Modulation of Vibrio mimicus hemolysin through limited
proteolysis by an endogenous metalloprotease
Tamaki Mizuno
1
, Syed Z. Sultan
1
, Yoshimi Kaneko
1
, Tomonaga Yoshimura
1
, Yoko Maehara
1
,
Hiroshi Nakao
1
, Tomofusa Tsuchiya
1
, Sumio Shinoda
2
and Shin-ichi Miyoshi
1
1 Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Japan
2 Faculty of Science, Okayama University of Science, Japan
Vibrio mimicus is a Gram-negative bacterium closely
related to Vibrio cholerae, the etiologic agent of the
epidemic cholera [1]. However, this species causes
sporadic gastroenteritis and food poisoning in
humans [1]. Because V. mimicus is ubiquitous in
fresh and brackish aquatic environments [2], infection
usually occurs after the consumption of raw fish
and ⁄or shellfish contaminated with the bacterium
[1,3].
Although several enterotoxic factors, including a
cholera toxin-like enterotoxin, have been isolated [4,5],
a heat-labile hemolysin, designated V. mimicus hemol-
ysin (VMH) [6], may be the most important virulence
factor of the pathogen. Purified VMH can induce fluid
accumulation when injected into a ligated ileal loop
[6,7] and specific antibody against VMH significantly
inhibits fluid accumulation [6,8]. A VMH-negative
mutant showed reduced ability to cause fluid accumu-
lation [8]. Moreover, the gene encoding VMH is pres-
ent in all strains from various clinical and
environmental sources [9]. Recently, in vitro studies
have demonstrated that the enterotoxic action
of VMH may be linked to the activation of cyclic
AMP-dependent and Ca
2+
-dependent Cl
)
secretory
Keywords
hemolysin; metalloprotease; processing;
Vibrio cholerae;Vibrio mimicus
Correspondence
S. Miyoshi, Graduate School of Medicine,
Dentistry and Pharmaceutical Sciences,
Okayama University, Tsushima-Naka,
Okayama 700 8530, Japan
Fax: +81 86 251 7926
Tel: +81 86 251 7966
E-mail: miyoshi@pharm.okayama-u.ac.jp
(Received 10 October 2008, revised 30
November 2008, accepted 3 December
2008)
doi:10.1111/j.1742-4658.2008.06827.x
Vibrio mimicus is a causative agent of human gastroenteritis and food
poisoning, and this species produces an enterotoxic hemolysin (V. mimi-
cus hemolysin) as a virulence determinant. Vibrio mimicus hemolysin is
secreted as an 80 kDa precursor, which is later converted to the 66 kDa
mature toxin through removal of an N-terminal propeptide via cleavage
of the Arg151–Ser152 bond. In this article, we investigate the role of
the endogenous metalloprotease (V. mimicus protease) in the maturation
of V. mimicus hemolysin. In vitro experiments using purified proteins
showed that, although it activated the precursor at the early stage via
cleavage of the Asn157–Val158 bond, V. mimicus protease finally con-
verted the activated and physiologically maturated toxin to a 51 kDa
protein through removal of the C-terminal polypeptide. This 51 kDa
derivative was unable to lyse erythrocytes because of its inability to bind
to the erythrocyte membrane. Vibrio mimicus protease-negative strains
were found to produce high levels of V. mimicus hemolysin at the loga-
rithmic phase of bacterial growth and maintained high hemolytic activity
even at the stationary phase. These findings indicate that, although it is
not directly related to toxin maturation in vivo,V. mimicus protease can
modulate the activity of V. mimicus hemolysin and ⁄or its precursor
through limited proteolysis.
Abbreviations
Boc, tert-butyloxycarbonyl; Cm
r
, chloramphenicol resistance; HIB, heart infusion broth; HU, hemolysin unit; MCA, 4-methylcoumaryl-7-amide;
PU, protease unit; PVDF, poly(vinylidene difluoride); VCC, Vibrio cholerae cytolysin; VMH, Vibrio mimicus hemolysin; VMH
N51
, 51 kDa
derivative; VMP, Vibrio mimicus protease.
