Clostridium septicum causes several life-threatening infections that, without treatment, are fatal. The most devastating of these diseases is non-traumatic gas gangrene. The only lethal factor secreted by C. septicum is alpha toxin. It is a cytolytic, pore-forming toxin that is produced as an inactive protoxin which requires proteolytic activation by normal cellular proteases such as furin. The investigators propose to continue the detailed study of the cytolytic mechanism of alpha toxin in order to gain insight into its biology and to explore ways that may be used to ameliorate its effect in vivo. They propose to: 1) identify the crucial residues of the propeptide of alpha toxin which facilitate its non-covalent interactions with the main body of the toxin and generate derivatives with a greater inhibitory activity towards alpha toxin, 2) identify the transmembrane domains(s) of alpha toxin, 3) identify the residues of the toxin involved in receptor binding, and 4) crystallize a more soluble derivative of alpha toxin and the complex of this derivative with one of the GPI-anchored receptors for alpha toxin. To achieve the first aim, the residues of the propeptide will be sequentially substituted with glycine, isolated and the affinity of the propeptide for the toxin determined.
In aim 2 they will utilize two approaches to map out the membrane-penetrating domains(s) of alpha toxin. The first approach will be to substitute suspected membrane-spanning residues of alpha toxin with cysteine purify these derivatives and then form channels with each toxin in a planar bilayer. Charged derivatives of the sulfhydryl-specific reagent methanethiosulfonate (MTS) are then introduced into aqueous phase on either side of the bilayer. The charged MTS reagent will cause a change in the channel conductance only if the cysteine has been substituted for a channel-lining residue. The same cysteine-substituted residues (in a cysteine-less derivative of alpha toxin) will also be modified with the environmentally sensitive fluorescent probe NBD and the fluorescence examined before and after the toxin have been allowed to insert into membranes. If alpha toxin interacts with the membrane via either an amphipathic beta sheet or an alpha helix they will observe a difference in the periodicity of the response from both assays. The receptor-binding domain has tentatively been localized to a region near C55 of alpha toxin. Thus, in aim 3, residues near C55 in the structural model of alpha toxin will be changed by in vitro mutagenesis to determine which residues participate in receptor binding.
In aim 4 they propose to crystallize alpha toxin and the alpha toxin-receptor complex. This is made possible by the availability of large quantities of a more soluble form of alpha toxin and one of its GPI-anchored receptors, the human folate receptor (hFR).

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI032097-08
Application #
6618132
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Program Officer
Korpela, Jukka K
Project Start
1993-01-01
Project End
2005-06-30
Budget Start
2003-07-01
Budget End
2005-06-30
Support Year
8
Fiscal Year
2003
Total Cost
$156,609
Indirect Cost
Name
University of Oklahoma Health Sciences Center
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
878648294
City
Oklahoma City
State
OK
Country
United States
Zip Code
73117
Kennedy, Catherine L; Lyras, Dena; Cordner, Leanne M et al. (2009) Pore-forming activity of alpha-toxin is essential for clostridium septicum-mediated myonecrosis. Infect Immun 77:943-51
Melton-Witt, Jody A; Bentsen, Lori M; Tweten, Rodney K (2006) Identification of functional domains of Clostridium septicum alpha toxin. Biochemistry 45:14347-54
Wichroski, Michael J; Melton, Jody A; Donahue, Carolyn G et al. (2002) Clostridium septicum alpha-toxin is active against the parasitic protozoan Toxoplasma gondii and targets members of the SAG family of glycosylphosphatidylinositol-anchored surface proteins. Infect Immun 70:4353-61
Sellman, B R; Tweten, R K (1997) The propeptide of Clostridium septicum alpha toxin functions as an intramolecular chaperone and is a potent inhibitor of alpha toxin-dependent cytolysis. Mol Microbiol 25:429-40
Gordon, V M; Benz, R; Fujii, K et al. (1997) Clostridium septicum alpha-toxin is proteolytically activated by furin. Infect Immun 65:4130-4
Ballard, J; Crabtree, J; Roe, B A et al. (1995) The primary structure of Clostridium septicum alpha-toxin exhibits similarity with that of Aeromonas hydrophila aerolysin. Infect Immun 63:340-4
Gottschalk, S; Tweten, R K; Smith, L C et al. (1995) Efficient gene delivery and expression in mammalian cells using DNA coupled with perfringolysin O. Gene Ther 2:498-503
Johnson, S; Driks, M R; Tweten, R K et al. (1994) Clinical courses of seven survivors of Clostridium septicum infection and their immunologic responses to alpha toxin. Clin Infect Dis 19:761-4
Ballard, J; Sokolov, Y; Yuan, W L et al. (1993) Activation and mechanism of Clostridium septicum alpha toxin. Mol Microbiol 10:627-34