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The cholera epidemic in Latin America, from 1991 to 1993, started in Peru and was caused by an El Tor O1 strain. Some clinical strains were obtained in northern Brazil at the beginning of the epidemic. A subgroup within these strains, coming from a cluster of small villages, proved to be different from the prevailing El Tor. DNA fingerprints by the randomly amplified polymorphic DNA (RAPD) method (5) showed a distinct profile; after further characterization, these strains were denominated the Amazonia variant of Vibrio cholerae, a group of O1 pathogenic strains with a similar RAPD pattern that do not belong to the El Tor or classical biotype (3, 4). These strains do not secrete the cholera toxin (i.e., are phenotypically CT) and do not produce other known virulence factors such as the toxin-coregulated pilus, the thermostable toxin, or the Zot toxin. It was found, however, that they secrete a cytotoxic vacuolating activity.

Vacuolation of mammalian cells in response to bacterial secreted proteins has been described for Helicobacter pylori (14), through the action of the secreted cytotoxin VacA (6). The aerolysin of Aeromonas hydrophila and the hemolysin of Serratia marcescens have also been recently described as causing cell vacuolation (1, 10).

In this paper we describe the vacuolating phenotype of V. cholerae supernatants on Vero mammalian cells. We also show the results of screening a number of V. cholerae strains for the presence of this vacuolating cytotoxin and demonstrate a correlation of this activity with the expression of hemolysin.

The vacuolating phenotype. V. cholerae Amazonia supernatants exhibited a cytotoxic and vacuolating activity when applied to Vero cells. The vacuoles started to appear at 2 h after addition of the supernatant and were quantified at 24 h. Vacuoles became very large, taking almost all of the cell space, with the nucleus still clearly visible. Figure 1B shows vacuolated cells, photographed under phase-contrast microscopy after fixation (22), compared to untreated cells (Fig. 1A). Under vacuolating conditions, cells retained their shape and adherence. The number of vacuoles per cell varied, from one large vacuole to 40 or 50 small and some large ones.

View larger version (142K): [in this window] [in a new window]   FIG. 1.   Vacuolation of mammalian cells caused by a cytotoxin secreted by V. cholerae. (A) Control normal Vero cells. (B) Vacuolated cells after treatment with the supernatant of a V. cholerae strain (Amazonia), seen by phase-contrast microscopy (magnification, ×320). (C and D) Vacuolated cells after a neutral red uptake assay, done after 24 h of incubation with the cytotoxin. The weak base neutral red is taken up by the vacuoles.

Cytotoxin quantification. Vacuolating activity was quantified by two different methods. One was direct observation with an inverted microscope. Vero cells were transferred to 96-multiwell plates (100 µl, 2 × 10³ cells/well) and incubated overnight (37°C, 5% CO₂). Cytotoxin dilutions in the same medium were prepared in a separate plate, from supernatants or concentrates, starting with a dilution of 1/20 and diluted twofold, generally up to 1/2,560. Cytotoxin dilutions were substituted for the old medium in the multiwell plates and were incubated at 37°C in the CO₂ incubator. The dilution with maximal vacuolation at 24 h was taken as a measure of activity. The second quantification method was that of neutral red uptake, extensively used in the study of vacuolation by the VacA cytotoxin of H. pylori (18). Figures 1C and D show examples of cells with the vacuoles filled with neutral red, photographed on a Zeiss Axioplan 2 microscope. The uptake of the weak base neutral red by the vacuoles indicates that they are acidic, as are vacuoles caused by VacA. The two quantification methods agreed with each other, and the dilution with maximal vacuolation was used as a measure for activity comparisons.

The vacuolating activity is protein mediated. Temperature sensitivity was tested by heating samples of a filtered concentrated supernatant for 30 min at different temperatures. Vacuolating activity was tested as described above. Vacuoles were detected with filtrates subjected to a temperature up to 50°C, although after treatment at this temperature the activity was already reduced about eightfold. With treatment above this temperature (60°C) no vacuolation or cytotoxicity was observed, which represents a decrease in activity of at least 20-fold. Proteinase K sensitivity was also tested. Supernatants were treated with 10 or 25 µg of proteinase K per ml at 37°C and tested at various times. Treatment with 10 µg/ml for 10 min reduced the activity 8-fold, while treatment with 25 µg/ml reduced it 16-fold. Other properties observed were a relative resistance to trypsin (only twofold decrease in activity after treatment with 500 µg/ml for 2 h at room temperature) and retention of the activity in ultrafiltration membranes (Centriplus; Millipore) with a cutoff size of 100 kDa, pointing to a protein or an oligomer with a high molecular weight, which we denote VcVac.

Bafilomycin A sensitivity tests. Vacuole production by VacA from H. pylori is inhibited by bafilomycin A (18), a specific inhibitor of vacuolar-type H⁺-ATPases at concentrations below 10 µM. Bafilomycin A was used to determine if the vacuolation caused by the V. cholerae cytotoxin was also inhibited by this compound. Vero cell monolayers were prepared in 96-well plates and incubated overnight as described above. Fresh medium was substituted for the spent medium (100 µl per well), and 1-µl aliquots of various concentrations of bafilomycin A in dimethyl sulfoxide were added. The final concentrations used were 100, 50, 25, and 0 nM. The cells were incubated for 30 min at 37°C; 10 µl from each dilution of the toxin being tested (1/20 to 1/2,560 of supernatants) was then added to the wells, in duplicate, and incubation was continued for 24 h. As a control, supernatants from VacA⁺ H. pylori were assayed for vacuolation of Vero cells in the presence and absence of bafilomycin A.