FEBS Journal 276 (2009) 825–834 ª2009 The Authors Journal compilation ª2009 FEBS 825
pathways [7,10]. VMH is a single-chain polypeptide
and is secreted from bacterial cells as an 80 kDa
(80 399 Da) precursor (pro-VMH), which is later con-
verted to the 66 kDa (65 997 Da) mature toxin
through removal of an N-terminal propeptide [11].
However, the proteolytic enzyme(s) mediating toxin
maturation has not been identified to date.
Vibrio mimicus protease (VMP) is another extracellu-
lar product of the species. This proteolytic enzyme is a
zinc metalloprotease; therefore, its activity is abolished
by incubation with zinc-specific chelating agents or
heavy metal ions [12]. In addition, VMP is immuno-
logically cross-reactive with metalloproteases from
other Vibrio species [13]. Chowdhury et al. [14] have
shown previously that VMP has no enterotoxic activ-
ity; however, it may activate pro-VMH because the
enterotoxic activity of the concentrated culture super-
natant is increased by treatment with VMP. By con-
trast, VMP may also function to inactivate the toxin,
as the purification of VMH was successful only in the
presence of a metalloprotease inhibitor [6]. In order to
explain this inconsistency, the proteolytic actions of
purified VMP against pro- and mature VMH were
investigated.
In this article, we show that VMP can convert pro-
VMH to the 66 kDa active toxin in vitro through
cleavage of the Asn157–Val158 bond in the N-terminal
propeptide. However, it finally truncates the C-termi-
nal 15 kDa polypeptide of the activated and physiolog-
ically mature toxin, causing inactivation of the
hemolysin. We also describe the steady production of
mature VMH by VMP-negative strains.
Results
Limited proteolysis of pro-VMH by VMP
As reported previously [12], VMP was purified from
the culture supernatant of strain E-33, an environ-
mental isolate. VMP thus purified was used to act
on pro-VMH, which was isolated from a periplasmic
fraction of an Escherichia coli transformant carrying
the vmh gene on a recombinant plasmid. When the
hemolytic activity was measured periodically with
horse erythrocytes, remarkable hemolytic activity
was detected after 30 min of incubation (Fig. 1A).
However, the activity gradually decreased on exten-
sion of the incubation period (Fig. 1A). SDS-PAGE
analysis revealed that pro-VMH (80 kDa) was
successively converted to the 66 and 51 kDa proteins
(Fig. 1B), suggesting that the intermediate product
(66 kDa) is active but the final product (51 kDa) is
inactive.
The analysis of the N-terminal amino acid sequence
showed that the 66 kDa intermediate protein was
derived via cleavage of an Asn157–Val158 bond
(Fig. 1C). However, in culture, maturation of pro-
VMH is achieved by hydrolysis of the Arg151–Ser152
bond [15]. Therefore, it can be concluded that,
although VMP initially activates pro-VMH in vitro,it
does not directly contribute to physiological toxin mat-
uration. The amino acid sequencing analysis also indi-
cated that the 51 kDa final product was derived from
the 66 kDa intermediate product via truncation of the
C-terminal 15 kDa polypeptide. Therefore, the C-ter-
minal polypeptide lost by the truncation may be essen-
tial for hemolytic activity. In order to confirm the
VMP-independent maturation of pro-VMH and the
A
B
C
Fig. 1. Proteolytic activation and inactivation of pro-VMH by VMP.
pro-VMH (6 lg) was incubated with or without purified VMP
(50 ng) at 37 C for the indicated periods in Tris ⁄HCl buffer (pH 7.5)
to a final volume of 35 lL. After incubation, hemolytic activity
towards horse erythrocytes was measured (A), and the molecular
weights of the proteins were estimated by SDS-PAGE (B). The
N-terminal amino acid sequence of the 66 kDa protein was
determined, and the cleavage site was identified (C).
