(Received for publication, September 24, 1996)
From the Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University, Yamadaoka 3-1, Suita, Osaka 565, Japan
Vibrio cholerae synthesizes a toxin
named El Tor cytolysin/hemolysin, which lyses erythrocytes and other
mammalian cells. This toxin is encoded by the hlyA gene and
is synthesized as a precursor form, prepro-HlyA. Prepro-HlyA consists
of, from the amino terminus of this protein, a signal peptide, a
pro-region, and a mature region. The pro-region is cleaved off
extracellularly resulting in activation. To analyze the role of the
pro-region, we substituted the native hlyA gene with the
pro-region-deleted hlyA gene
(hlyApro). The hemolytic activity of the
mutant organism was markedly decreased; the product of the
hlyA
pro gene, secreted in the periplasm, was
degraded. To compare their abilities to form tertiary structure, the
purified mature- and pro-HlyA were denatured and then renatured by
reducing the concentration of denaturant; the denatured pro-HlyA
recovered almost all activity while the mature-HlyA was not renatured.
The sequences of the pro-region and a molecular chaperone, Hsp90, were
similar. The pro-region expressed in Escherichia coli
containing the hlyA
pro gene increased the
cytolytic activity. The purified pro-region peptide also facilitated
renaturation of the denatured mature HlyA. These results suggest that
the pro-region possibly guides the folding of the cytolysin similar to
a molecular chaperone; the pro-region and molecular chaperones share
common function and structure.
Vibrio cholerae O1 biotype El Tor, which is the causative agent of the current seventh cholera pandemic, secretes a cytolytic toxin (V. cholerae El Tor cytolysin/hemolysin). The cytolysin lyses erythrocytes and other mammalian cells and exhibits enterotoxicity in experimental diarrhea models (1). Thus, the cytolysin may contribute to the pathogenesis of gastroenteritis caused by V. cholerae strains, especially the strains not producing cholera toxin, the major cause of cholera diarrhea.
El Tor cytolysin/hemolysin is encoded by hlyA and synthesized as an 82-kDa precursor form (prepro-HlyA) (2). The prepro-HlyA consists of, from the amino terminus, a signal peptide (25 residues), a pro-region (132 residues), and a mature region (584 residues) (2). The signal peptide is cleaved during secretion through the bacterial inner membrane, resulting in pro-HlyA. Following secretion through the outer membrane, a 15-kDa pro-region of pro-HlyA is cleaved, generating mature HlyA. Removal of the pro-region to yield mature HlyA results in a more than ten-fold increase in cytolytic activity (2). The role of the pro-region, however, in production and the secretory process is not known.
It is well known that proteases are produced in a precursor form and
processed to active enzymes by releasing the pro-region of the
precursor protein. Recently, the pro-regions of a few serine proteases,
namely, subtilisin E of Bacillus subtilis (3), -lytic protease of Lysobacter enzymogenes (4), and carboxypeptidase Y of Saccharomyces cerevisiae (5), have been shown to have a
chaperone-like activity with their mature regions in vitro. To distinguish the pro-regions of these proteases from molecular chaperones, they are called intramolecular chaperones; of importance is
that these N-terminal peptides possess chaperone-like activity that
only functions in folding of the precursor protein of which they are a
part (6, 7). The roles of intramolecular chaperones in vivo,
where and how they work in the cell, however, are still not well
understood. Further, it has been reported that the isolated pro-regions
of subtilisin E and
-lytic protease guide the folding of the matured
enzyme intermolecularly as well as intramolecularly (3, 4, 7).
In this paper, we show that the pro-region of El Tor cytolysin works as a chaperone for the cytolysin, a protein that is biologically and physically different from the serine proteases. The pro-region seems to act like a molecular chaperone (e.g. heat shock protein 90 (Hsp90)). Moreover, we present evidence suggesting that the isolated pro-region of HlyA acts like an intermolecular chaperone in facilitating the folding of cytolysin.
