INFECTION AND IMMUNITY,
Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Sept. 2000, p. 5096–5106 Vol. 68, No. 9
Construction and Phenotypic Evaluation of a Vibrio vulnificus
vvpE Mutant for Elastolytic Protease
KWANG CHEOL JEONG,1HYE SOOK JEONG,1JOON HAENG RHEE,2SHEE EUN LEE,2
SUN SIK CHUNG,2ANGELA M. STARKS,3GLORIA M. ESCUDERO,3
PAUL A. GULIG,3AND SANG HO CHOI1*
Department of Food Science and Technology, Institute of Biotechnology, Chonnam National University, Kwang-Ju,
500-757,1and Department of Microbiology, Chonnam National University Medical School, Kwang-Ju,
500-190,2South Korea, and Department of Molecular Genetics and Microbiology,
College of Medicine, University of Florida, Gainesville, Florida 32610-02663
Received 25 April 2000/Accepted 6 June 2000
Vibrio vulnificus is an opportunistic gram-negative pathogen that commonly contaminates oysters. Predis-
posed individuals who consume raw oysters can die within days from sepsis, and even otherwise healthy people
are susceptible to serious wound infection after contact with contaminated seafood or seawater. Numerous
secreted and cell-associated virulence factors have been proposed to account for the fulminating and destruc-
tive nature of V. vulnificus infections. Among the putative virulence factors is an elastolytic metalloprotease. We
cloned and sequenced the vvpE gene encoding an elastase of V. vulnificus ATCC 29307. The functions of the
elastase were assessed by constructing vvpE insertional knockout mutants and evaluating phenotypic changes
in vitro and in mice. Although other types of protease activity were still observed in vvpE mutants, elastase
activity was completely absent in the mutants and was restored by reintroducing the recombinant vvpE gene.
In contrast to previous characterization of elastase as a potential virulence factor, which was demonstrated by
injecting the purified protein into animals, inactivation of the V. vulnificus vvpE gene did not affect the ability
of the bacteria to infect mice and cause damage, either locally in subcutaneous tissues or systemically in the
liver, in both iron-treated and normal mice. Furthermore, a vvpE mutant was not affected with regard to
cytolytic activity toward INT407 epithelial cells or detachment of INT407 cells from culture dishes in vitro.
Therefore, it appears that elastase is less important in the pathogenesis of V. vulnificus than would have been
predicted by examining the effects of administering purified proteins to animals. However, V. vulnificus utilizes
a variety of virulence factors; hence, the effects of inactivation of elastase alone could be masked by other
compensatory virulence factors.
The pathogenic marine bacterium Vibrio vulnificus is the
causative agent of food-borne diseases such as life-threatening
septicemia and possibly gastroenteritis in individuals with un-
derlying predisposing conditions such as liver damage, excess
levels of iron, and immunocompromised conditions (2, 14).
Wound infections result from exposure to seawater or from the
handling of shellfish contaminated with V. vulnificus. Mortality
from septicemia is very high (?50%), and death may occur
within 1 to 2 days after the first signs of illness (14, 47). Several
potential virulence factors including an endotoxin, a polysac-
charide capsule (46, 55, 57), iron-sequestering systems (19, 54),
a cytolytic hemolysin (43, 53), an elastase (16, 24, 36), a phos-
pholipase A2(48), and other exotoxins have been identified for
V. vulnificus. However, to date, only the capsule (55) and iron-
sequestering systems (19) have been confirmed as virulence
factors by using the molecular version of Koch’s postulates (6,
11), in which mutations are constructed in genes encoding
putative virulence factors, followed by complementation of any
observed attenuating phenotypes. It is interesting that a mu-
tation in the cytolytic hemolysin of V. vulnificus exhibited no
attenuating effect in mouse models of disease (52). Most re-
cently, a pleiotropic mutation in a gene encoding prepilin pep-
tidase was shown to significantly attenuate the virulence of
V. vulnificus in mice (37). The prepilin peptidase mutant was
defective in the secretion of cytolysin, elastase, chitinase, and
probably other proteins, so it is difficult to assign the attenua-
tion to a particular effector protein.
The proteolytic activity of V. vulnificus has been charac-
terized as elastase, collagenase, and caseinase (16, 24). The
V. vulnificus elastase activity is from a neutral metalloprotease,
and the characteristics of the protease as a potential virulence
factor have been studied primarily using the purified protein in
animal models (17, 23, 25–28, 32). Injection of purified elastase
could reproduce many of the observed aspects of disease
caused by V. vulnificus, including dermonecrosis, destruction of
tissues, edema, and ulceration. These diverse activities are
believed to be caused by the proteolytic degradation or inac-
tivation of biologically important host proteins and immune
system components such as collagen, fibrin, and complement.
Conversely, increased vascular permeability is stimulated by
the activation of Hagemann factor and prekallikrein directly by
the elastase, leading to the production of bradykinin (23, 30,
32). Additionally, the activity of the elastase toward the host
iron-binding proteins is involved in the utilization of heme and
iron (34, 35). More direct evidence for the relevance of these
diverse biological and biochemical activities during infection is
based on injecting biochemical inhibitors of protease activity or
neutralizing antibodies during infection of experimental ani-
mals (20, 27). Additionally, some data have been reported on
undefined chemically induced mutants deficient in the produc-
tion of elastase (27). However, no definitive analysis of the role
of the V. vulnificus elastase by means of the construction of a
defined mutation has been reported.
* Corresponding author. Mailing address: Department of Food Sci-
ence and Technology, Institute of Biotechnology, Chonnam National
University, Kwang-Ju, 500-757, South Korea. Phone: 82-62-530-2146.
Fax: 82-62-530-2149. E-mail: email@example.com.
Recently the gene that encodes a V. vulnificus protease was
cloned and sequenced (4, 5). The deduced gene product was
predicted to be a 609-amino-acid polypeptide, with the mature
protease having a molecular mass of 45 kDa and consisting of
413 amino acids generated by deletion of the N-terminal 196
amino acids. By using the mature protease purified from re-
combinant Escherichia coli, two functional domains, a 35-kDa
N-terminal domain required for catalytic activity and a 10-kDa
domain required for attachment to the substrate, were identi-
To study the role of the elastase in the pathogenesis of
infection, we constructed by allelic exchange two V. vulnificus
mutants that no longer produced elastase. Using both iron-
treated and normal mice, we observed no alteration in viru-
lence as determined by levels of local and systemic infection or
histopathology. Furthermore, the ability of V. vulnificus to lyse
or cause the detachment of cultured epithelial cells was not
affected by the protease mutation.
MATERIALS AND METHODS
Strains, plasmids, media, and culture conditions. The strains and plasmids
used in this study are listed in Table 1. E. coli strains used for plasmid DNA
replication or conjugational transfer of plasmids were grown in Luria-Bertani
(LB) broth or on LB broth containing 1.5% (wt/vol) agar. Nutrient agar plates
supplemented with 1.5% (wt/vol) skim milk were used for screening E. coli
transformants carrying and expressing the recombinant V. vulnificus elastase
gene. Unless noted otherwise, V. vulnificus strains were grown in LB medium
supplemented with 2.0% (wt/vol) NaCl (LBS). For mouse and cell culture ex-
periments, V. vulnificus strains were grown in LB broth containing 0.85% (wt/vol)
NaCl (LBN). For 50% lethal dose (LD50) experiments, vibrios were grown in
brain heart infusion broth containing 2.5% (wt/vol) NaCl (BHI-N). When re-
quired, appropriate antibiotics were added to the media as follows: ampicillin at
100 ?g/ml, chloramphenicol at 10 ?g/ml, kanamycin at 50 ?g/ml, and tetracycline
at 10 ?g/ml. All medium components were purchased from Difco (Detroit,
Mich.), and chemicals were purchased from Sigma (St. Louis, Mo.).