Fragmentation of Vibrio mimicus hemolysin T. Mizuno et al.
826 FEBS Journal 276 (2009) 825–834 ª2009 The Authors Journal compilation ª2009 FEBS
inactivation of mature VMH with VMP, we carried
out a series of experiments.
Detection of the sequence and disruption of the
metalloprotease gene (vmp)
The properties of purified VMP are very similar to
those of the proteases termed ‘vibriolysin’
(EC 3.4.24.25), the thermolysin-like metalloproteases
from Vibrio species, including V. cholerae. However,
cloning of the gene (vmp) encoding the enzyme has
not been performed to date. For the detection of the
complete sequence of the vmp gene, oligonucleotide
primers for PCR amplification were designed from
the highly conserved regions in the genes of vibrio-
lysin. Although the PCR experiment was performed
with several V. mimicus isolates, the expected PCR
product was obtained only for an environmental iso-
late, strain ES-39. The nucleotide sequence of the
PCR product (910 bp) was determined, and inverse
PCR was carried out to amplify the full vmp gene
(1833 bp). The nucleotide sequence of the vmp gene
of strain ES-39 was determined and deposited in the
DDBJ ⁄EMBL ⁄GenBank database under accession
number AB435238. Later, PCR experiments per-
formed with the set of primers designed from the
revealed sequence of the vmp gene showed that all
V. mimicus strains tested possessed the gene (data
not shown). The homology of the deduced amino
acid sequence of the VMP precursor (611 amino
acids) was as high as 88.9% to the V. cholerae
metalloprotease sequence and 70% to other vibrio-
lysin sequences. In addition, VMP was found to
contain the consensus zinc-binding motifs, His1-Glu-
X-X-His and Gly-X-X-Asn-Glu25-X-X-Ser-Asp [16].
Taken together, it can be concluded that VMP is
also a member of the vibriolysins.
Although detection of the sequence of the vmp ORF
was successfully performed using strain ES-39, the cul-
ture supernatant of this isolate showed no proteolytic
activity. Therefore, another environmental isolate,
strain ES-37, which exhibited high proteolytic and
hemolytic activity, was used for the construction of a
vmp mutant. The vmp gene was disrupted by single
crossover homologous recombination using the recom-
binant suicide vector from pKTN701, in which a chl-
oramphenicol resistance (Cm
r
) marker and a fragment
of vmp ORF were inserted. One suitable mutant, strain
FMP-37, which revealed the Cm
r
phenotype and no
proteolytic zone on the skimmed milk agar plate was
isolated. The disruption of the vmp gene was confirmed
by PCR and RT-PCR for the detection of the vmp
gene and vmp mRNA, respectively.
Production of mature VMH by VMP-negative
strains
The culture supernatants of strains ES-37 (vmp
+
)
and FMP-37 (vmp::Cm
r
) were collected periodically,
and the hemolytic activity was measured using
horse erythrocytes. Culture supernatants from both
strains showed similar hemolytic activity (Fig. 2A)
until the late logarithmic phase (16 h of cultivation).
Western blot analysis of the VMH antigen in the
culture supernatants from different times of cultiva-
tion indicated conversion of the 80 kDa pro-toxin to
the 66 kDa toxin (Fig. 2C). In addition, N-terminal
amino acid sequencing analysis indicated specific
hydrolysis of the Arg151–Ser152 bond in either
strain. Strain ES-39 (VMP
)
) was also tested for
hemolysin production, and this VMP-negative strain
was also found to produce a sufficient amount of
mature hemolysin. Therefore, it was concluded that
the maturation of pro-VMH was independent of
VMP.
At the stationary phase (24 or 32 h of cultivation),
the hemolytic activity in the culture supernatant
of strain ES-37 was drastically reduced (Fig. 2A).
