The Shiga toxin family consists of Shiga toxin, produced by Shigella dysenteriae 1, and the Shiga-like toxins (SLTs), produced by Escherichia coli. Only the production of moderate to high levels of Shiga toxin and SLT in vitro has been implicated in pathogenesis. All Shiga toxin molecules are composed of an enzymatic (A) subunit and multiple copies of a binding (B) subunit. The long term goal of this proposal is to investigate the regulation, assembly, and export of Shiga toxin.
Four specific aims are proposed to achieve this goal: 1) The amount of Shiga toxin A and B subunits produced by S. dysenteriae will be measured and compared to the 5B:IA stoichiometry of the holotoxin. The effect that inducing overproduction of the binding subunit will be assessed using a B subunit gene (stxB) clone. Competitive binding assays will be used to investigate the hypothesis that free B subunits affect toxin levels in vivo by competing with the holotoxin for binding sites. 2) The following models for the transcriptional regulation of the stxA and stxB genes will be investigated using Northern blot, primer extension, and mutational analyses: i) a bicistronic mRNA with independent ribosome binding sites (rbs) for the stxA and stxB genes is transcribed from a promoter 5' to stxA; ii) the stxA and stxB genes are transcribed from separate promoters; or iii) endonucleolytic processing of a bicistronic mRNA which influences transcript stability may regulate toxin synthesis. 3) Site-directed mutagenesis and gene fusion will be used to investigate the hypothesis that the putative stxB gene rbs is responsible for overproduction of the B polypeptide. This model will be tested in vivo by assessing the virulence of E. coli strains expressing the mutated slt-II operon for mice. 4) The capacity of B subunits to form pentamers which may modulate virulence by competing with the holotoxin for receptor binding sites will be assessed using the stxB clone. The region of the A subunit which is required for holotoxin assembly will be established by constructing protein fusions. Ultimately, the A subunit residues which are required for holotoxin assembly will be identified using site-directed mutagenesis. This information will be used to investigate A subunit translocation domain(s).

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29AI029929-05
Application #
2065325
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1992-02-01
Project End
2000-01-31
Budget Start
1996-02-01
Budget End
2000-01-31
Support Year
5
Fiscal Year
1996
Total Cost
Indirect Cost
Name
Wayne State University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
City
Detroit
State
MI
Country
United States
Zip Code
48202
Skinner, L M; Jackson, M P (1998) Inhibition of prokaryotic translation by the Shiga toxin enzymatic subunit. Microb Pathog 24:117-22
Skinner, L M; Jackson, M P (1997) Investigation of ribosome binding by the Shiga toxin A1 subunit, using competition and site-directed mutagenesis. J Bacteriol 179:1368-74
Begum, D; Jackson, M P (1995) Direct detection of Shiga-like toxin-producing Escherichia coli in ground beef using the polymerase chain reaction. Mol Cell Probes 9:259-64
Jemal, C; Haddad, J E; Begum, D et al. (1995) Analysis of Shiga toxin subunit association by using hybrid A polypeptides and site-specific mutagenesis. J Bacteriol 177:3128-32
al-Jaufy, A Y; King, S R; Jackson, M P (1995) Purification and characterization of a Shiga toxin A subunit-CD4 fusion protein cytotoxic to human immunodeficiency virus-infected cells. Infect Immun 63:3073-8
al-Jaufy, A Y; Haddad, J E; King, S R et al. (1994) Cytotoxicity of a shiga toxin A subunit-CD4 fusion protein to human immunodeficiency virus-infected cells. Infect Immun 62:956-60
Habib, N F; Jackson, M P (1993) Roles of a ribosome-binding site and mRNA secondary structure in differential expression of Shiga toxin genes. J Bacteriol 175:597-603
Haddad, J E; Jackson, M P (1993) Identification of the Shiga toxin A-subunit residues required for holotoxin assembly. J Bacteriol 175:7652-7
Begum, D; Strockbine, N A; Sowers, E G et al. (1993) Evaluation of a technique for identification of Shiga-like toxin-producing Escherichia coli by using polymerase chain reaction and digoxigenin-labeled probes. J Clin Microbiol 31:3153-6
Haddad, J E; al-Jaufy, A Y; Jackson, M P (1993) Minimum domain of the Shiga toxin A subunit required for enzymatic activity. J Bacteriol 175:4970-8

Showing the most recent 10 out of 11 publications