Measurement of cell growth and enzyme activities. For comparison of the
growth rates and protease activities of parental, wild-type V. vulnificus ATCC
29307 and its elastase mutant, KC64, 50-ml cultures of nutrient broth in 250-ml
Erlenmeyer flasks were inoculated with an initial cell density (measured as the
optical density at 600 nm [OD600]) of approximately 0.005 and were incubated at
30°C with shaking. The inocula were from late-exponential-phase cultures in
LBS. Samples of 5 ml were removed at the indicated times for determination of
cell density, total protease activity, and elastase activity. Bacterial growth was
monitored by measuring the OD600of cultures. For measurements of enzyme
activities in supernatants, bacterial cells were removed from cultures by centrif-
ugation at 10,000 ? g for 5 min and the supernatants were filtered through
0.45-?m-pore-size filters. Minor modifications of procedures described previ-
ously (8, 44) were used for determination of total protease activity, defined as a
casein-hydrolyzing activity, and of elastase activity. For total protease activity, the
reaction was initiated by addition of 100 ?l of filtered supernatant as an enzyme
source to 2 ml of 0.25-mg/ml azocasein in 10 mM Tris-HCl buffer (pH 7.5). After
incubation for 1 h at 30°C, the reaction was stopped by addition of 2 ml of 8%
(wt/vol) trichloroacetic acid. The reaction mixture was clarified by centrifugation,
and 2 ml of the supernatant was transferred to a new tube. Color development
was enhanced by addition of 2 ml of 0.5 M sodium hydroxide, and the absorbance
at 400 nm was measured. For elastase activity, 100 ?l of enzyme source was
added to 1 ml of a solution containing 20 mg of elastin-Congo red/ml in 10 mM
sodium phosphate (pH 7.0). The resulting reaction mixture was incubated for 4 h
at 37°C, and the absorbance at 495 nm was determined. One unit of enzyme
activity is defined as an increase in absorbance of 0.001 per hour for total
protease activity and of 0.01 per hour for elastase activity. The mean of triplicate
results was used.
DNA techniques, cloning, and sequencing. Isolation of genomic or plasmid
DNA and transformation of E. coli strains were carried out according to the
procedures outlined by Sambrook et al. (42). Restriction enzymes and DNA-
modifying enzymes were used as recommended by the supplier (Promega, Mad-
ison, Wis.). Primary DNA manipulations were carried out in E. coli DH5?, and
restriction mapping was used to confirm that transformants contained the ap-
For cloning the protease gene, genomic DNA isolated from V. vulnificus
ATCC 29307 was partially digested with Sau3AI. DNA fragments in the 2- to
6-kb range were purified from agarose gels by using the Geneclean II kit (Bio
101, Inc., Vista, Calif.) and used for ligation into pUC18 digested with BamHI.
Competent cells of E. coli DH5? were transformed with the ligation products,
and the transformants exhibiting clear zones around colonies on nutrient agar-
skim milk plates were identified. One such clone contained a 2.5-kb insert and
was named pKC980.
The nucleotide sequence of the entire 2.5-kb insert of pKC980 was determined
by primer walking using the dideoxy-chain termination method with Top DNA
polymerase (Bioneer, Seoul, Korea). Comparisons of nucleotide and amino acid
sequences were conducted using BLAST (basic local alignment search tool) (1,
Insertional inactivation of the elastase gene by allelic exchange. To construct
mutant V. vulnificus strains deficient in the metalloprotease, a mutation was
introduced into the vvpE gene carried by pKC980. The 5? end and 3? end of the
DNA insert of pKC980, corresponding to the regions encoding the N-terminal 24
amino acids and the C-terminal 388 amino acids of the elastase, respectively,
were removed by digestion with PstI and HindIII. The overhang ends were blunt
ended using the Klenow fragment of DNA polymerase I. The resulting 0.6
kb-DNA fragment was isolated from an agarose gel and was ligated into the
allelic exchange suicide vector pNQ705 (22), which had been linearized with
EcoRV. The resulting plasmid, pKC9844, encodes the 5?- and 3?-truncated vvpE
gene as depicted in Fig. 1A. Since pNQ705 has the R6K ? origin for replication
requiring the ? protein (15) and can replicate only in bacterial hosts carrying the
TABLE 1. Bacterial strains and plasmids used in this study
Strain or plasmidRelevant characteristicsa
Reference or source
Clinical isolate; virulent
Clinical isolate; virulent
ATCC 29307 vvpE::pKC9844; elastase deficient
MO6-24/O vvpE::pKC9844; elastase deficient
supE44 ?lacU169 (?80 lacZ ?M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1
?(lac pro) argE(Am) recA56 gyrA rpoB ?pir; host for ?-requiring plasmids
thi thr leu tonA lacY supE recA::RP4-2-Tc::Mu ?pir; Kmr; host for ?-requiring plasmids;
Cloning vector; R6K ? ori (requires ?); oriT of RP4; Cmr
IncP ori; broad-host-range vector; oriT of RP4; Tcr
pUC18 with 2.5-kb partial Sau3A fragment encoding vvpE; Apr
pNQ705 with 0.6-kb HindIII-PstI fragment encoding internal coding sequence of vvpE;
for allelic exchange; Cmr
pRK415 with 2.5-kb EcoRI-SalI fragment carrying vvpE from pKC980; Tcr
aApr, ampicillin resistant; Cmr, chloramphenicol resistant; Kmr, kanamycin resistant; Tcr, tetracycline resistant.
VOL. 68, 2000EVALUATION OF A VIBRIO VULNIFICUS MUTANT5097
pir gene encoding the ? protein, the pKC9844 allelic exchange recombinant was
transformed into E. coli SY327?pir (22).
The truncated vvpE gene was then introduced into the V. vulnificus chromo-
some by homologous recombination as follows. pKC9844 was transformed into
E. coli SM10?pir, which supplies tra gene products to mobilize pKC9844 for
conjugation viathe RP4 originof
SM10?pir(pKC9844) was used as a conjugational donor to deliver pKC9844 into
the wild-type V. vulnificus strains ATCC 29307 and MO6-24/O. Conjugation was
carried out by methods described previously (45), with slight modifications.
Briefly, the recipient strains, V. vulnificus ATCC 29307 and MO6-24/O, and the
donor strain, SM10?pir(pKC9844), were grown overnight on agar plates, and
cells were recovered with sterile cotton swabs. Both cell masses were spotted and
mixed on LB agar. The mixture of cells was incubated for at least 8 h, suspended
in 1 ml of saline, and then spread on TCBS (thiosulfate citrate bile salts) agar (to
select against the E. coli donor) supplemented with skim milk (as an indicator for
protease activity) and chloramphenicol (to select for pKC9844). The desired
transconjugants were selected by chloramphenicol resistance and screened for
the inability to exhibit clearing of the skim milk. Potential mutants were subse-
quently tested for lack of elastase activity. The V. vulnificus elastase mutants
chosen for further analysis were named KC64 and CMM111, derived from the
parental strains ATCC 29307 and MO6-24/O, respectively.