Presence of the vacuolating activity in other V. cholerae strains. The 20 Amazonia strains that we tested produce the vacuolating activity. In addition, 37 other strains were tested (Table 1): 18 El Tor, 3 Gulf of Mexico El Tor, 6 classical, and 10 environmental strains from the state of Rio de Janeiro, Brazil (C. Barros and A. Coelho, unpublished data). Vacuolating activity was found in five of the El Tor strains, the three Gulf of Mexico strains, and seven of the environmental strains. All strains positive for vacuolation were known to be hemolytic. This prompted us to test the hypothesis that the hemolysin is involved in the vacuolating phenotype.

Presence of the hlyA gene and hemolytic activity in the Amazonia strains. Primers surrounding a central portion of the hlyA gene of the El Tor strain N16961 were used to amplify the same region from several Amazonia strains. A fragment with the predicted size (0.6 kb) was amplified (data not shown). Our first report on these strains described them as nonhemolytic (4) due to inadequate filtration of the supernatants used in tube tests (20). In this study they were found to be hemolytic both on 5% sheep blood plates (PML Microbiologicals) and in tube tests (20) done after filtering with low-protein-binding filters (data not shown).

DNA sequencing. The sequence of the complete hlyA gene from Amazonia strain FG1066 was obtained by automated laser fluorescence sequencing of three overlapping PCR fragments, amplified with the Expand High Fidelity PCR system (Boehringer). The size of the gene is 2,226 bp as for other El Tor and environmental hemolysins. A comparison was made to the sequence of the hemolytic El Tor strain N16961, obtained from The Institute for Genomic Research website (http://www.tigr.org). Fifty-five base substitutions were found, a 2.5% difference. The hemolysin protein sequence, obtained by translation of the DNA sequence, has 741 amino acids. Six amino acid changes are present, all in the carboxy-terminal half of the sequence, the region purportedly associated with hemolytic activity (2).

Construction of hlyA insertion knockout mutants of an Amazonia strain. To confirm that the hemolysin is required for cytotoxic vacuolating activity, the hlyA gene was disrupted by insertion of a suicide plasmid. A 0.6-kb central fragment of the hemolysin gene of Amazonia strain FG1066 was amplified by PCR with the primers hemf (5'-GAGCGTAATGCGAAGAATGC-3') and hemr (5'-GAGTCAGGTTTTGGTTACAGG-3') and cloned in the suicide plasmid pKAS32 (21). The resulting plasmid, pFG300, was introduced by mating into V. cholerae Amazonia strain FG1106, a streptomycin-resistant (Sm^r) derivative of FG1066.

Reversion of the insertion mutations restores the vacuolation activity and hemolysis. Homologous recombination leading to reversion of the insertion mutants FG1153 was obtained by two methods. In the first, a few spontaneous revertants of the nonhemolytic strain appearing on sheep blood plates as hemolytic colonies were purified. In another approach not dependent on the hemolytic phenotype, the insertion mutant FG1153 was plated on streptomycin (1 mg/ml), and bacteria that had lost the wild-type rpsL allele present on the inserted suicide vector were selected. Six of these revertants were tested for the vacuolating cytotoxin activity and hemolysis. The results for the cytotoxin test in Table 3 show a restoration of the activity after excision of the inserted plasmid. Hemolytic activity was also fully restored (data not shown).

Cloning of the hlyA region of the Amazonia strain. A 6.5-kb PstI DNA fragment from Amazonia strain FG1066 was cloned into the positive kanamycin resistance (Km^r) selection vector pKil194T (9). E. coli DH5 was electroporated with the ligation products, and recipients were selected by plating on Luria broth (LB) supplemented with kanamycin. Colonies were then screened on sheep blood agar. One hemolytic colony carried a plasmid, pFG350, with the 6.5-kb fragment previously found in other strains to carry the hlyA and hlyB genes (16). This insert hybridized on a Southern blot to a probe consisting of the whole hlyA gene (data not shown). The E. coli strain carrying this plasmid was grown in the conditions described for cytotoxin production, and filtered supernatants and concentrates were prepared. Vacuolating activity in the supernatant was at the lower limit of detection and was found only in two out of four experiments at the 1/20 dilution, but the concentrates from the four experiments contained a vacuolating activity with an average maximum at a 1/30 dilution (Table 3). This activity was not found in the parental strain DH5 tested in the same experiments.

Complementation of the hlyA insertion mutant for VcVac production and hemolysis. Plasmid pFG350 was transferred into the Ap^r V. cholerae hlyA insertion mutant strain FG1153 to test for complementation of the hlyA mutation. The transfer was done in a triparental mating with EK222, DH5/pFG350, and FG1153. The colonies obtained on LB-kanamycin-ampicillin plates were tested on blood agar plates, as Km^r is present both on the conjugative plasmid pRK2013 and on plasmid pFG350 with the hlyA gene. The vast majority of the colonies obtained were hemolytic (>90%). Plasmid preparations confirmed the presence of the 11-kb pFG350 in these transconjugants (FG1229). Supernatants and concentrates were obtained from six such cultures and tested for vacuolation (Table 3). Complementation occurred, and these strains had a high level of VcVac, yielding higher titers (1/40,960 from the concentrate) than the original Amazonia strains in the two experiments done. This is probably due to an increased copy number of the hlyA gene.

Vacuolation with purified El Tor hemolysin. Purified El Tor hemolysin (kindly provided by A. Zitzer) was tested in our cell system to determine whether it would produce the same vacuolation effect. Twofold dilution series from the original preparation were tested. Hemolysin was very cytotoxic to the cells at a high concentration, giving rise to vacuoles similar to the ones described above, in dilutions of approximately 1/800. This corresponds to 0.5 ng of purified El Tor HlyA.

Nucleotide sequence accession number. The sequence reported can be found in GenBank under accession no. AF194418.