacterium diphtheriae and Mycobacterium tuberculosis . FEMS Microbiol. Lett.: (In press).
Jobling, M.G., and R.K. Holmes. Activation of second messenger pathways by ADP-ribosylation of G-proteins. In T. Proft, ed., Microbial Toxins: Molecular and Cellular Biology . Horizon Bioscience, Wymondham , UK . (In press).
Oram, D.M., and R.K. Holmes. Toxins that interfere with protein synthesis. In T. Proft, ed., Microbial Toxins: Molecular and Cellular Biology . Horizon Bioscience, Wymondham , UK . (In press).
Spinler, J.K., and R.K. Holmes. Corynebacterium diphtheriae - molecular detection of diphtheria toxin. In J. Fuchs and M. Podda, eds., Encyclopedia of Medical Genomics and Proteomics . Marcel Dekker, Inc., New York . (In press).
CU Faculty PubMed Publications |
|
All PubMed Publications |
Back to Top