Southern blot analysis. Approximately 10 ?g of genomic DNA isolated from
V. vulnificus ATCC 29307 and its protease mutant, KC64, was digested com-
pletely with EcoRI and separated on a 0.7% (wt/vol) agarose gel. After transfer
to a nitrocellulose membrane (Schleicher and Schuell, Keene, N.H.), the DNA
was fixed by UV irradiation and hybridized with a 0.6-kb PstI-HindIII DNA
probe representing the internal sequences of the vvpE coding region. The probe
was labeled with [?-32P]dCTP using the Prime-a-gene labeling system (Pro-
mega). The prehybridization and hybridization solutions consisted of 6? SSC
(1? SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 5? Denhardt’s reagent,
0.5% (wt/vol) sodium dodecyl sulfate (SDS), and 100 ?g of denatured, frag-
mented salmon sperm DNA/ml. Prehybridization and hybridization were done
for 2 and 16 h, respectively, at 65°C. After hybridization, the membranes were
washed at room temperature for 2 h in 0.1? SSC–0.4% SDS and then at 68°C for
1 h in the same solution. The blot was exposed using a phosphorimage analyzer
(BAS1500 model; Fuji Photo Film Co. Ltd., Tokyo, Japan).
Analysis of expression of vvpE mRNA and VvpE protein from insertion mu-
tants. (i) Northern blot analysis of vvpE mRNA. For Northern blot analysis, a
1.2-kb HindIII DNA fragment derived from the region of vvpE encoding the
C-terminal 389 amino acids was labeled and used as a probe. Total cellular RNA
from cultures of KC64 and ATCC 29307 grown to an OD600of 1.5 was isolated
by using the Trizol reagent kit according to the manufacturer’s specifications
(GIBCO-BRL, Gaithersburg, Md.) and was suspended in diethyl pyrocarbonate-
treated water. RNA was separated by agarose gel electrophoresis, transferred to
a nylon membrane, and hybridized as described previously (33), with slight
modifications. Briefly, 5.5 ?l of RNA (approximately 20 ?g) was mixed with 1 ?l
of 10? MOPS buffer [1? MOPS buffer is 0.02 M 3-(N-morpholino)propanesul-
fonic acid (pH 7.0), 5 mM sodium acetate (pH 5.2), and 1 mM EDTA], 3.5 ?l of
37% (wt/vol) formaldehyde, and 10 ?l of deionized formamide. The mixture was
heated for 10 min at 60°C and then mixed with 10 ?l of loading buffer (50%
[vol/vol] glycerol, 0.2% [wt/vol] xylene cyanole, 0.2% [wt/vol] bromophenol blue).
The RNA was separated by electrophoresis in a 1.2% (wt/vol) agarose gel
containing 1.1% (wt/vol) formaldehyde and 1? MOPS buffer. The gel was
soaked for 45 min in 10? SSC, and the separated RNA was transferred to a
nylon membrane (Nytran; Schleicher and Schuell) and fixed by UV irradiation.
The membrane was hybridized and developed by the same procedure as that
used for Southern blot analysis, except that the32P-labeled 1.2-kb HindIII DNA
probe was used.
(ii) Western blot analysis of VvpE protein. Wild-type and VvpE?mutant V.
vulnificus strains were grown in LBN broth to an OD420of approximately 3.25.
Cells were harvested by centrifugation, and supernatants were filter sterilized
through 0.22-?m-pore-size filters. Supernatants were concentrated 200-fold by
precipitation with trichloroacetic acid and suspension in water. Cells and con-
centrated supernatants were boiled in SDS-polyacrylamide gel electrophoresis
(PAGE) sample buffer (18). Equivalent amounts of cells and supernatants of the
various strains were electrophoresed by SDS-PAGE (4 to 15% [wt/vol] acryl-
amide, Tris-HCl precast Ready Gel; Bio-Rad, Hercules, Calif.), and proteins
were transferred to polyvinylidene difluoride membranes (Immobilon-P; Milli-
pore Corp., Bedford, Mass.) using Towbin transfer buffer (49). One of the
duplicate membranes was stained with Coomassie brilliant blue, and the second
was examined for the presence of VvpE protein by Western blot analysis using
the immunoglobulin G (IgG) fraction of rabbit anti-V. vulnificus elastase serum,
kindly provided by Shin-Ichi Miyoshi (27). Briefly, the gel was presoaked with
phosphate-buffered saline (PBS) containing 0.05% (wt/vol) Tween 20 (PBS-T)
and then incubated with a 1:500 dilution of the anti-elastase serum in PBS-T for
1.5 h at room temperature. The membrane was washed three times with PBS-T
and then incubated with goat anti-rabbit IgG conjugated with alkaline phospha-
tase (Sigma). After three more washes, the antibody-antigen complexes were
visualized by using the 5-bromo-4-chloro-3-indolylphosphate (BCIP)-nitroblue
tetrazolium (NBT) substrate (Sigma).
Subcutaneous infection of mice. We examined the virulence of V. vulnificus
ATCC 29307 and KC64 using both iron dextran-treated and normal ICR mice
transfer,oriT(21,45). E. coli
(specific pathogen free; Harlan Sprague-Dawley, Indianapolis, Ind.), as de-
scribed elsewhere (46a). Briefly, female mice from 7 to 11 weeks of age were
housed under specific-pathogen-free conditions. For experiments involving iron
treatment, mice were injected intraperitoneally with 250 ?g of iron dextran
(Sigma)/g of body weight immediately before subcutaneous (s.c.) injection into
the lower back with bacterial cells suspended in buffered saline containing 0.01%
(wt/vol) gelatin (BSG). Between 15 and 24 h later, the mice were euthanized with
carbon dioxide, the s.c. lesion was removed for enumeration of bacterial CFU
and histology, and a portion of the liver was removed for enumeration of CFU.
To enumerate CFU in tissues, samples were weighed, suspended in BSG, and
homogenized in glass tissue homogenizers. Homogenates were diluted in BSG
and plated on LBN. When mice died before harvest, they were assigned the
highest value obtained for the same tissue and the same strain in the same
experiment. Occasionally, mice were unaffected by the particular V. vulnificus
strains examined. These apparent inoculation failures are reported as such.
Bacterial cultures were initially grown as overnight, static LBN cultures at room
temperature. On the day of the infection, the overnight cultures were diluted
1:20 into fresh prewarmed (37°C) LBN and incubated with shaking until the
OD420reached 1.0 to 1.5. The cells were harvested by centrifugation and then
suspended and diluted in BSG for inoculation. For iron dextran-treated mice,
bacterial inocula of ATCC 29307 and KC64 ranged from 84 to 206 CFU per
mouse for one experiment and from 530 to 560 CFU per mouse for a repetition.
For normal mice, inocula were approximately 2 ? 107CFU per mouse for one
experiment and 2 ? 106to 4 ? 106CFU per mouse for a repetition. For
MO6/24-O and CMM111, inocula for iron dextran-treated mice ranged from 100
to 900 CFU/mouse among three different experiments. All manipulations of mice
were approved by the University of Florida Institutional Animal Care and Use
Histological analysis. Skin lesions were fixed in buffered 10% (vol/vol) forma-
lin, embedded in paraffin, and sectioned at 5 ?m at the University of Florida
Department of Pathology, Immunology, and Laboratory Medicine Diagnostic
Referral Laboratory. Sections were stained with hematoxylin and eosin. Initial
examination by the veterinary pathologist for lesion characteristics and severity
was conducted in a blinded manner.
