Affinity filters, a new approach to the isolation of tox mutants of Vibrio cholerae.

Abstract

We have devised a novel plate assay method for detecting mutants of Vibrio cholerae altered in the production of cholera toxin (tox mutants). Colonies replicated from a master plate are grown on the surface of a cellulose filter disc to which ganglioside-albumin conjugates have been attached. Toxin secreted by the colonies is tightly bound to the ganglioside filters. After removal of the cells by washing, the bound toxin may be detected by treating the filters with radioactively labeled antibodies against either whole toxin or one of its constituent polypeptide chains, followed by autoradiography.Colonies producing significantly greater or lesser amounts of toxin than the parental type are easily recognized and can be shown in liquid culture to have the corresponding hypertoxinogenic or hypotoxinogenic phenotype. This method, termed "the ganglioside filter assay," is applicable to screening large numbers of colonies and should facilitate isolation of various specific classes of mutants in cholera toxin production. In modified form the method will be applicable to various systems in which mutants of secreted proteins are sought.Isolation of bacterial mutants defective in the production of protein toxins (tox mutants) has proven to be laborious, primarily because toxin production usually confers no known selective advantage for bacterial growth or survival in vitro. Generally, large numbers of clones from mutagenized populations of bacteria must be tested for production of toxic material or specific antigen. Screening for these properties has been greatly facilitated in some instances by assay methods performed either directly on colonies growing in agar [e.g., immunoprecipitin assays (1)] or in microtiter plates [e.g., toxicity or immunological assays (2)] but there is a great need for improved methods in this area.Cholera toxin (choleragen), a multimeric protein of molecular weight about 84,000, consists of a B protomer containing five noncovalently linked chains (molecular weight, 11,590) perhaps in the form of a ring, and an A protomer containing two peptides, A1 (molecular weight, 24,000) and A2 (molecular weight, 9700) linked by a single disulfide bridge (3, 4). The B protomer has been shown to have a high affinity for the oligosaccharide moiety of ganglioside GM1, and there is evidence to indicate that this ganglioside may serve as the cell surface receptor for cholera toxin (5). Free B protomer, which has been termed "choleragenoid," is often found in culture fluids in addition to whole toxin and has been shown to block competitively the action of cholera toxin on intact cells (5). The A1 chain promotes the stimulation of adenylate cyclase activity in erythrocyte ghosts and certain other systems by a NAD-dependent reaction (6). A similar reaction in intestinal epithelium is believed to be responsible for the massive loss of fluid into the intestinal lumen, which gives rise to the major symptoms of clinical cholera (7). The A2 subunit is required for reassembly of choleragen from its subunits and ...

Structure of partially‐activated E. coli heat‐labile enterotoxin (LT) at 2.6 Å resolution

“…For LT and CT both proteolysis of the A subunit and reduction of the 187-l 99 disulfide bond are required for activation [6,7]. In determining the mechanistic significance of these activation steps it may be instructive to consider what is known of the activation process for other related toxins.…”

Botulinum ADP‐ribosyltransferase activity as affected by detergents and phospholipids

“…The column was then eluted with a linear gradient of NaCI (1.2 1; 0-200 mM) in TED containing 1% sodium cholate and aprotinin and followed by further elution with 500 ml of 250 mM NaC1 in the same solution. The activity of C3 substrates was eluted with NaC1 at 250 mM, while IAP substrates were recovered from the column at about 125 mM NaCI [24]. The C3 substrate-rich fractions (240 ml) from the DEAESephacel were concentrated and then fractionated on a column (3.2 × 90 cm) of Sephacryl S-300HR (Pharmacia LKB) in TMDG (20 mM Tris-HCl, pH 8.0, 1 mM MgCI/, 1 mM dithiothreitol and 1 ,aM GDP) containing 100 mM NaCI, aprotinin and 1% sodium cholate.…”

“…The enzymatic activity of bacterial toxins dependent on ADP-ribosylation for their action appears to be latent; upon mild denaturation by detergents, protease digestion, or the disulfide bond reduction of native protein, there is a large increase in the enzymatic activity. For example, activation of cholera toxin by thiol results from reduction of a single disulfide bond between the two peptides in the A subunit [24]. Action of ATP to stimulate lAP-catalyzed ADP-ribosylation appears to be due to the dissociation of the A protomer, enzymatically active subunit ($1), from the binding component of B oligomer [4].…”

A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR

“…Bacterial strains were maintained at -70°C in LB medium containing 25% (vol/vol) glycerol (19). LB, M9 minimal, and CYE media were prepared as described previously (16,19). M9 minimal medium contained 0.4% glycerol as the carbon source and was supplemented as indicated with 25 mM asparagine, arginine, glutamate, and serine.…”