The long term goals of this project are to determine molecular mechanisms for virulence regulation in pathogenic bacteria and to develop new methods to treat bacterial infections. The investigators will study Corynebacterium diphtheriae, a paradigm for toxin-mediated bacterial infections, and Mycobacterium tuberculosis, a prototype for intracellular bacterial infections. These very different bacterial pathogens produce closely related, iron-activated, global regulatory proteins that govern virulence: the diphtheria toxin repressor (DtxR) and the iron-dependent regulator (IdeR), respectively. The investigators will determine the molecular basis for function of DtxR, IdeR and the homologous regulator SirR from Staphylococcus epidermidis. The investigators will use structure-based design to develop new antimicrobial drugs called """"""""super-activators"""""""" that will target DtxR, IdeR or their homologs; activate them by iron-independent mechanisms; and inhibit production of virulence factors that are negatively regulated by iron- and DtxR-related repressors. The development of IdeR as a novel target for antimicrobial therapy could address the urgent global need for improved treatment of tuberculosis. The investigators will characterize the genes and gene products that are iron-regulated and under control of DtxR and IdeR, both to provide new insights into the pathogenesis of diphtheria and tuberculosis and for development of additional classes of antimicrobial agents.
Specific Aim I will analyze structure and function of DtxR, IdeR and SirR. The investigators will investigate the molecular basis for repressor-operator interactions, for iron-independent super-repressor activity, and for domain function in biological activity of these regulatory proteins.
Specific Aim 2 will characterize the DtxR and IdeR regulons in C. diphtheriae and M. tuberculosis. The investigators will develop an allelic exchange system for C. diphtheriae, characterize the DtxR and IdeR regulons by proteomic and molecular genetic methods, assess physiological functions of DtxR and IdeR domain 3, and investigate atypical phenotypes among clinical isolates of C. diphtheriae.
Specific Aim 3 will develop super-activators of DtxR and IdeR by structure-based design. The investigators will design combinatorial peptide libraries, test them for super-activator function, identify individual peptides with activity, determine the structural basis for that activity, and develop better super-activators by iterative application of these methods. The investigators will also use molecular genetic methods to identify novel mechanisms for super-repressor activity and new lead compounds for development as tools against these bacterial infections.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Program Officer
Klein, David L
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University of Colorado Denver
Schools of Medicine
United States
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Jobling, Michael G (2016) The chromosomal nature of LT-II enterotoxins solved: a lambdoid prophage encodes both LT-II and one of two novel pertussis-toxin-like toxin family members in type II enterotoxigenic Escherichia coli. Pathog Dis 74:
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Qian, Yilei; Lee, John H; Holmes, Randall K (2002) Identification of a DtxR-regulated operon that is essential for siderophore-dependent iron uptake in Corynebacterium diphtheriae. J Bacteriol 184:4846-56
Feese, M D; Ingason, B P; Goranson-Siekierke, J et al. (2001) Crystal structure of the iron-dependent regulator from Mycobacterium tuberculosis at 2.0-A resolution reveals the Src homology domain 3-like fold and metal binding function of the third domain. J Biol Chem 276:5959-66
Lee, J H; Holmes, R K (2000) Characterization of specific nucleotide substitutions in DtxR-specific operators of Corynebacterium diphtheriae that dramatically affect DtxR binding, operator function, and promoter strength. J Bacteriol 182:432-8
Pohl, E; Holmes, R K; Hol, W G (1999) Crystal structure of the iron-dependent regulator (IdeR) from Mycobacterium tuberculosis shows both metal binding sites fully occupied. J Mol Biol 285:1145-56
Pohl, E; Holmes, R K; Hol, W G (1999) Crystal structure of a cobalt-activated diphtheria toxin repressor-DNA complex reveals a metal-binding SH3-like domain. J Mol Biol 292:653-67
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Pohl, E; Qui, X; Must, L M et al. (1997) Comparison of high-resolution structures of the diphtheria toxin repressor in complex with cobalt and zinc at the cation-anion binding site. Protein Sci 6:1114-8

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