Bacterial strains and plasmids
used in this study are listed in Table I. V. cholerae N86 was the original strain used in this study.
Escherichia coli HB101, SM10pir and BL21(DE3)
were used for general manipulation of plasmids, mobilization of plasmid into V. cholerae, and expression of fusion protein,
respectively.
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Luria-Bertani (LB) broth or LB agar was used for general bacterial growth (8). To observe hemolysis of colonies on agar plates, LB medium containing 1.5% agar and 1.5% (v/v) washed fresh sheep erythrocytes (SB-LBA) were used. For Western blot analysis and pulse-chase experiments, syncase medium with 3% glycerol (9, 10) and M9 minimal medium with 0.1% casamino acids (Difco) (2) were used, respectively. In the construction of mutant clones though conjugation, thiosulfate citrate bile salt (TCBS) agar medium (Nissui Seiyaku, Tokyo, Japan) was used for counterselection of E. coli.
Chemicals, Recombinant DNA Techniques and Protein PurificationEnzymes and chemicals were purchased from standard commercial sources. Manipulation of DNA was carried out as described previously (8). Site-directed mutagenesis was performed by the method of Kunkel (11, 12). El Tor cytolysin (mature HlyA) was purified from the supernatant of V. cholerae N86, as previously reported (10). Antibodies against purified mature HlyA were affinity purified from rabbit antiserum, as described previously (10). Purification of fusion peptides was performed using pET Expression System 25b (Novagen, Madison, WI).
Gene ReplacementReplacement of a native gene on V. cholerae chromosome with a mutated gene was carried out by
double-crossover homologous recombination as described previously (13).
Briefly, E. coli SM10pir harboring a
recombinant plasmid having a mutant gene on a suicide vector, pKY719,
was conjugated with V. cholerae. Conjugation was performed
on nitrocellulose membranes overlaid on LB agar plates. Following
incubation for 2 h at 37 °C, the mated bacteria were washed
from the membranes and inoculated onto TCBS agar medium plates
containing 5 µg/ml chloramphenicol. Following incubation overnight at
37 °C, the chloramphenicol-resistant V. cholerae
colonies, resulting from single-crossover events, were isolated. One of
the isolates was cultured in LB broth without antibiotics. Colonies
resulting from the second crossover were tested for chloramphenicol
sensitivity on LB agar plates with 5 µg/ml of chloramphenicol.
Chloramphenicol-sensitive colonies were confirmed for the presence of
about a 400-base pair deletion by Southern blot hybridization.
One hundred µl of an overnight culture in LB broth were inoculated into 10 ml of syncase medium (9) supplemented with 3% glycerol in Petri dishes. After a 9-h stationary incubation at 30 °C, cells were collected by centrifugation at 5,000 × g for 5 min. The precipitated cell pellet was suspended with 1 ml of 2,000 unit/ml polymyxin B for 30 min at 4 °C (14) to stimulate the release of periplasmic proteins. The bacterial cells were removed from the supernatant by centrifugation. The culture supernatant, separated from the cell fraction, was precipitated by addition of 1 ml of 100% (w/v) trichloroacetic acid. Following centrifugation for 15 min at 1,500 × g, the precipitate was suspended in 1 ml of 10 mM phosphate-buffered saline (PBS),1 pH 7.0. Electrophoresis on 10% polyacrylamide gel and Western blot analysis were carried out as described previously (2).