LD50determination. Bacteria were grown in BHI-N broth overnight at 25°C.
The following day, 0.1 ml of the culture was inoculated into 100 ml of BHI-N
broth and shaken at 25°C. After 4 h of cultivation, bacterial cells were harvested
by centrifugation and suspended in PBS to appropriate concentrations. Groups
of six to eight normal female CD-1 mice (specific pathogen free; 8 weeks old;
Daehan Animal Co., Taejon, Korea) were injected intraperitoneally with 0.2 ml
of serial dilutions of bacterial suspensions. The infected mice were observed for
48 h, and the LD50s were calculated by the method of Reed and Muench (40).
Cell culture experiments. To examine the effects of the vvpE mutation on the
ability of V. vulnificus to damage epithelial cells, we performed three different
assays using INT407 intestinal epithelial cells. Bacterial cells for infection were
grown as described above for animal infection, except that the bacteria were
ultimately suspended in cell culture medium, Dulbecco’s modified Eagle medium
(DMEM) containing 10% (vol/vol) fetal calf serum (Life Technologies/BRL),
instead of BSG. INT407 cells grown in DMEM containing 10% fetal calf serum,
100 U of penicillin/ml, and 100 ?g of streptomycin/ml were seeded to achieve
approximately 70% confluence in 24-well culture plates (Costar) for the day of
infection. One hour before infection, antibiotic-containing medium was replaced
with antibiotic-free medium. The INT407 cells were infected at a multiplicity of
infection of 5 and allowed to incubate at 37°C for 1 h, at which time gentamicin
was added to a final concentration of 100 ?g/ml to kill the bacteria. This was
done to prevent the bacteria from rapidly multiplying in the cell culture medium
and destroying the monolayers before cytopathology could be examined. Bacte-
rial cells and any extracellular products remained in the cell culture wells for the
duration of the experiment. Twenty-four hours later, the infected cell cultures
were assayed for lysis of host cells by using the Cytotoxicity Detection Kit
(Boehringer Mannheim), killing and/or destruction of the cell monolayer with
the CellTiter 96 AQueous Cell Proliferation Assay kit (Promega), and detach-
ment of host cells by a modification of a crystal violet staining technique (41).
The Cytotoxicity Detection Kit measures lactate dehydrogenase (LDH) activ-
ity released into the culture supernatant by lysed cells. At harvest time, a sample
of supernatant from experimental wells (either infected or uninfected) was cen-
trifuged at 250 ? g for 10 min to remove cells and debris. To measure total LDH
activity in either infected or uninfected wells, Triton X-100 was added to the
wells containing host cells and the remaining supernatant at a final concentration
of 1% (vol/vol) to lyse host cells. The colorimetric assay to measure LDH activity
was then performed according to the manufacturer’s instructions, by reading the
A490. For each well, we computed the LDH activity in the supernatant divided by
the total LDH activity in the well. The percent LDH release by uninfected cells,
approximately 15%, was subtracted from the percent LDH release in V. vulni-
ficus-infected wells. Triplicate wells were run for each sample, and the experi-
ment was performed at least twice.
The CellTiter Proliferation Assay measures nonspecific dehydrogenase activity
of metabolically active cells that remain attached to the culture dish. After the
24-h treatment period, the wells were rinsed twice with Hanks balanced salt
solution (HBSS) to remove dead and detached cells. DMEM (0.5 ml) containing
100 ?g of gentamicin/ml was added back, followed by 0.1 ml of the tetrazolium
reagent. The reaction was developed and the A490was read according to the
5098JEONG ET AL.INFECT. IMMUN.
manufacturer’s instructions. We report the percent loss of viable and attached
cells in infected wells compared to that in uninfected wells. Triplicate wells were
run for each sample, and the experiment was performed three times.
The crystal violet staining assay measures the amount of cell mass remaining
attached to culture dishes after infection. This assay was originally developed to
measure the effects of tumor necrosis factor alpha on cell lines (41). After the
24-h treatment period, wells were rinsed twice with PBS, followed by staining
with 0.167% (wt/vol) crystal violet diluted in PBS. After 10 min the wells were
rinsed with PBS until no further leaching of crystal violet was observed. The
crystal violet stain in attached cells was released by rinsing the wells with 95%
(vol/vol) ethanol. The relative concentration of crystal violet was measured by the
A490. As for the CellTiter Proliferation Assay, we calculated the percentage of
INT407 cells detached from infected wells compared with that for uninfected
wells. Triplicate wells were run for each sample, and the experiment was per-
Nucleotide sequence accession number. The nucleotide sequence of V. vulni-
ficus ATCC 29307 vvpE was submitted to GenBank and was assigned accession
Cloning and sequencing analysis of vvpE from V. vulnificus
ATCC 29307. A fragment of genomic DNA from V. vulnificus
ATCC 29307 that conferred elastolytic activity on E. coli
DH5? was cloned as described in Materials and Methods,
yielding plasmid pKC980. The nucleotide sequence of the
2.5-kb DNA insert containing the vvpE structural gene and the
upstream regulatory region was determined, revealing a coding
region consisting of 1,830 nucleotides. The deduced amino
acid sequence revealed a protein of 609 amino acids with a
theoretical molecular mass of 65,965 Da and a pI of 6.07. The
nucleotide sequence of V. vulnificus ATCC 29307 vvpE was
submitted to GenBank and was assigned accession number
The N-terminal 20-amino-acid sequence previously deter-
mined from purified metalloprotease of V. vulnificus by
Kothary and Kreger (17) is present within the deduced 609-
amino-acid polypeptide encoded by vvpE. The two amino acid
sequences are nearly identical, with the initial residue of the
Kothary and Kreger sequence corresponding to our Ala resi-
due at deduced position 197. There was a single difference at
amino acid residue 200, with an Asn in the Kothary and Kreger
sequence and an Asp in VvpE. Thus the 609-amino-acid VvpE
polypeptide is presumed to be processed by deleting the N-
terminal 196 amino acids to form the mature protein. Further-
more, the N-terminal amino acids show the typical pattern of
putative leader peptides, as observed in other metalloproteases
(12). The signal peptide is predicted to be cleaved between
Ala24 and Ala25 based on the von Heijne method (51). The
mature elastase protein is therefore deduced to consist of 413
amino acids, with a calculated molecular weight of 45,430 Da.
This result was not unexpected, since many other bacterial
metalloproteases have been shown to rely on signal and leader
sequences to aid in transport across the bacterial membrane, in
addition to undergoing other stages of maturation. Further-
more, the calculated molecular weight and that observed by
Western blot analysis (see below) are in good agreement with
those observed for the purified metalloprotease of V. vulnificus
A database search for similar sequences revealed two other
protease genes, vvp and empV, cloned from V. vulnificus strains
YJ061 and CKM-1, respectively, whose nucleotide sequences
showed high levels of identity with the vvpE sequence. The
vvpE coding sequence is between 97 and 99% identical with
those of empV and vvp, respectively. The deduced amino acid
sequence of VvpE was 98% (599 of 609 amino acids) and 96%
(570 of 589 amino acids) identical to those of the EmpV and
Vvp proteases of V. vulnificus, respectively. Furthermore, the
amino acid sequence of VvpE contains the conserved regions,
such as zinc-binding residues (H-343-X-V-S-H-347) and ac-
tive-site residues (V-425-H-X-X-S-G-X-X-N-X-A-X-Y-437),
observed in many other extracellular zinc-containing metallo-
proteases of Vibrio spp. (12). All of this information confirmed
that the vvpE gene encodes the metalloprotease gene of V.
vulnificus ATCC 29307.
