Diphtheria toxin (DT) and the family of heat-labile enterotoxins (LTs) are well studied virulence factors of Corynebacterium diphtheriae and Escherichia coli, respectively. Each toxin has a B domain that binds to plasma membrane receptors and an A domain that mediates toxicity. DT ADP-ribosylates elongation factor 2, thereby inhibiting protein synthesis and killing target cells. LTs ADP-ribosylate the regulatory protein G, and activate adenylate cyclase. The long term objectives are to understand the genetics, regulation, and mode of action of these toxins at the molecular level. Such knowledge is essential for understanding pathogenesis of diphtheria and enterotoxic E. coli diarrhea and for development of improved vaccines, immunotoxins, hybrid toxins, or related biologic products involving these toxins.
The specific aims of this proposal are to: 1) determine the mechanisms by which iron regulates production of DT and the activity of the iron uptake system in C. diphtheriae. The investigators will analyze the tox promoter sequence that interacts with the E. coli Fur protein, clone and characterize the tox repressor from C. diphtheria, isolate and characterize ferric uptake regulation (fur) mutants of C. diphtheriae, and determine whether tox repressor activity is mediated by the putative fur gene product in C. diphtheriae. 2) characterize the interactions of DT with its cellular receptor. Dr. Holmes and colleagues have developed monoclonal antibodies to membrane components of human cells that block intoxication by DT and they will characterize their mode of action. They will also purify DT receptor molecule(s), clone and characterize and corresponding cDNA(s), determine whether DT-resistant cells that lack functional receptors have genes or membrane components homologous with those for DT receptors, and characterize the binding domains of DT and its receptor. 3) analyze structure-function relationships of LTs. They will compare purified type I and type II LTs in intestinal assays in several animal species to determine whether their different receptor specificities correlate with differences in biologic activity. They will use genetic complementation with cloned A and B subunit genes, directed mutagenesis of cloned genes, chemical modification of purified toxins, and synthetic oligopeptides to analyze the molecular determinants of biologic activity of representative type I and type II LTs.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI014107-15
Application #
3125645
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1976-09-30
Project End
1995-03-31
Budget Start
1991-04-01
Budget End
1992-03-31
Support Year
15
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Henry M. Jackson Fdn for the Adv Mil/Med
Department
Type
DUNS #
City
Rockville
State
MD
Country
United States
Zip Code
20817
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:
Jobling, Michael G; Gotow, Lisa F; Yang, Zhijie et al. (2015) A mutational analysis of residues in cholera toxin A1 necessary for interaction with its substrate, the stimulatory G protein Gs?. Toxins (Basel) 7:919-35
Spinler, Jennifer K; Zajdowicz, Sheryl L W; Haller, Jon C et al. (2009) Development and use of a selectable, broad-host-range reporter transposon for identifying environmentally regulated promoters in bacteria. FEMS Microbiol Lett 291:143-50
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
Goranson-Siekierke, J; Pohl, E; Hol, W G et al. (1999) Anion-coordinating residues at binding site 1 are essential for the biological activity of the diphtheria toxin repressor. Infect Immun 67:1806-11
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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