Pulse-Chase ExperimentA pulse-chase experiment was carried out basically by the method described previously (2). In brief, 50 µl of an overnight culture in LB broth were inoculated into 5 ml of M9 minimal medium supplemented with 0.1% casamino acids. After incubation for 6 h at 37 °C, cells were precipitated by centrifugation and suspended in sulfur-free M9 medium. Cells were then incubated for 1 h to induce sulfur starvation, and 1 mCi of a [35S]methionine and [35S]cysteine mixture was then added, followed by further incubation. After 3 min, 500 µl of a mixture containing nonradioactive methionine (50 mM) and cysteine (100 mM) were added. At appropriate periods, 1.5 ml of the culture medium were transferred into ice-chilled centrifugation tubes containing 30 µl of 0.5 M EDTA and centrifuged immediately. To extract the periplasmic proteins, the precipitated cell pellet was immediately suspended in 100 µl of Buffer A (20 mM Tris-HCl, pH 7.5, 0.1 M NaCl, 10 mM EDTA) with 2,000 unit/ml of polymyxin B and incubated for 30 min at 4 °C. The solution was then centrifuged to remove the precipitated cells, and the supernatant was used as the periplasmic sample.
Culture supernatant (0.3 ml) was mixed with 30 µl of 100% (w/v) trichloroacetic acid and centrifuged. The precipitate was suspended in 100 µl of Buffer A, followed by heating at 95 °C for 3 min to ensure solubilization, and used as the supernatant sample.
Both samples (100 µl) were diluted with 900 µl of Buffer B (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5% Triton X-100, 0.05% bovine serum albumin and 5 mM EDTA), and undissolved materials were removed by centrifugation. The sample was mixed with 10 µl of the antibodies and kept at 0 °C overnight. The mixture was added to 20 µl of a Sepharose CL4B gel coupled with protein A (50% (v/v) suspension) and incubated for 2 h at 0 °C. The mixture was centrifuged, and the supernatant was discarded. The precipitate was washed five times with Buffer B, suspended in 20 µl of SDS-polyacrylamide gel electrophoresis sample buffer, heated for 3 min at 95 °C, and centrifuged (5,000 × g). 10 µl of the supernatant were applied to SDS-polyacrylamide gels.
Denaturation and Renaturation ProcedureDenaturation and renaturation were achieved essentially by the methods previously reported (3, 4, 5, 15). In brief, denaturation was carried out by addition of 120 µl of 8 M guanidine HCl to 40 µl of 1.25 nmol/ml of purified pro- or mature HlyA to give a final guanidine HCl concentration of 6 M. The mixure was incubated at 20 °C for 2 h. Renaturation was accomplished by addition of 9440 µl of renaturing buffer (10 mM sodium phosphate buffer, 0.15 M NaCl and 0.1% bovine serum albumin, 5 mM 2-mercaptoethanol, pH 7.0) to give a final guanidine HCl concentration of 0.1 M. Following incubation for 30 min at 20 °C, hemolytic activity was determined.
Assay for Hemolytic ActivityHemolytic activity was determined as described previously (16). Briefly, sheep erythrocytes were washed in PBS three times and adjusted to a hematocrit of 2% with PBS. Sixty µl of serially diluted samples with PBS were mixed with an equal volume of the erythrocyte suspension and incubated for 2 h at 37 °C. After centrifugation at 2,000 × g for 30 s, the supernatant (100 µl) of the reaction mixtures was taken for spectrophotometric measurement of released hemoglobin at 540 nm on a 96-well plate by Multiskan Mcc/340 (Labosystems, Tokyo, Japan). One hemolytic unit (HLU) is defined as the activity required to lyse 50% of cells in 0.1 ml of 1% sheep erythrocytes in phosphate-buffered saline at 37 °C for 2 h.
To investigate
the role of the pro-region of El Tor cytolysin of V. cholerae, we constructed the pro-region-deleted gene
(hlyApro) from the structural gene
(hlyA) for El Tor cytolysin (2, 17, 18) (Fig.
