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Vibrio cholerae organisms belonging to serogroups O1 and O139 are the causative agents of epidemics of the disease cholera. The massive diarrhea produced during the disease is attributed to the cholera enterotoxin (CT). Deletion mutants of V. cholerae O1 strains deficient in production of the CT molecule or its subunits, A and B, however, still have been shown to induce mild to moderate diarrhea in volunteers (12). The search for a cause of the diarrhea due to these strains resulted in the discovery of additional toxins of V. cholerae O1, such as hemolysin-cytolysin, zonula occludens toxin, and accessory cholera enterotoxin, etc., which have secretogenic effects on the intestinal mucosa (9). V. cholerae is known to produce several hemolysins; the best studied among them, the El Tor hemolysin, an hlyA gene product, has been purified, characterized, and suggested to be a virulence factor contributing to cholera pathogenesis (7, 14, 16). Clinical isolates of V. cholerae non-O1 are also known to produce a thermolabile hemolysin which is biologically, physicochemically, and antigenically similar to El Tor hemolysin and is capable of inducing fluid accumulation in ligated intestinal loops of adult rabbits (8).

Recently, we purified and characterized a hemolysin from a V. cholerae O139 strain which also showed high phospholipase C activity (15). Association of phospholipase C enzymatic activity with the V. cholerae hemolysin molecule had not been indicated previously. The bifunctional hemolysin-phospholipase C (HlyPC) molecule of V. cholerae O139free from CT and of molecular mass of 67 kDa and pI 6.4showed enterotoxic activity, as evidenced by fluid accumulation in the ligated rabbit ileal loop and in the intestines of suckling mice. The objective of the present study was to raise monoclonal antibodies (MAbs) against the purified bifunctional HlyPC molecule of V. cholerae O139 and to use them to study the interrelationship of the hemolytic and enzymatic activities of the HlyPC molecule vis-à-vis its enterotoxic property.

HlyPC was purified from strain CO55/5, a clinical isolate of V. cholerae O139 which had undergone four serial passages in ligated rabbit intestinal loops (15) to increase its hemolysin production. Spontaneous mutants of CO55/5, deficient in either hemolytic (Hly) or phospholipase C (PC) activity, were selected by screening of single colonies of CO55/5. The hemolytic activity of the culture supernatant was assayed according to the method of Tikoo et al. (19) with a 2% rabbit erythrocyte suspension followed by spectrophotometric measurement of the released hemoglobin at 540 nm. To isolate PC mutants of CO55/5, the phospholipase C activity was monitored according to the spectrophotometric method of Berka and Vasil (3) with the specific substrate p-nitrophenyl phosphoryl choline.

HlyPC, Hly, and PC were purified from CO55/5 and its PC and Hly mutants, respectively, by Biogel P-100 chromatography of the ammonium sulfate precipitate of the culture supernatant of the respective strains, followed by chromatofocusing on a PBE 94 (Pharmacia) column, as detailed previously (15). Figure 1 represents the purification of PC. HlyPC and Hly were similarly purified, but separate representative data are not shown.

View larger version (30K): [in this window] [in a new window]   FIG. 1.   Purification of PC from Hly mutant of CO55/5. (A) Fractionation of ammonium sulfate precipitate of culture supernatant on Biogel P-100 regular column previously equilibrated and eluted with Tris-EDTA-azide buffer, pH 8. , absorbance at 280 nm; , PC activity of each fraction. (B) Chromatofocusing of Biogel P-100 column eluate of V. cholerae O139 phospholipase C on a PBE 94 column with 0.0025 M imidazole-HCl (pH 7.4) as start buffer and polybuffer 74-HCl (pH 4.0) as eluent. , pH; , optical density (O.D.) at 280 nm; , PC activity.

Antibodies. Two stable hybrids, 3H7 and 4C1, were raised by fusion of spleen cells of HlyPC-immunized BALB/c mice with hypoxanthine-guanine phosphoribosyl transferase-deficient mouse myeloma cells (P3 × 63; Ag 8.653) by standard procedures; ascites were induced as previously described (18). Polyclonal antibodies against HlyPC were raised in rabbits (15).

SDS-PAGE and immunoblotting. The three purified proteins, HlyPC, Hly, and PC, moved as 67-kDa molecules in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10) (Fig. 2A) and reacted as well-defined similar single bands with polyclonal anti-HlyPC rabbit sera in immunoblotting by standard methods (21). The monoclonal ascitic fluids 3H7 and 4C1 reacted with HlyPC and Hly proteins but did not identify the PC protein in the immunoblot (Fig. 2B).

View larger version (60K): [in this window] [in a new window]   FIG. 2.   (A) SDS-PAGE of purified HlyPC, Hly, and PC in the absence of mercaptoethanol. Lane 1, molecular mass marker; lane 2, HlyPC; lane 3, Hly; lane 4, PC. (B) Immunoblot after SDS-PAGE. Lane C, Crude HlyPC (ammonium sulfate precipitate); lane 1, HlyPC; lane 2, Hly; Lane 3, PC. a, developed with polyclonal anti-HlyPC serum; b, developed with MAb 3H7 ascitic fluid.

Hemolytic, enzymatic, and enterotoxic activities of purified Hly-PC, Hly, and PC in the presence of Fab fragments. The hemolytic activities of Hly and HlyPC were effectively inhibited by treatment with Fab fragments of both the MAbs and the polyclonal anti-HlyPC rabbit sera (Fig. 3). The phospholipase C activity of HlyPC and PC were also inhibited by the polyclonal anti-HlyPC Fab fragment but not by the Fab fragment of the MAbs (Fig. 4). Neutralization of only the hemolytic activity of the HlyPC molecule by the MAbs without affecting the phospholipase C activity indicates that the epitopes involved in the two activities are different; the functions reside in two separate domains of the same molecule and are independent of each other.

View larger version (20K): [in this window] [in a new window]   FIG. 3.   Effects of various concentrations of Fab fragments of different antibodies on the hemolytic activities of Hly (A) and HlyPC (B). In the reaction mixtures, the amounts of HlyPC or Hly and of the undiluted Fab fractions were in a ratio of 1:1. In subsequent reaction mixtures, the amounts of Fab proteins varied according to the dilution. Protein in 100 µl of undiluted Fab fractions: polyclonal, 120 µg; 3H7, 200 µg; 4C1, 125 µg.

View larger version (24K): [in this window] [in a new window]   FIG. 4.   Effects of various concentrations of Fab fragments of different antibodies on the phospholipase C activities of HlyPC and PC. In the reaction mixtures, the amounts of HlyPC or PC and of the undiluted Fab fractions were in a ratio of 1:1. In subsequent reaction mixtures, the amounts of Fab proteins varied according to the dilution. Protein in 100 µl of undiluted Fab fractions: polyclonal, 120 µg. Phospholipase C from C. perfringens (Sigma) was used as a standard. Units of enzyme activity were calculated by comparing the release of p-nitrophenol by the sample with that of the standard enzyme, of known enzymatic activity.

hlyA ctx V. cholerae

hlyA ctx