Construction of V. vulnificus isogenic mutants deficient in
elastase activity. We used standard suicide vector methods to
insertionally inactivate the vvpE genes of the wild-type V. vulni-
ficus strains ATCC 29307 and MO6-24/O. Plasmid pKC9844,
encoding 5?- and 3?-truncated vvpE, was integrated into the
genomes of V. vulnificus ATCC 29307 and V. vulnificus MO6-
24/O by a single homologous recombination event, leading to
a partial diploid of the vvpE gene consisting of two mutant
genes truncated in the N terminus- or C terminus-coding re-
gions separated by intervening vector DNA (Fig. 1A). The
insertional disruption of vvpE in the mutants was confirmed by
Southern blot analysis (Fig. 1B). When wild-type V. vulnificus
ATCC 29307 genomic DNA digested with EcoRI was hybrid-
ized with the internal coding sequence probe, a 10-kb band was
observed. A representative strain of the caseinase-negative
transconjugants, KC64, was chosen from those insertional mu-
tants whose EcoRI-digested genomic DNA produced bands of
approximately 3.5 and 7.0 kb hybridizing with the32P-labeled
vvpE probe, as shown in Fig. 1B. Since plasmid pKC9844
carries three EcoRI sites from the vector, pNQ705, this pattern
of hybridization confirms that the vvpE gene of KC64 was
disrupted by insertion of vector DNA of pKC9844, as depicted
in Fig. 1A. Similarly, the vvpE mutant strain CMM111 was
constructed from parental strain MO6-24/O.
To determine the stability of the insertional mutation, V.
vulnificus KC64 was grown overnight without chloramphenicol
selection. The inserted plasmid DNA was stably maintained, as
determined by maintenance of chloramphenicol resistance (all
of more than 500 colonies tested) and Southern analysis (data
not shown). The mutation was also stably maintained during
growth in mice, since most, if not all, colonies of KC64 isolated
from infected mice retained chloramphenicol resistance and
lack of elastase activity (data not shown).
Total protease and elastase activities of V. vulnificus KC64.
The mutant strain KC64 was characterized for total protease
and elastase activities relative to those of the wild-type strain
ATCC 29307. For ATCC 29307 both total protease activity and
elastase activity were produced during growth at mid-exponen-
tial phase and reached a maximum during stationary phase
(Fig. 2A). The disruption of vvpE in KC64 resulted in a com-
plete loss of elastase activity and also reduced the production
of total protease activity. The residual level of total protease
activity in KC64 corresponded to approximately one-third that
in the wild type (Fig. 2B). These data demonstrated that the
vvpE gene encoded the elastase activity of V. vulnificus. The
observation that the mutant still exhibited protease activity
revealed the existence of at least one more protease being
produced by V. vulnificus ATCC 29307. Therefore, we have
designated vvpE to represent V. vulnificus elastase, in order to
differentiate it from the other genes encoding other potential
proteases of V. vulnificus.
Although it seemed unlikely that the decrease in protease
activity by two-thirds resulted from polar effects of the vvpE
insertional mutation on downstream genes, this possibility
could not be ruled out a priori. Therefore, we investigated
whether reintroduction of recombinant vvpE could comple-
ment the lack of elastase activity of KC64 cells. A 2.5-kb
EcoRI-SalI fragment carrying vvpE was isolated from pKC980
and was subcloned into vector pRK415 (13) digested with
HindIII. Since pRK415 has an IncP1 origin and RP4 oriT, the
VOL. 68, 2000 EVALUATION OF A VIBRIO VULNIFICUS MUTANT 5099
resulting plasmid, pRKY980, was mobilizable into KC64 by
conjugation. The protease activity of KC64(pRKY980) was
restored to a level comparable to the wild-type level of ATCC
29307 (Fig. 2C). Therefore, the decreased protease activity of
KC64 resulted from inactivation of functional vvpE rather than
from any polar effects on any genes downstream of vvpE.
Confirmation of lack of vvpE mRNA and VvpE protein in
mutant strains. Because the vvpE mutants were created by
insertional inactivation of the wild-type gene by using a suicide
plasmid encoding a DNA fragment internal to the coding se-
quence, it was possible, though unlikely, that one of the two
resulting partial genes, that encoding the C terminus, could
still be expressed and yield a mature peptide. To examine this
possibility, we performed both Northern and Western blot
analyses of the parental and mutant strains. Northern blot
analysis using a probe to the 3? end of the vvpE coding se-
quence identified a vvpE transcript of approximately 2.0 kb
produced by the wild-type strain ATCC 29307 but not by the
mutant strain KC64 (data not shown). Western blot analysis of
parental and vvpE mutants for both sets of V. vulnificus strains
demonstrated that no anti-elastase-reactive proteins were pro-
duced by the mutants in either culture supernatants (Fig. 3) or
whole cells (data not shown). In addition to the lack of elastase
activity (Fig. 2), these combined results confirm that the vvpE
gene disruptions eliminate all detectable expression of the
vvpE gene and VvpE protein. This result was not unexpected
given that the mutant copy of the vvpE gene encoding the
C-terminal portion of VvpE had no promoter, ribosome-bind-
ing site, or start codon.
vvpE is not essential for virulence of V. vulnificus in mice.
Several studies have demonstrated numerous activities of met-
alloproteases of V. vulnificus when they are injected into ani-
mals (17, 23, 25–28, 32). The effects observed were consistent
with the pathogenesis of infection in animals, including vaso-
FIG. 1. Construction of vvpE isogenic mutant by insertional mutagenesis. (A) Homologous recombination between strain ATCC 29307 and plasmid pKC9844
resulted in insertional mutation of vvpE in strain KC64. Dashed lines, the V. vulnificus chromosome; solid line, the plasmid sequence; open rectangles, the target vvpE
gene; solid rectangles, the truncated vvpE gene; large X, genetic crossover. Abbreviations: E, EcoRI; cat, chloramphenicol acetyltransferase gene. (B) Southern blot
analysis of strain KC64, generated by homologous recombination. Genomic DNAs from strain ATCC 29307 and strain KC64 were digested with EcoRI and hybridized
to a32P-labeled DNA probe consisting of a HindIII-PstI fragment internal to the vvpE coding sequence. The positions of hybridization are indicated in kilobases at
the right. Details of the procedure are described in Materials and Methods.
FIG. 2. Growth kinetics and activities of total protease and elastase in V. vulnificus strains. V. vulnificus strains were grown in nutrient broth as described in Materials
and Methods. Samples removed at the indicated times from cultures of strains ATCC 29307 (A), KC64 (B), and KC64(pRKY980) (C) were analyzed. I, cell density;
E, total protease activity; F, elastolytic protease activity.
5100JEONG ET AL.INFECT. IMMUN.
dilation, increased vascular permeability, edema, and necrosis.
To examine the effects of the lack of elastase activity on the
virulence of V. vulnificus during infection, we used both iron
dextran-treated and normal mice inoculated s.c. with either
wild-type V. vulnificus ATCC 29307 or the VvpE?strain KC64.
