TEXT

Top Abstract Text References

Vibrio cholerae utilizes a complex array of structural and regulatory elements in the pathogenesis and immunogenicity of cholera. Although site-specific deletion mutagenesis of the genes encoding cholera toxin (11, 18) resulted in engendering significant protective immunity and the dramatic elimination of the symptoms of cholera gravis, clinical studies consistently reported a residual degree of diarrhea and malaise among vaccinees, indicating that a secretogenic activity remained (11, 16, 18, 21). The extent and intensity of symptoms among vaccinees appeared to be dependent on the dose, the study population, and the vaccine strain under investigation, although little is known of the bacterial factors or the host secretogenic mechanism involved (16, 21-25). Identifying and attenuating the cause of vaccine reactogenicity while preserving immunogenicity remains a major objective of oral vaccine development (19). Characterizing virulence determinants may also contribute to a better understanding of Vibrio pathogenesis and host responses.

Numerous model systems are available for the identification, assay, and characterization of secretogenic activities. Morphological studies on isolated mammalian tissue, such as the Chinese hamster ovary (CHO) cell assay, have provided useful information on nonlethal virulence factors from several bacterial pathogens because these tests do not require an endpoint of cell death (9). A wide range of whole animal systems, including the infant mouse model (13), has contributed to the identification and characterization of numerous virulence determinants from enteric pathogens.

In this study, we assayed culture supernatants from several ctxA-negative V. cholerae strains for cytotonic activity on cultured CHO cells. A CHO elongation activity expressed by the cholera vaccine strain CVD 103-HgR was purified to electrophoretic homogeneity by using (NH₄)₂SO₄ fractionation and four chromatographic steps. Physical characteristics of the protein were determined, including the M_r subunit structure, stability, and amino-terminal sequence. The purified protein was named secreted CHO cell elongating protein (S-CEP). A partially purified cytotonic protein preparation induced fluid accumulation (FA) in the infant mouse model. The identification and description of a novel cytotonic protein in V. cholerae raises questions about its expression and activity in the human intestine and its possible role in vaccine reactogenicity.

The following El Tor biotype strains of O serogroup 1 V. cholerae were studied: JBK 70 (a ctxAB mutant derivative of Inaba strain N16961 [11]) and three environmental isolates from Brazil, namely 8731, 1074, and 1196 (14). The classical biotype strains studied were CVD 103, CVD 103-HgR, and CVD 103-HgR2, all three of which are ctxA mutants of Inaba V. cholerae strain 569B (8). Overnight cultures in Luria broth (10 ml) were inoculated into 1-liter volumes of casamino-yeast extract broth (30 g of casamino acids [Difco; Becton Dickinson, Franklin Lakes, N.J.], 4 g of yeast extract [Difco], 0.5 g of K₂HPO₄ dissolved in 1 liter of H₂O, pH 6.8) in 2-liter flasks and incubated at 37°C in a rotary shaker for 18 h. Cultures were harvested by centrifugation (8,300 × g for 20 min at 4°C), and supernatants were sterilized by filtration (0.22-µm pore size) and stored at 0 to 4°C.

Both culture supernatants and chromatographic fractions (prepared as described below) were subjected to the CHO tissue culture assay conducted as reported elsewhere (9, 13). Briefly, CHO cells were cultured to confluence in Eagle minimal essential medium containing Hanks base salts (Sigma Chemical Co., St. Louis, Mo.) supplemented with 10% fetal bovine serum and 10% tryptose phosphate broth. After incubation at 37°C for 48 h in a humid atmosphere containing 5% CO₂, monolayers of CHO cells were released by treatment with 0.025% trypsin containing 1 µM EDTA. CHO cells were resuspended and seeded into fresh microtiter plates at a density of approximately 1,000 cells per well. The CHO cells were treated with a test or control sample (20 µl) and examined microscopically after 24 h of incubation. One CHO cell unit was defined as the reciprocal of the dilution that caused elongation of 50% of the cells in the well.

V. cholerae classical strain CVD 103-HgR was selected as the source of cytotonic agent because of its relatively high level of activity compared with other organisms tested (data not shown), its role as a vaccine, and the extensive literature on this organism. An additional advantage in studying this strain is its deficiency in the expression of HlyA and engineered deletion of CtxA, which would otherwise exert, respectively, powerful cytolytic and cytotonic effects on CHO cells (5, 6, 8, 10, 15). The absence of cholera toxin and hemolysin activity removed a mask and thereby revealed a remaining CHO cell cytotonic activity which was purified in this study.

Cytotonic toxin purification. Culture supernatants of V. cholerae CVD 103-HgR were subjected to five steps of protein purification, (i) ammonium sulfate fractionation, (ii) anion exchange, (iii) cation exchange, (iv) hydrophobic interaction, and (v) gel filtration chromatography, to yield electrophoretically homogeneous protein bearing a single, unique amino-terminal sequence. Purification proceeded as follows.

View larger version (10K): [in this window] [in a new window]   FIG. 1.   Molecular mass estimation by gel exclusion chromatography. The molecular mass of S-CEP was estimated from the elution profile of standard proteins with known masses. An estimate of 75,000 was made for the molecular mass of the active form of the native toxin.

Protein analysis. Protein concentrations were estimated either by using the Bio-Rad Protein Assay kit (Bio-Rad, Richmond, Calif.) (3) or by using visual estimations of sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) gels stained with Coomassie brilliant blue. The subunit composition of S-CEP was investigated by using PAGE (8 to 25% [wt/vol] total acrylamide) in the presence of SDS according to the manufacturer's instructions (PhastSystem; Amersham Pharmacia Biotech). Analysis of bulked active fractions from Superose-12 fractionation revealed an homogeneous, electrophoretically pure protein band migrating with an M_r of 79,000 (Fig. 2), with no visible evidence of copurifying subunit polypeptides. When compared to the elution pattern of the S-CEP activity directly from gel filtration chromatography (active moiety of M_r of 75,000), the estimated molecular masses were sufficiently similar to support a model of S-CEP as a toxin active as a single major polypeptide chain.

View larger version (83K): [in this window] [in a new window]   FIG. 2.   SDS-PAGE analysis of purified S-CEP. Purified S-CEP was analyzed under denaturing conditions in gradient (8 to 25% total acrylamide) gels. A single discernable band of Mr = 79,000 was identified, suggesting a single major active polypeptide species.