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Applied and Environmental Microbiology, August 2001, p. 3707-3711, Vol. 67, No. 8
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.8.3707-3711.2001
Purification and Characterization of a
Vulnificolysin-Like Cytolysin Produced by Vibrio
tubiashii
Mahendra H.
Kothary,1,*
Rachel B.
Delston,2
Sherill K.
Curtis,2
Barbara A.
McCardell,1 and
Ben D.
Tall2
Divisions of Virulence
Assessment1 and Microbiological
Studies,2 Center for Food Safety and Applied
Nutrition, U.S. Food and Drug Administration, Washington, D.C. 20204
Received 15 February 2001/Accepted 15 May 2001
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ABSTRACT |
An extracellular cytolysin from Vibrio tubiashii was
purified by sequential hydrophobic interaction chromatography with
phenyl-Sepharose CL-4B and gel filtration with Sephacryl S-200. This
protein is sensitive to heat and proteases, is inhibited by
cholesterol, and has a molecular weight of 59,000 and an isoelectric
point of 5.3. In addition to lysing various erythrocytes, it is
cytolytic and/or cytotoxic to Chinese hamster ovary cells, Caco-2
cells, and Atlantic menhaden liver cells in tissue culture. Lysis of erythrocytes occurs by a multihit process that is dependent on temperature and pH. Twelve of the first 17 N-terminal amino acid residues
(Asp-Asp-Tyr-Val-Pro-Val-Val-Glu-Lys-Val-Tyr-Tyr-Ile-Thr-Ser-Ser-Lys) are identical to those of the Vibrio vulnificus cytolysin.
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TEXT |
Vibrio tubiashii is a
marine organism that causes bacillary necrosis in larval and juvenile
bivalve mollusks (24, 25). The disease is characterized by
a rapid onset of symptoms, such as a generalized reduction in larval
motility, an increase in larval quiescence, and extensive soft tissue
necrosis. The pathogen has been isolated from hard clam larvae,
juvenile hard clams, and Eastern oyster spat and larvae (7, 12,
25). V. tubiashii has been isolated from diseased
mollusks in the United States and United Kingdom and has been
associated with red tides caused by Mesodinum rubrum along
the northwest coast of Spain (12, 23, 24, 25). In spite of
the economic importance of V. tubiashii in the cultivation
of bivalve mollusks, nothing is known about the virulence mechanisms of
this pathogen. Romalde et al. (23) reported that culture
supernatants of the pathogen exhibited cytotoxicity towards fathead
minnow peduncle cells and mouse lung fibroblasts in tissue culture. We
describe the purification and properties of a cytolysin that lyses
various types of erythrocytes and Chinese hamster ovary (CHO) cells and
is cytotoxic to human intestinal (Caco-2) cells and fish (Atlantic
menhaden liver [AML]) cells in tissue culture.
Cytolysin production and purification.
Two V. tubiashii strains (ATCC 19105 and ATCC 19109) were obtained from
the American Type Culture Collection (Manassas, Va). Both strains were
confirmed to be V. tubiashii using biochemical tests and
were stored at 
70°C. The ATCC 19105 frozen culture was rapidly
thawed and inoculated onto two plates containing Trypticase soy agar
(BBL, Cockeysville, Md.) supplemented with 1% NaCl. The plates were
incubated at 30°C for 16 to 18 h, and the bacteria were
harvested in 5 ml of Casamino Acids-yeast extract broth (3% Casamino
Acids, 0.4% yeast extract, 0.05% K2HPO4 [pH
7.4] supplemented with 1% NaCl). A 2-liter flask containing 500 ml of
Casamino Acids-yeast extract broth was inoculated with the seed culture
suspension (25 optical density units at 650 nm; ca. 1010
CFU), and the culture was incubated for 7 h at 37°C on a rotary shaker at 100 rpm. Culture supernatant fluids (stage 1) were recovered by centrifugation at 16,000 × g (20 min). Disodium
hydrogen phosphate and sodium chloride were dissolved in the stage 1 preparation to final molarities of 0.067 and 0.077 M, respectively, and
the pH was adjusted to 7.0 with concentrated HCl. The preparation was
then subjected to hydrophobic interaction chromatography using a column
(1.6 by 30 cm) of phenyl-Sepharose CL-4B (Amersham Pharmacia Biotech,
Piscataway, N.J.) equilibrated with phosphate-buffered saline (PBS)
(0.067 M Na2HPO4-0.077 M NaCl, pH 7.0). The
column was washed first with PBS, then with PBS diluted (1:10) with
water, and finally with 25% ethylene glycol in diluted PBS. Washing
the column with 50% ethylene glycol in diluted PBS eluted the
cytolysin. Peak fractions having hemolytic activity were pooled (stage
2). The stage 2 preparation was subjected to gel filtration using a
column (2.6 by 94 cm) of Sephacryl S-200 (Amersham Pharmacia Biotech)
equilibrated with PBS. Peak fractions having activity were pooled
(stage 3).