1). Restriction enzyme MscI sites were
created at both ends of the pro-region by site-directed mutagenesis,
resulting in pNK100. The DNA encoding the pro-region (amino acids
26-157) was removed by digestion with MscI and
self-ligation of pNK100, resulting in pNK101. This deletion does not
change the coding frame, and the amino acid residues constituting the
pre-mature junction (Ala-Asn) are the same as those of the pre-pro and
pro-mature junctions. The NruI-AccI fragment of
pNK101 was then exchanged with the NruI-AccI
fragment of hlyA in pNK105, resulting in pNK106. This
exchange was carried out because pNK105 has longer upstream and
downstream adjacent regions of V. cholerae chromosome than pNK101 does, which would give it an advantage in the homologous recombination studies. The SacI-BamHI fragment of
pNK106, having hlyA
pro with adjacent regions,
was subcloned into the SacI and BamHI sites of an
R6K-ori suicide vector, pKY719 (19), resulting in
pNK109.
The plasmid, pNK109, was introduced into E. coli
SM10pir, which was mated with V. cholerae N86.
V. cholerae containing a single-crossover recombinant of
hlyA were isolated first; second crossover recombination
events were confirmed by Southern blot hybridization. We selected two
isogenic clones generated from a single-crossover intermediate clone,
one having the native hlyA gene (N86-RV, revertant to the
same genotype as the parent) and the other lacking the coding sequence
for the pro-region (N86hlyA
pro). N86hlyA
pro showed no significant differences
in growth compared with the parent N86 or the isogenic revertant,
N86-RV. The hemolytic activity of N86hlyA
pro
on the SB-LBA medium was significantly decreased compared with N86 and
N86-RV. The hemolytic activity in the supernatant of
N86hlyA
pro was only 7% of N86 and N86-RV (data not shown).
The specific activity of the pro-region-cleaved
mature HlyA is much higher than that of pro-HlyA (2). However, the
hlyApro, which lacked the sequences coding for
the pro-region, expressed much weaker hemolytic activity than did the
wild-type parent. Therefore, we investigated the fate of the product
of this mutant gene in N86hlyA
pro. Pulse-chase
analysis showed no HlyA
Pro in the culture medium of
N86hlyA
pro although native HlyA of N86-RV was
secreted within a few minutes (Fig. 2A). On
the other hand, in the periplasmic fraction of
N86hlyA
pro, several 30-40-kDa degradation
products, which were not seen in N86-RV, appeared rapidly (Fig.
2A). Western blot analysis gave similar results. HlyA
Pro
was not secreted into culture medium although some degradation products
were evident (Fig. 2B). These data indicate that the HlyA
pro-region is essential for secretion and that HlyA
Pro stays in the
periplasmic space where it is degraded.
Denatuation of Pro- and Mature HlyA
As misfolded or denatured
proteins are considered to be more susceptible to proteases and
degraded more easily than folded proteins (20), we suspected that
HlyAPro might be defective with regard to folding. We compared the
renaturation abilities of denatured pro- and mature HlyA proteins.
First, to test the influence of denaturant on activities of both
toxins, guanidine HCl was added to each toxin. Following incubation for
2 h at 20 °C, hemolytic activities were determined. Hemolytic
activities of both toxins decreased similarly with increasing
concentration of guanidine HCl; both toxins lost almost all in the
activities when the concentration of guanidine HCl was 1 M
(data not shown).
To denature
the pro- and mature HlyA completely, both toxins were incubated with 6 M guanidine HCl for 2 h. After denaturation, the
mixtures were quickly diluted with renaturing buffer. After incubation
for 30 min at 20 °C, hemolytic activity was determined (Fig.
3). After dilution of guanidine HCl in the samples, the specific activities of the pro-HlyA increased. When the final concentration of guanidine HCl was decreased to 0.1 M, the
activity of the denatured pro-HlyA was recovered completely. On the
contrary, the activity of denatured mature HlyA was not recovered even
when the concentration of denaturant was decreased to 0.1 M.
As trypsin activates pro-HlyA by removal of the pro-region (21), the influence of the denaturation and renaturation on activation was investigated. Pro-HlyA and mature HlyA were denatured in 6 M guanidine HCl. These solutions were then diluted to give a final concentration of guanidine HCl of 0.1 M, and treated with trypsin to activate pro-HlyA. The activity of the renatured and activated pro-HlyA was 222 HLU/nmol, which was comparable with untreated pro-HlyA (232 HLU/nmol) (Table II). However, the recovery of activity with the mature HlyA was poor (<10 HLU/nmol, less than 3% of undenatured control). These results suggest that the mature HlyA is unable to fold itself and that the HlyA pro-region may contribute to the refolding of the denatured toxin.