By contrast, the samples from strains FMP-37 and
ES-39 retained a high level of hemolytic activity
(Fig. 2A). The proteolytic activity in the culture
supernatant from strain ES-37 increased significantly
at this phase of growth (Fig. 2B). However, the
samples from strains FMP-37 and ES-39 possessed
negligible proteolytic activity, indicating that VMP is
the major protease of the species (Fig. 2B). As
shown in Fig. 2C, western blot analysis revealed
that protease production may result in the appea-
rance of the 51 kDa derivative. By contrast, the
15 kDa polypeptide, assumed to be removed from
VMH, could not be detected. These findings
demonstrate that VMP cleaves mature VMH into
the 51 kDa inactive fragment and several smaller
peptides.
It should be noted that, although the proteolytic
activity towards azocasein was negligible, strain
FMP-37 (vmp::Cm
r
) might produce another
proteolytic enzyme by which mature VMH is
converted to the 55 kDa form (Fig. 2C). In addition,
the VMH preparation obtained from the culture
supernatant of the wild-type strain, cultivated in
the presence of a VMP inhibitor, contained a small
amount of the 55 kDa protein (Fig.3B). These
results suggest that the elimination of the proteolytic
activity of VMP may result in the transcription of a
silent gene encoding a substitute proteolytic enzyme,
as documented in other Vibrio species [17,18].
T. Mizuno et al. Fragmentation of Vibrio mimicus hemolysin
FEBS Journal 276 (2009) 825–834 ª2009 The Authors Journal compilation ª2009 FEBS 827
Limited proteolysis and inactivation of VMH
VMH was purified from the culture supernatant of
strain E-33 in the presence of an inhibitor for VMP, as
documented previously [6]. As shown in Fig. 3A,
although the hemolytic activity of mature VMH was
lost gradually, even in the absence of VMP, the activ-
ity was decreased more rapidly by incubation with
purified VMP. The inactivation of the toxin was
accompanied by the appearance of a 51 kDa derivative
(Fig. 3B). This derivative was termed ‘VMH
N51
’
because amino acid sequencing analysis revealed that
the 51 kDa protein was the N-terminal fragment
derived after removal of the C-terminal 15 kDa poly-
peptide. The 55 kDa product of proteolysis obtained
in the absence of VMP activity (Fig. 3B) was also
demonstrated to be the N-terminal derivative of
mature VMH by amino acid sequencing.
The cholera toxin-negative strains of V. cholerae
have been reported to produce an ortholog of VMH,
V. cholerae cytolysin (VCC), as an enterotoxic factor
[19,20]. This ortholog consists of three functional
domains: the N-terminal cytolysin, the central b-trefoil
lectin-like and the C-terminal b-prism lectin domain.
Olson and Gouaux [21] reported that b-octyl glucoside
interacted with three amino acid residues in the C-ter-
minal b-prism lectin domain of VCC. For VMH [6,22],
as well as for VCC [23,24], a carbohydrate moiety,
A B
C
Fig. 2. Extracellular processing of pro-VMP by V. mimicus strains. Each bacterial strain was cultivated in 5 mL of HIB supplemented with
0.5% NaCl at 37 C for the indicated periods. Thereafter, the hemolytic activity towards horse erythrocytes (A) and the proteolytic activity
towards azocasein (B) were measured. For western blot analysis of the VMH antigen, proteins in the culture supernatant were precipitated
and concentrated by the addition of trichloroacetic acid. An aliquot of the concentrated preparation, which was equivalent to 0.2 mL of the
culture supernatant, was subjected to SDS-PAGE using a PhastGel Gradient 10–15. After SDS-PAGE, the separated proteins were trans-
ferred to a PVDF membrane, and the VMH antigens were detected with IgG antibody against purified VMH.
Fragmentation of Vibrio mimicus hemolysin T. Mizuno et al.