Hemolytic activity of the cytolysin was measured using sheep
erythrocytes (Colorado Serum Company, Denver, Colo.) by a method previously described (3). Briefly, the cytolysin
preparations were diluted to 0.5 ml with PBS and added to 0.5 ml of PBS
containing 1 mg of bovine serum albumin (BSA) (Sigma, St. Louis, Mo.)
per ml. To this mixture, 1 ml of washed erythrocyte suspension in PBS
(0.7%, vol/vol) was added, and the tube was incubated at 37°C for 30 min. Unlysed erythrocytes were pelleted by centrifugation, and the
absorbance of the supernatant was measured at 545 nm. One hemolytic
unit (HU) is defined as the amount which causes the release of 50% of
the hemoglobin in the standardized erythrocyte suspension.
Analysis of the stage 3 preparation using the PhastSystem (Amersham
Pharmacia Biotech) showed that the cytolysin was homogeneous
by sodium
dodecyl sulfate-polyacrylamide gel electrophosporesis
(SDS-PAGE) in an
8 to 25% gradient gel (Fig.
1) and by
thin-layer
isoelectric focusing in a pH 3 to 9 gel (Fig.
2). The molecular
weight of the denatured
reduced cytolysin, as estimated by the
relative mobility method of
Weber et al. (
26), was 59,000. This
molecular weight is
much higher than the apparent molecular weight
(<10,000) of the native
protein as determined by gel filtration
(
1). Similar
observations have been made for the hemolysin
of non-O1
Vibrio
cholerae and vulnificolysin of
Vibrio vulnificus,
which
also exhibit very low molecular weights by gel filtration
but which
have molecular weights of 60,000 and 56,000, respectively,
as
determined by SDS-PAGE (
6,
11,
18,
27). The
V. tubiashii cytolysin has an isoelectric point of 5.3, which is
similar to
that of the non-O1
V. cholerae hemolysin
(
18) but is different
from that of vulnificolysin
(
6).
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FIG. 1.
SDS-PAGE of V. tubiashii cytolysin
preparations. Lanes: 1, molecular mass markers (values at left are in
kilodaltons); 2, stage 1 (4 µg); 3, stage 2 (0.7 µg); 4, stage 3 (0.5 µg). The gel was stained with Coomassie brilliant blue R.
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FIG. 2.
Analytical thin-layer isoelectric focusing of V. tubiashii cytolysin. Lanes: 1, pI markers; 2, stage 3 (1 µg).
The arrowhead indicates the location of the hemolysin isoform. The gel
was stained with Coomassie brilliant blue R.
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The quantitative results of the purification are summarized in Table
1. The amount of protein in the
preparations was estimated
by the method of Bradford (
4),
using a standard (BSA) and reagent
that were purchased from Bio-Rad
Laboratories (Richmond, Calif.).
About 30% of the cytolysin was
recovered in a purified state.
The amount of protein in this
preparation was only 41 µg, but
the specific activity was very high
(167,683 HU per mg of protein).
Strong binding of the protein to
phenyl-Sepharose CL-4B during
hydrophobic interaction chromatography
suggests that the cytolysin
is extremely hydrophobic. The hydrophobic
nature of the protein
was also apparent during the gel filtration
chromatography stage
of the purification process: the cytolysin eluted
from the column
in a very broad peak with an apparent molecular weight
of less
than 10,000 (results not shown). This elution pattern is
suggestive
of an interaction between the gel and the protein molecule.