In iron dextran-treated mice, s.c. injection of approximately
102CFU of either strain resulted in extremely ill mice and
occasional death within 24 h postinoculation. There was no
difference between the counts of ATCC 29307 and KC64 bac-
teria recovered from s.c. lesions, which were as high as 108
CFU/g of tissue for both strains (Fig. 4A). The s.c. lesions
produced by the parent and metalloprotease mutant strains
were indistinguishable at the gross (Fig. 5) and histological
(Fig. 6) levels from those observed in our analysis of other
virulent V. vulnificus strains in this model (46a). In Fig. 5 it is
clear that both ATCC 29307 and KC64 produced extensive
hemorrhagic and edematous lesions, with dilation of the asso-
ciated vasculature. The regional lymphatics were also inflamed
(Fig. 5). Histopathological findings included extensive edema
of the dermis and edema and necrosis of s.c. tissue (Fig. 6),
with extension of infection through the dermis up to the epi-
dermis. It is notable that, despite the red coloration of the
lesions, intact red blood cells were rarely observed outside of
blood vessels, most likely because they had been lysed by the
hemolytic activity of the vibrios. Bacteria were easily observed
in the s.c. lesion, with necrotic or degenerating polymorpho-
nuclear leukocytes nearby. We also occasionally observed
perivascular infection by either of the V. vulnificus strains lo-
calized to dilated blood vessels. To examine the effects of the
vvpE mutation on the spreading of the infection beyond the
site of inoculation, we examined the CFU in the livers of the
s.c. inoculated mice. As was seen for infection of s.c. tissues,
there was no significant difference in CFU per gram of liver
tissue between the wild-type strain ATCC 29307 and the
VvpE?mutant KC64, with yields of approximately 106CFU/g
of tissue (Fig. 4A). Therefore, the elastase also appeared to be
dispensable for vibrios reaching and multiplying in the liver.
Since it had been proposed that the metalloprotease could
remove iron from transferrin (35) and increase the utilization
of heme (34), we also examined the effects of the metallopro-
tease mutation on disease in non-iron-treated mice. To cause
disease in this model, 106to 107CFU of either strain had to be
injected s.c.; however, s.c. lesions were still observed with levels
of infection indistinguishable between the parent and protease
mutant (Fig. 4A). Recovery of bacteria from livers was mostly
undetectable for either strain in non-iron-treated mice, in spite
of the relatively high levels of s.c. infections. Therefore, in the
mouse model of infection by s.c. inoculation, in which both
FIG. 3. Lack of secretion of VvpE by mutant V. vulnificus. Parental and vvpE
mutant V. vulnificus strains were grown in LBN to late-exponential-phase growth.
Cells (not shown) and supernatants were then examined for the presence of
VvpE elastase protein by Western blot analysis using a rabbit anti-V. vulnificus
elastase antibody (27), as described in Materials and Methods. Lane A, 46-kDa
molecular mass marker; lanes B and D, the wild-type parental strains ATCC
29307 and MO6-24/0, respectively; lanes C and E, the corresponding VvpE?
mutant strains KC64 and CMM111, respectively.
FIG. 4. Lack of effect of the vvpE mutation on infection of mice by V. vulnificus. Mice were either injected intraperitoneally with iron dextran (? Fe) or not
(?Fe) immediately before s.c. inoculation of the wild-type strain V. vulnificus ATCC 29307 (VvpE?) or the elastase mutant strain KC64 (VvpE?) (A) or the wild-type
V. vulnificus strain MO6-24/O (VvpE?) or the elastase mutant strain CMM111 (VvpE?) (B). Fifteen to 24 h later, CFU in skin lesion and liver were quantified as
described in the text. Error bars, standard deviations. (A) For VvpE?infection of iron-treated mice, n ? 8, plus 1 unaffected mouse; for VvpE?infection of iron-treated
mice, n ? 10; for infections of non-iron-treated mice, n ? 10. The differences in CFU per gram of tissue between the VvpE?and VvpE?strains were not significant
in any pair (P ? 0.2). (B) Combined results of three experiments are shown. For VvpE?infection of iron-treated mice, n ? 10, plus 2 unaffected mice; for VvpE?
infection of iron-treated mice, n ? 10, plus 3 unaffected mice. The differences in CFU per gram of tissue between the VvpE?and VvpE?strains were not significant
in any pair (P ? 0.75).
VOL. 68, 2000 EVALUATION OF A VIBRIO VULNIFICUS MUTANT 5101
local and systemic aspects of infection are measured, the VvpE
metalloprotease was completely dispensable for disease.
To examine the effects of the vvpE mutation in the MO6-
24/O background, we examined infection of s.c. inoculated,
iron dextran-treated mice. As shown in Fig. 4B, there was no
significant difference in either skin lesion or liver CFU between
the parental strain MO6-24/O and the vvpE mutant CMM111.
We also examined the effects of the vvpE mutation on the
lethality of both the ATCC 29307 and MO6-24/O strain back-
grounds in non-iron-treated mice (Table 2). The intraperito-
neal LD50s of the vvpE mutant strains were very similar to
those of the respective parent strains. Therefore, the lack of a
significant change in the mouse virulence phenotype caused by
the vvpE mutation is observed in both V. vulnificus strain back-
The VvpE metalloprotease has no phenotype in interactions
of V. vulnificus with epithelial cells in vitro. Since many of the
biological activities of protease from V. vulnificus are associ-
ated with effects on host cells, we examined the parental strain,
ATCC 29307, and the protease mutant, KC64, for different
effects on INT407 intestinal epithelial cells. We measured the
abilities of the strains to lyse the epithelial cells by release of
LDH from the host cells. As shown in Fig. 7, KC64 exhibited
cytolytic activity similar to that of ATCC 29307, with approx-
imately 70% LDH release higher than the background of un-
infected cells, which release approximately 15% of total LDH
(P ? 0.00005 compared with either V. vulnificus strain). To
examine the ability of the bacteria to cause the detachment of
host cells from culture dishes without necessarily lysing them,
we used the CellTiter Proliferation Assay on vibrio-infected
cell cultures. This assay produced somewhat variable results;
however, there was no consistent difference between the abil-
ities of the protease mutant and the wild-type parent to detach
INT407 cells after 24 h of interaction (Fig. 7). In other repe-
titions of the experiment (not shown), the protease mutant
showed either lower or higher activity than the parent strain in
causing detachment of INT407 cells. Finally, we examined the
abilities of the V. vulnificus strains to cause the detachment or
release of the host cells from the cell culture plates, known
effects of proteases, using an assay that measures crystal violet
staining of the INT407 cells remaining attached to culture
dishes (41). In agreement with results obtained using the Cell-
Titer Proliferation Assay, ATCC 29307 and KC64 caused sim-
ilar levels of detachment after 24 h of treatment, approximately
80 to 85% (Fig. 7). Therefore, using these in vitro cell culture
assays, we could not identify a virulence-associated phenotype
for the VvpE elastolytic protease by comparing the wild-type
parent, ATCC 29307, with its isogenic metalloprotease mutant,
Disease caused by infection with V. vulnificus is remarkable
for the invasive nature of the infection, ensuing severe tissue
damage, and rapidly fulminating course. Understanding the
molecular pathogenesis of this multifaceted host-pathogen in-
teraction is critical for the development of improved treatment
and prevention, as well as elucidating how certain bacteria can
circumvent host defenses, multiply in the host, and cause such
extensive damage. The characterization of somatic as well as
secreted products of V. vulnificus has yielded a large list of
putative virulence attributes, whose known functions are easily
imagined to explain the pathology of disease. These putative
virulence factors include a carbohydrate capsule, lipopolysac-
charide, a cytolysin/hemolysin, an elastolytic metalloprotease,
iron-sequestering systems, a lipase, and pili. However, only the
capsule and iron acquisition systems have been confirmed to be
essential for virulence by the use of the molecular version of
Koch’s postulates (19, 55). Notably, a null mutation in the gene
encoding hemolysin had no effect on virulence in mice (52). A
FIG. 5. Dorsal view of gross pathology of s.c. lesions caused by infection with
VvpE?and VvpE?V. vulnificus strains. Mice were injected intraperitoneally
with iron dextran. Immediately afterwards mice were injected s.c. in the lower
right dorsal quadrant with 102CFU of either the parental V. vulnificus strain
ATCC 29307 (B) or the VvpE?mutant KC64 (C). Control mice (A) received no
further injections after iron dextran. Between 15 and 24 h later, mice were
euthanized, and the skin was peeled back from head to tail to reveal s.c. tissues.