828 FEBS Journal 276 (2009) 825–834 ª2009 The Authors Journal compilation ª2009 FEBS
including ganglioside, has been suggested to be a com-
ponent of the binding site on erythrocyte membranes.
Therefore, it was considered that the ability of
VMH
N51
to associate with erythrocyte membranes
might be abolished. To clarify this point, a preparation
of VMH or VMH
N51
was incubated with horse eryth-
rocyte ghosts, and proteins associated with the ghosts
were detected by western blot analysis using the anti-
body against VMH. In contrast with the significant
binding of VMH, the 66 kDa protein, negligible bind-
ing of VMH
N51
was observed (Fig. 4A). It was noted
that, although there was a very small amount in the
VMH preparation (Fig. 3B), the 55 kDa N-terminal
derivative could be detected by western blot analysis.
This may indicate that this derivative has high reactiv-
ity to the antibody or associates with the membranes
very tightly.
Sheep erythrocytes are less sensitive to VMH
because of the presence of fewer toxin-binding sites on
the membrane [6]. Therefore, less binding of VMH, as
well as of the 55 kDa derivative, was observed. Mean-
while, VMH
N51
revealed weak but significant associa-
tion with sheep erythrocytes (Fig. 4B) in spite of the
absence of hemolytic activity (Fig. 5A). This suggests
the presence of a second binding site on the sheep
erythrocyte membrane.
Inhibitory effect of the 51 kDa derivative
The hemolytic activity of the culture supernatant of
strain ES-37 (vmp
+
) was drastically decreased at 32 h
of cultivation (Fig. 2A). However, the culture superna-
tant was found to contain a significant amount of
mature VMH (Fig. 2C). Thus, it was hypothesized that
the proteolytic derivative VMH
N51
might block the
hemolytic action of the mature toxin. As shown in
Fig. 5A, when allowed to act on horse erythrocytes in
the presence of a 30-fold concentrated amount of
VMH
N51
, hemolysis by VMH was blocked almost
completely. This result indicates that, in spite of its
inability to bind to the membrane, VMH
N51
may inter-
fere with the hemolytic process of VMH on the horse
erythrocyte membrane. However, supporting experi-
ments, using recombinant VMH
N51
, are needed to
eliminate the possibility that small fragments, derived
from the C-terminal 15 kDa polypeptides present in
the VMH
N51
preparation, may also inhibit the hemo-
lytic activity of mature VMH.
By contrast, when allowed to act on sheep erythro-
cytes, about 50% of the activity was retained
(Fig. 5A). VMH is a member of the pore-forming
A
B
SDS-PAGE
Fig. 3. Limited proteolysis and inactivation of VMH by VMP. VMH
(6 lg) was incubated with or without purified VMP (50 ng) at 37 C
for the indicated periods in Tris ⁄HCl buffer (pH 7.5) to a final vol-
ume of 35 lL. After incubation, the hemolytic activity towards
horse erythrocytes was measured (A), and the molecular weights
of the proteins were estimated by SDS-PAGE (B).
A
B
Fig. 4. Binding of VMH and its derivatives to erythrocyte mem-
branes. VMH (3 lg) or VMH
N51
(10 lg) was allowed to act on horse
(A) or sheep (B) erythrocyte ghosts at 37 C for 1 h in Tris ⁄HCl buf-
fer (pH 7.5) to a final volume of 20 lL. Thereafter, the erythrocyte
ghosts were collected, rinsed twice with buffer and treated with
2% SDS at 100 C for 3 min. The SDS-treated samples were sub-
jected to SDS-PAGE, the proteins separated were transferred to a
PVDF membrane and the VMH antigens were detected using the
IgG antibody against purified VMH.
T. Mizuno et al. Fragmentation of Vibrio mimicus hemolysin
FEBS Journal 276 (2009) 825–834 ª2009 The Authors Journal compilation ª2009 FEBS 829