The
hydrophobic nature of the protein may also have been responsible
for the loss of activity that was observed for the various methods
(ammonium sulfate precipitation, ultrafiltration, dialysis, and
lyophilization) we employed to isolate and concentrate the cytolysin
from the culture supernatant fluids.
Cytolysin inactivation.
The cytolysin lost its activity when
it was incubated at temperatures of 37 to 100°C for 30 min (Table
2). The losses of activity at 37 and
56°C were reduced to 0 and 94%, respectively, when 0.25 mg of BSA
per ml was added to the cytolysin solution. Thus, the hemolytic assay
was routinely carried out at 37°C for 30 min in the presence of BSA.
The cytolysin was stable when incubated at 4°C for 24 h in
buffers with pH values of 5 to 10 but lost all its activity at pH 4. The cytolysin was sensitive to digestion with chymotrypsin and
subtilisin but not to trypsin and papain. Sensitivity to chelating
agents (EGTA and EDTA), dithiothreitol (DTT), cholesterol, and mixed
gangliosides was determined by incubating the cytolysin with the
reagents for 30 min at 27°C. The organic solvents in which the
cholesterol and mixed gangliosides were dissolved were evaporated with
a stream of nitrogen, and the reagents were suspended in 0.5 ml of PBS
and sonicated for 20 s using a sonicator equipped with a microtip
(Tekmar Co., Cincinnati, Ohio). Cytolysin (0.5 ml) was then added to
the sonicated suspension, and the mixture was incubated at 27°C for
30 min. Addition of 0.1 µg of cholesterol resulted in a loss of 85%
of the cytolysin's activity; amounts greater than 1 µg inhibited
100% of the activity. The addition of 1 mM EGTA and 1 mM EDTA to the
reaction mixture and preincubation of the cytolysin with 10 mM DTT and
mixed gangliosides did not affect the cytolysin's activity. Results
are similar to those reported for vulnificolysin, except that a much
smaller amount of cholesterol (1 µg compared to 100 µg) is required
to inactivate the V. tubiashii cytolysin (6).
The proteases, EGTA, EDTA, DTT, cholesterol, and mixed gangliosides
were purchased from Sigma.
Variables influencing erythrocyte lysis.
The effects of
temperature, pH, and toxin concentration on erythrocyte lysis were
examined by incubating tubes (six for each parameter) containing
erythrocyte suspension (final concentration of 0.35%, vol/vol) and the
cytolysin and removing one tube every 10 min. The unlysed erythrocytes
were centrifuged, and the absorbance of the supernatant was measured.
The effects of temperature (27, 32, 37, 42, and 47°C) and pH (7, 8, 9, and 10) were examined by using 2 HU of the cytolysin, while the
effect of cytolysin concentration was determined with 0.5, 1.0, 2.0, and 3.0 HU. The effect of erythrocyte concentration on hemolysis was
examined by incubating 2 HU with different amounts (0.175 to 3.5%
final concentration) of sheep erythrocytes. Optimal hemolysis was
observed at temperatures of 37 to 47°C; however, the rate of
hemolysis was maximal at higher temperatures. The optimal pH for
hemolysis was 7 to 8. The rate and amount of hemolysis were dependent
on the amount of cytolysin (Fig. 3A). An
increase in the number of erythrocytes (0.175 to 1.05%) resulted in an
initial increase of total absorbance (hemolysis) followed by a gradual
decrease in the hemolysis (Fig. 3B). However, the percentage of
hemolysis (lysed erythrocytes as a proportion of the total
erythrocytes) decreased with increased erythrocyte concentration.
Results indicate that in the presence of an excess of erythrocytes, the
amount of cytolysin that binds to an erythrocyte is not enough to cause
its lysis. Further, these results suggest that more than one molecule
of the cytolysin is required to lyse an erythrocyte. Such multihit
processes have been described for various other cytolysins and
hemolysins (6, 10, 13-15, 17, 21).
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FIG. 3.
Effects of V. tubiashii cytolysin and
erythrocyte concentrations on cytolysin-induced lysis of erythrocytes.