In panels B and C a large, edematous, hemorrhagic lesion is visible unilaterally
in the area surrounding the injection site. The regional lymph node is inflamed
(white arrows), and the localized vasculature is dilated. Some lesion material is
adherent to the underlying musculature over the lower back, as well.
5102JEONG ET AL.INFECT. IMMUN.
prepilin peptidase, whose function is essential for the secretion
of numerous proteins by V. vulnificus including elastase, he-
molysin, and chitinase, has also been shown to be essential for
virulence in mice (37). However, which of the observed or as
yet unidentified secreted proteins affected by the prepilin pep-
tidase mutation are responsible for the attenuation is un-
known. In the present study we focused on analysis of the
elastase, since numerous studies have shown that injection of
the protease into experimental animals can reproduce many
aspects of the disease process observed during experimental
infection with viable V. vulnificus.
Elastase, a metalloprotease with a broad substrate specific-
ity, including biologically important host molecules, has been
suggested to be an important virulence factor of various hu-
man-pathogenic bacteria (12). The well-characterized elastase
of Pseudomonas aeruginosa is capable of degrading or inacti-
vating elastin, collagen, immunoglobulins, serum complement
factors, and some plasma proteins (7, 39). This enzyme is
important for massive tissue destruction, which may aid the
bacteria in invading the host (7, 39). There have been several
different lines of evidence leading to the hypothesis that elas-
tase is an important, if not an essential, component of virulence
for V. vulnificus during infection of animals. Many of these
studies involved injection of purified protein into animals, with
the production of specific symptoms or pathology observed
during infection. For example, injection of elastase resulted in
dermonecrosis and toxicity in mice (17). Edema can be caused
by combined increases in vasodilation and vascular permeabil-
ity. V. vulnificus elastase can affect vascular permeability by
stimulating histamine release from mast cells (27) and by stim-
ulating the production of bradykinin (23), which acts on the
vascular endothelium. Interestingly, elastase stimulates the
bradykinin pathway in two different steps: first in the initiation
events by activation of Hagemann factor and later by the cleav-
age of prekallikrein to kallikrein (30, 32). Most recently, Miyo-
shi and colleagues (26) examined in great detail the pathology
associated with the injection of elastase in order to understand
the mechanism of edema and hemorrhage. They concluded
that the elastase degraded type IV collagen of the basement
membrane underlying the vascular endothelium. The lack of
support for the endothelium, coupled with the increase in
vascular permeability due to stimulation of histamine release
and bradykinin production, the decrease in clotting due to
stimulation of fibrinolysis, and necrosis of the vascular endo-
thelium, could all contribute to the massive edema and hem-
orrhage observed during infection. Consistent with this hy-
pothesis, administration of antibodies to collagen decreased
the effects of infection on the vasculature.
In contrast to the reported effects of administering V. vulni-
ficus protease in experimental animals, the analysis of down-
FIG. 6. Histopathology of s.c. lesions caused by infection with VvpE?and VvpE?V. vulnificus strains. Mice were injected intraperitoneally with iron dextran.
Immediately afterwards, mice were injected s.c. in the lower right dorsal quadrant with 102CFU of either the parental V. vulnificus strain ATCC 29307 (B) or the VvpE?
mutant KC64 (C). Control mice (A) received no further injections after iron dextran. Between 15 and 24 h later, mice were euthanized, and a tissue sample from the
lesion was fixed in formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin, as described in the text. In all panels, the dermis is labeled with
the latter “d.” In panel A the dermis is intact, with thick, intensely staining, and dense collagen fibers. In panels B and C the dermis is edematous, as evidenced by the
less-intense, open staining. Infection by both strains of V. vulnificus extended into the dermis. The s.c. region beneath the cutaneous muscle layer is labeled with the
letter “s.” In panel A the tissue is populated with healthy cells with well-defined nuclei. In panels B and C the tissues contain necrotic cells with condensed nuclei, fibrin
deposition, and numerous vibrios.
TABLE 2. Effect of the vvpE mutation on the lethality
of V. vulnificus to normal mice
Wild typevvpE mutantb
ATCC 29307 (n ? 8)
MO6-24/O (n ? 10)
1.8 ? 107
2.8 ? 106
5.8 ? 107
3.8 ? 106
aParental V. vulnificus strain background. n, number of mice for each inocu-
lation group, ranging from 104to 109CFU in 10-fold increments.
bThe vvpE mutants of ATCC 29307 and MO6-24/O are KC64 and CMM111,
respectively (see Table 1).
VOL. 68, 2000EVALUATION OF A VIBRIO VULNIFICUS MUTANT 5103
regulation of elastase activity by host protease inhibitors has
complicated the overall picture. For example, elastase is inhib-
ited by ?2-macroglobulin, normally found in plasma (16, 28,
30). It was originally proposed that the presence of ?2-macro-
globulin in plasma would restrict the biological action of the
protease to the interstitial tissues. However, elastase activity
could not be detected in interstitial fluids, even in the presence
of high levels of V. vulnificus infection (3). It was later found
that leakage of plasma into tissues, i.e., edema formation,
caused the inhibition of elastase by ?2-macroglobulin (28, 29).
Therefore, it appeared that the success of the protease in
causing edema could ultimately result in elimination of its
Another likely unrelated function of elastase is the release of
or acquisition of heme from heme-binding host proteins and
iron from transferrin (34, 35). This was shown directly by
demonstrating that the protease could degrade hemoglobin. In
contrast to the wild-type parent, protease-deficient mutants
were unable to grow in synthetic medium containing haptoglo-
bin-hemoglobin unless V. vulnificus protease was added. Sim-
ilarly, protease-deficient mutants could not acquire iron from
As opposed to the studies described above, in which the
biochemical activities of elastase were examined in vitro or in
which the biological activities were examined in animal models,
further evidence supporting a role for elastase in virulence was
provided by inhibiting its function in vivo by administration of
biochemical inhibitors of the enzyme, antibodies neutralizing
the protease, or mutant vibrios. For example, administration of
soybean trypsin inhibitor or anti-metalloprotease antibodies
inhibited increased vascular permeability during infection (27).
Furthermore, Miyoshi and Shinoda (27) constructed ni-
trosoguanidine-induced mutant V. vulnificus strains that were
defective in protease activity. These mutant strains also caused
less vascular permeability in rat skin and exhibited lower levels
of virulence, as measured by CFU in tissues or LD50. However,
the exact nature of the mutations was not determined, and, as
described above for the prepilin peptidase which exhibits nu-
merous effects on protein secretion, it is possible that factors
other than metalloprotease were affected by the mutations.
