Abstract
Two hybrid clones producing monoclonal antibodies (MAbs) raised against the purified enterotoxic hemolysin-phospholipase C (HlyPC) bifunctional molecule of a Vibrio cholerae O139 strain were used to study its enterotoxicity in relation to its hemolytic and enzymatic activities. Fab fragments of MAbs from ascites produced by the two hybrids neutralized the hemolytic activity of HlyPC, leaving the enzymatic activity unaffected. In ligated rabbit ileal loop and infant mouse intestine, the Fab fragments of the MAbs were not able to neutralize the enterotoxicity of HlyPC, suggesting that PC rather than Hly is the enterotoxic moiety of the molecule. The enterotoxicity of the purified PC molecule isolated from an Hly− spontaneous mutant of the HlyPC-producing parent strain further confirms this contention. The Hly molecule isolated from a PC− mutant was not diarrheagenic.
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 O139—free from CT and of molecular mass of 67 kDa and pI 6.4—showed 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.
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).
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.
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.
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.
The enterotoxicity of purified HlyPC was completely neutralized in rabbit ileal loop (4) (Table 1) and the infant mouse model (2) (Table 2) by the Fab fragment of the polyclonal anti-HlyPC rabbit antibody, which inhibited in vitro both the hemolytic and enzymatic activities of the molecule. The Fab fragment of the MAbs, which had no effect on the phospholipase C activities of HlyPC and PC in vitro, failed to neutralize the enterotoxic effects of the molecules in the animal models. This indicates that the hemolytic and the enterotoxic properties of the bifunctional molecule are unrelated to each other and that the phospholipase C activity is responsible for the enterotoxicity. The purified hemolysin from the PC− mutant was devoid of enterotoxicity. These observations suggest strongly that the Hly moiety of HlyPC alone is not diarrheagenic and that enterotoxicity is due to PC.
TABLE 1.
Effects of antibodies on fluid accumulation in rabbit ileal loop with HlyPC, PC, and Hly
| Loop injection (μg of protein) | V/L ratio (mean ± SD)a |
|---|---|
| HlyPC (250) | 1.03 ± 0.11 |
| HlyPC + Fab of polyclonal anti-HlyPC (250) | 0 |
| HlyPC + Fab of polyclonal anti-HlyPC (25) | 0 |
| HlyPC + Fab of polyclonal anti-HlyPC (2.5) | 0 |
| HlyPC + Fab of polyclonal anti-HlyPC (0.25) | 0.94 ± 0.12 |
| HlyPC + Fab of polyclonal anti-HlyPC (0.025) | 1.04 ± 0.11 |
| PC (125) | 1.10 ± 0.14 |
| PC + anti-CT serum (200) | 1.12 ± 0.16 |
| PC + Fab of polyclonal anti-HlyPC (125) | 0 |
| PC + Fab of polyclonal anti-HlyPC (12.5) | 0 |
| PC + Fab of polyclonal anti-HlyPC (1.25) | 0 |
| PC + Fab of polyclonal anti-HlyPC (0.125) | 1.0 ± 0.13 |
| PC + Fab of polyclonal anti-HlyPC (0.0125) | 1.02 ± 0.12 |
| PC + Fab of MAb 3H7 (125) | 1.12 ± 0.18 |
| PC + Fab of MAb 4C1 (125) | 1.14 ± 0.15 |
| Hly (500) | 0 |
Volume of fluid (in milliliters) accumulated per unit of length (in centimeters) of rabbit gut. Each result is the mean ± standard deviation of five individual experiments.
TABLE 2.
Effects of antibodies on fluid accumulation in suckling mouse intestines with HlyPC, PC, and Hly
| Oral administration (μg)a | F/A ratio (mean ± SD)b |
|---|---|
| HlyPC (100) | 0.092 ± 0.002 |
| HlyPC (100) + Fab of polyclonal anti-HlyPC serum (100) | 0.009 ± 0.002 |
| HlyPC (100) + Fab of MAb 3H7 (100) | 0.092 ± 0.001 |
| PC (75) | 0.094 ± 0.002 |
| PC (75) + Fab of polyclonal anti-HlyPC serum (75) | 0.093 ± 0.001 |
| PC (75) + Fab of MAb 3H7 (75) | 0.095 ± 0.001 |
| PC (75) + Fab of MAb 4C1 (75) | 0.097 ± 0.001 |
| Hly (100) | 0.006 ± 0.002 |
One hundred micrograms of HlyPC and 75 μg of PC exhibited comparable enzyme activities.
F/A ratio is weight of intestine/weight of rest of the body. F/A ratio of mice fed 0.1 ml of 0.15 M phosphate-buffered saline (pH 7.4), 0.018. Each result is the mean ± standard deviation of five individual experiments.
The absence of correlation of the hemolytic activity of El Tor hemolysin with its enterotoxic activity was also suggested previously by other workers. By observing the effects of oral administration of ΔhlyA Δctx V. cholerae O1 El Tor strains to volunteers, Kaper et al. (9) had indications that hemolysin was probably not diarrhoeagenic. Alm et al. (1) had noted that the hemolytic and enterotoxic activities of El Tor hemolysin occur at two separate sites of the same molecule: the C-terminal end of the molecule is associated with hemolytic activity, while the enterotoxic activity lies in the N-terminal end. Relying on the above observation, they further suggested that in the ΔhlyA Δctx strain of V. cholerae O1 fed to volunteers by Kaper et al., the hemolytic activity was deleted but the enterotoxic activity was retained in the mutants. These observations of Kaper et al. and Alm et al. lend strong support to our present findings that hemolysin acquires its enterotoxic property only when it is associated with phospholipase C as a bifunctional molecule.
The observation that the hemolysin gene, hlyA, and that for phospholipase C occur in adjacent positions in the V. cholerae O1 and O139 chromosome in a conserved manner (6) hints at the possibility that association of hemolysin with phospholipase C activity as a bifunctional molecule may not be an isolated incidence but occurs more widely than previously thought among V. cholerae of different serogroups.
Bacterial phospholipases C have been implicated in the pathogenicity of a number of bacteria (20). The enzyme acting on the lipid bilayer of the mammalian cell membrane results in considerable accumulation of the end product, viz., diacylglycerol, which then stimulates the arachidonic acid cascade by more than one route. The activation of the arachidonic acid cascade by Clostridium perfringens alpha toxin, the most studied bacterial phospholipase C, leads to production of prostaglandins which induce chloride ion secretion in rat colonic cells via activation of chloride ion channels (5). The nonhemolytic Bacillus cereus phospholipase C has also been reported to activate both the arachidonic acid cascade and prostaglandin formation in several cell types (11). The Pseudomonas aeruginosa phospholipase C triggers the production of thromboxanes, leukotrienes, and prostaglandins (13). Leukotrienes C4 and D4 are known to increase vascular permeability and to promote exudation of fluid into the extravascular space (17, 22). Stimulation of the arachidonic acid cascade, followed by an increase in prostaglandins leading to activation of chloride channels and fluid secretion, as observed with other bacterial phospholipase C enzymes, is a highly probable mechanism involved in the expression of the enterotoxic properties of the HlyPC and PC molecules of V. cholerae O139, and it should be studied further.
Acknowledgments
We thank Sujit Chowdhury and Pronob De for assistance during animal experiments.
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