Erythrocyte samples (1 ml) were incubated with the cytolysin, and the
mixture was centrifuged to pellet unlysed erythrocytes. The absorbance
of the supernatant fluids was measured at 545 nm and compared with that
of the control. (A) Effect of cytolysin concentration. The control for
this experiment was a saponin-lysed 0.35% (vol/vol) erythrocyte
suspension. (B) Effect of erythrocyte concentration. The reaction
mixture contained 2 HU of cytolysin and different amounts of
erythrocytes. The controls for this experiment were saponin-lysed 0.175 to 3.5% (vol/vol) erythrocyte suspensions.
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The ability of the cytolysin to bind to the sheep erythrocytes was
examined by incubating 1, 2, and 4 HU with the erythrocytes
(0.35%,
vol/vol) at 4 and 37°C for 1, 2, and 4 min and measuring
the amount
of activity associated with the erythrocytes. Briefly,
after the
incubation period, the erythrocytes were removed by
centrifugation,
resuspended in 1.5 ml of PBS and 0.5 ml of PBS-BSA,
and incubated at
37°C for 30 min. Erythrocytes were then centrifuged,
and the
absorbance of the supernatant was measured at 545 nm.
Results indicate
that the cytolysin bound to erythrocytes much
more rapidly at 37°C
than 4°C (Table
3). At 4°C, there was
no
significant difference between the amounts of toxin bound at 1
and 4 min. In contrast, prolonged incubation at 37°C resulted
in more of
the toxin binding to the erythrocytes. These results
suggest that
binding of the
V. tubiashii cytolysin to erythrocytes
is a
temperature-dependent step. In comparison, the binding of
vulnificolysin to erythrocytes is independent of temperature
(
6).
Biological activity.
The cytolysin was examined for its
activity against various erythrocytes and tissue culture cells. Blood
samples from goat, rabbit, calf, goose, chicken, horse, and guinea pig
were obtained from the Colorado Serum Company. The cytolysin was active
against erythrocytes from all seven animal species tested (Table
4). Erythrocytes from sheep and goat were
the most sensitive, while those from guinea pig and horse were the
least sensitive.
The effects of the toxin against CHO cells, Caco-2 cells, and AML cells
in tissue culture were examined using a method previously
described for
a CHO cell assay (
16). CHO cells were grown in
Eagle's
minimum essential medium (Sigma) supplemented with 10%
fetal calf
serum (Gibco BRL, Life Technologies, Rockville, Md.)
and 10% tryptose
phosphate broth, while the Caco-2 cells were
grown in Dulbecco's
modified Eagle's medium (Gibco BRL) supplemented
with 20% fetal calf
serum, 1 mM sodium pyruvate, 200 mM
L-glutamine,
and 1 mM
nonessential amino acids. AML cells were grown in a medium
previously
described by Faisal et al. (
5). Briefly, for the
assay,
the cells were grown to confluence in the growth medium,
harvested, and
resuspended at a concentration of 10,000 cells
per ml of the growth
medium containing 1% fetal calf serum. A
100-µl aliquot (1,000 cells) of the cell suspension was added
to each well in a microtiter
plate. The cytolysin was diluted
2- to 256-fold, and 10 µl of the
dilution was added to each well.
The cells were incubated either at
37°C (CHO and Caco-2) or at
26°C (AML) in an incubator with 5%
CO
2 and examined microscopically
after 2, 4, 6, 8, and
22 h for cytolytic and/or cytotoxic effects.
Activity was visible
within 4 to 6 h of incubation of the cells
with the cytolysin. The
affected CHO cells were lysed, but the
Caco-2 and AML cells appeared as
very small rounded cells that
had lost their characteristic morphology.
The minimum amounts
of toxin that exhibited cytolytic and/or cytotoxic
activity towards
ca. 50% of the CHO, Caco-2, and AML cells were 0.01, 0.02, and
0.1 HU,
respectively.
The suckling mouse assay, as previously described (
16),
was used for determining whether the cytolysin has the potential
to
cause diarrhea in this model. Pregnant ICR mice were ordered
from
Harlan Sprague-Dawley (Indianapolis, Ind.). Studies were
carried out in
accordance with an Institutional Animal Care and
Use Committee-approved
protocol. Purified cytolysin tested at
100 HU (0.6 µg) per mouse
failed to elicit any fluid. This result
suggests either that the
cytolysin does not play a role in the
ability of the organism to induce
fluid accumulation or that a
larger amount of the cytolysin is required
for a positive response.