What was needed was the construction of a defined mutation
in the elastase gene.
We therefore undertook this study to identify the function of
the metalloprotease during an infectious process, rather than
the artificial system of injecting purified protein, by construct-
ing isogenic metalloprotease mutants of V. vulnificus ATCC
29307 and MO6-24/O and applying the molecular version of
Koch’s postulates (6, 11). When the isogenic vvpE mutants
were compared with the parental strain for virulence in s.c.
inoculated mice, the mutants did not show any significant dif-
ferences in any aspect of the disease process (Fig. 4 through 6;
Table 2). The morbidity of iron dextran-treated mice inocu-
lated with ?103CFU of the VvpE?and VvpE?strains was
identical, and the gross pathology (Fig. 5) and histopathology
(Fig. 6) of the s.c. lesions were indistinguishable. Consistent
with our other studies (Starks et al., submitted), s.c. lesions
were extremely edematous, with extensive necrosis of host
tissue and death of phagocytes in regions of infection. The
extension of infection upwards into the dermis from the s.c.
inoculation site was the same for the wild type and the VvpE?
mutant, suggesting that the collagenase activity of the metal-
loprotease was not necessary for the spread of the infection
through the skin layers. These results argue that previously
characterized effects on host cells and vasculature observed
during V. vulnificus infection and reproduced by injection of
purified metalloprotease do not, in fact, require the production
of elastolytic metalloprotease in vivo during an infection. Fur-
thermore, the quantitation of infection in s.c. tissues, which
most likely reflects the growth of V. vulnificus due to degrada-
tion of host tissues, leakage of fluids from the vasculature into
tissues, and putative destruction of proteinaceous antimicro-
bial effectors such as complement, was not significantly differ-
ent between the VvpE?and VvpE?strains (Fig. 4). In addi-
tion to the putative role of the metalloprotease in localized
infection, we also examined the role of VvpE in the systemic
spread of V. vulnificus to deeper tissues such as the liver. As
shown in Fig. 4, there was again no significant difference in
CFU recovered between wild-type and VvpE?strains. Impor-
tantly, colonies of the VvpE?mutant recovered from all tis-
sues of mice retained their caseinase-negative phenotype, as
observed upon plating on skim milk agar media. This control
was essential, since the mutation we constructed could revert
by excision of the suicide plasmid pKC9844 from the vvpE
The metalloprotease has been reported to aid in the acqui-
sition of iron and heme from host proteins (34, 35). We con-
sidered the possibility that using the iron dextran-treated
mouse model could preclude these iron-associated functions
from being important due to excess levels of iron. Therefore,
we repeated s.c. inoculation of the ATCC 29307 pair of strains
using normal mice. Lack of iron treatment required an in-
crease in inocula to 106to 107CFU, and the resulting pathol-
ogy was greatly reduced compared with that in iron-treated
mice. However, s.c. lesions still contained equivalent bacterial
counts regardless of whether the parent or the mutant strain
was used (Fig. 4A). The added pressure of normal levels of
iron did not enable the detection of a virulence phenotype for
the VvpE elastase in s.c. inoculated mice.
In agreement with our results obtained by infection of mice,
FIG. 7. Lack of effect of the vvpE mutation on the cytopathology of V.
vulnificus in INT407 cells. INT407 intestinal epithelial cells were infected at a
multiplicity of infection of 5 for 1 h before addition of gentamicin at 100 ?g/ml.
Twenty-four hours later, lysis was measured using LDH release into culture
supernatant; values are expressed as percent release from vibrio-infected cells
compared with total LDH (Triton X-released) of vibrio-infected cells, with the
background lysis (15%) of uninfected cells subtracted. The proliferation assay
measures metabolically active cells remaining attached to culture dishes; values
are expressed as percent loss compared with uninfected wells. The crystal violet
assay measures percent loss of crystal violet-staining material for vibrio-infected
cell cultures compared with uninfected wells. Triplicate wells of each experiment
were run, and these assays were repeated at least once. The values for the VvpE?
and VvpE?strains were not significantly different in any of the three assays (P ?
5104 JEONG ET AL.INFECT. IMMUN.
no significant differences were observed between the effects of
wild-type and VvpE?mutant V. vulnificus ATCC 29307 on
INT407 cells in culture (Fig. 7). The experiments were de-
signed to assay effects on the host cells by vibrio products
produced during an initial 1-h infection period, in which the
bacteria rapidly multiply in the tissue culture medium. Twenty-
four hours after infection, similar levels of lysis, death, and
detachment for the epithelial cells were caused by the wild-
type and VvpE?strains (Fig. 7). It should be noted that we
examined effects on epithelial cells, not vascular endothelial
cells, which could be more relevant during infection. However,
the in vivo infection data, both quantitative CFU and qualita-
tive pathology, failed to support a differential effect of the
elastase on virulence. It is likely that the effects on epithelial
cells that we observed for both strains were due to the action
of the hemolysin/cytolysin. These results demonstrate that the
vvpE gene is not essential for virulence of V. vulnificus in these
animal and cell culture models.
The major problem to be addressed is the discrepancy be-
tween our infection experiments and those studies that relied
primarily on the injection of proteins into animals. First, it is
noteworthy that our vvpE mutant, which lacked elastase activ-
ity, exhibited residual total-protease activity, revealing the ex-
istence of at least one more gene for protease. Although other
explanations are possible, the lack of significant difference in
virulence between the vvpE mutant and the wild-type parent
could be related to the presence of this other protease(s) of V.
vulnificus. We find it difficult to imagine that the effects of
inactivation of elastase were completely compensated by ex-
pression of the other protease(s) in V. vulnificus; however,
analysis of this hypothesis awaits identification and mutation of
the gene(s) encoding the other protease(s). We therefore cau-
tion that our results demonstrate that the metalloprotease
encoded by the vvpE gene of V. vulnificus is dispensable for
virulence in mice; however, we cannot make any conclusions as
to the role of proteases in general. Additionally, the cytolysin/
hemolysin has the potential of duplicating several aspects of
the activity of the elastase, including stimulation of histamine
release, vascular permeability, and toxicity (10, 38, 56). The
virulence of most organisms is multifactorial, and backup or
redundant virulence factors have often been identified. The
best-known examples involve adherence factors, such that in
order to observe effects of inactivation of specific adhesins, the
specific mutations must be examined in the background of
mutations in the redundant systems (50). Whether the elasto-
lytic protease is completely dispensable in the mouse model of
infection or whether redundant and fully compensatory viru-
lence factors exist in V. vulnificus remains to be determined.
However, our results strongly underscore the necessity of ex-
amining putative virulence attributes by using the molecular
version of Koch’s postulates (6, 11) in addition to injecting
purified bacterial products into animal or cell culture systems.
We are indebted to D. Milton for providing the plasmid pNQ705
and the E. coli strains with ? pir. We thank Shin-Ichi Miyoshi,
Okayama University, for kindly providing rabbit anti-V. vulnificus elas-
tase antibody. We thank Trenton R. Schoeb for his expertise and
assistance in examining histopathological results and Thomas J. Doyle
for his expert technical assistance with animal and cell culture exper-
This study was supported by a grant to J.H.R. and S.H.C. from the
KRF (1998-019-F00032), Republic of Korea. Research in the labora-
tory of P.A.G. was supported by the USDA (USDA-NRICGP
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Editor: J. T. Barbieri
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