For example, 0.5 µg of the El Tor-like
hemolysin of non-O1
V. cholerae was sufficient for a
positive response, but a higher
dose (2 µg) of the hemolysin produced
by
Vibrio metschnikovii was required to induce a significant
amount of fluid in suckling
mice (
9,
19).
N-terminal amino acid sequence.
The cytolysin preparation was
concentrated using a ProSpin cartridge (Applied Biosystems, Foster
City, Calif.), and the excised ProSpin membrane was sequenced by Edman
degradation using a model 477A protein sequencer (Applied Biosystems).
The presence of only one sequence further confirmed that the purified
preparation was homogeneous. The same sequence was obtained when a
Coomassic brilliant blue R (Sigma)-stained cytolysin band was excised
from a Western blot and sequenced. The first 17 N-terminal amino acids
of the cytolysin are shown in Table 5. A
search of the protein database revealed that the sequence had homology
only to vulnificolysin (6, 28); 12 of these 17 amino acids
are identical to those of vulnificolysin, and 3 other amino acids are
logical substitutions.
PCR.
Due to the high degree of homology between the N-terminal
amino acid sequences of the V. tubiashii and the V. vulnificus cytolysins, we examined both strains of V. tubiashii for the presence of the cytolysin gene of V. vulnificus by PCR. Two V. vulnificus strains (C7684 and
MO6-24) were used as positive controls. A colony from each strain was
picked from a plate containing Trypticase soy agar and 1% NaCl,
suspended in 25 µl of 0.5 M NaOH, and incubated at room temperature
for 30 min. After the addition of 25 µl of Tris-HCl (pH 7.5) and 450 µl of sterile distilled water, the lysates were frozen at 20°C
until needed. PCR primers based on sequences within the V. vulnificus cytolysin gene, as described by Hill et al.
(8), were used to generate a 519-bp product. PCR was performed in 50-µl volumes containing 45 µl of Platinum PCR
supermix (Life Technologies), primers (500 ng), and 5 µl of cell
lysate as a DNA template. Following an initial 5-min holding period at 94°C, each of the subsequent 30 PCR cycles consisted of 1.75 min at
94°C, 2 min at 60°C, and 2 min at 72°C. Analysis of the two V. tubiashii strains did not yield the expected 519-bp
product. Results suggest that the homology between the hemolysins at
the amino acid level (at least at the N-terminal end) does not extend to the DNA sequence using the primers specified by Hill et al. (8). It is quite possible that some homology may be
present in the remaining sequence, and the use of different primers may detect both of the pathogens.
Various vibrios cause fatal diseases in both marine and freshwater fish
(
2). For example,
Vibrio anguillarum, V. vulnificus, Vibrio ordalii, Vibrio damsela, V. cholerae, and
Vibrio
parahaemolyticus cause systemic infections in edible finfish,
crustaceans, and
shellfish. Extracellular proteins, especially
hemolysins, produced
by many of the bacterial pathogens are postulated
to play an important
role in the pathogenesis of disease in fish. Some
of these hemolysins
have been purified and tested for their virulence
in fish (
20,
22). However, the role of bacterial virulence
factors in diseases
of mollusks has not been studied. The cytolytic and
cytotoxic
properties of the
V. tubiashii cytolysin may be
important in the
mortality of larval and juvenile bivalve mollusks.
Interestingly,
the cytolysin has various physicochemical properties,
including
an N-terminal amino acid sequence, that are similar to those
of
the vulnificolysin of
V. vulnificus, a human and fish
pathogen
(
6).
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FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Virulence Assessment (HFS-327), Center for Food Safety and Applied
Nutrition, U.S. Food and Drug Administration, 200 C Street, S.W.,
Washington, DC 20204. Phone: (202) 205-4454. Fax: (202) 205-4939. E-mail: mkothary{at}cfsan.fda.gov.
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Applied and Environmental Microbiology, August 2001, p. 3707-3711, Vol. 67, No. 8
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.8.3707-3711.2001
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