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Molecular chaperones (22, 23) are considered to
mediate the folding of newly synthesized proteins in vivo
and are also known to promote folding of denatured proteins in
vitro. If the pro-region and molecular chaperones have similar
effects, some structural homology among these proteins would be
expected. We compared the amino acid sequence of the pro-region with
those of chaperones in EBI and SWISS-PROT data bases. A highly
homologous region was observed between the pro-region and a heat shock
protein, Hsp90, which is a member of a family of molecular chaperones
(15) found in Escherichia coli from humans (24, 25, 26) (Fig. 4). The homologous region found in Hsp90 is highly
conserved among the Hsp90 family proteins (24, 25, 26). In the HlyA
pro-region, this homologous region constitutes about one-half (68 residues) of the entire pro-region (132 residues) and is located in the central section. This homologous region had 34% identity and 63% similarity to Hsp90.
Expression System of Pro-peptide Fusion Protein
To examine
whether the HlyA pro-region can interact intermolecularly with its
target, mature region, similar to native molecular chaperones, we
constructed a pro-region expression system (Fig. 5). The
pro-region of the hlyA gene was subcloned into the
BamHI and HindIII sites of an expression vector,
pET25b(+) in frame, resulting in pNK118. E. coli BL21(DE3)
harboring pNK118 expressed a 30-kDa protein (Pro-peptide) after
induction with 1 mM
isopropyl-1-thio--D-galactopyranoside. This molecular
size agreed with the estimated size (31 kDa) of the pro-region fusion
product of pNK118 (data not shown).
In Vivo Complementation of HlyA
pNK118 (pro) was introduced into E. coli BL21(DE3) harboring pNK108 (hlyApro)
by transformation. The transformants were cultured in LB broth
containing 100 µg/ml of ampicillin and 50 µg/ml of kanamycin, and
the Pro-peptide encoded on pNK118 was induced with 1 mM
isopropyl-1-thio-
-D-galactopyranoside. After a 30-min
induction, the cells were sonicated and centrifuged, and the hemolytic
activities of the supernatants were determined. The hemolytic activity
of E. coli harboring pNK107 (hlyA) and pNK118
(pro) simultaneously was 0.1 HLU/ml. E. coli
harboring both pNK108 (hlyA
pro) and the vector
plasmid, pET25b(+), gave no detectable hemolytic activity. E. coli harboring pNK108 (hlyA
pro) and
pNK118 (pro), however, had about 40% of the activity (0.04 HLU/ml) compared with the control, E. coli harboring pNK107
(hlyA) and pNK118. This indicates that the expression of
Pro-peptide complemented the defective cytolysin (HlyA
Pro) in
vivo.
To investigate if the isolated pro-region may support
formation of active cytolysin in vitro, the Pro-peptide was
overexpressed and purified from E. coli BL21(DE3) harboring
pNK118 using a pET expression system. To rule out possible influence of
the Pro-peptide on hemolytic activity of native toxin, mature HlyA was
mixed with Pro-peptide, incubated for 1 h, and the hemolytic
activity was determined. The hemolytic activity of the mature HlyA was
unaffected even when 100-fold concentration of the Pro-peptide
molecules was added (Fig. 6).
mature HlyA was denatured with 6 M guanidine HCl and diluted 60 times with renaturing buffer with and without the Pro-peptide and incubated for 1 h (Fig. 6). When denatured cytolysin was diluted without Pro-peptide, little activity was recovered. Depending on the concentration of the Pro-peptide in the renaturation solution, however, hemolytic activity was recovered. More than 80% activity was restored when the molar ratio of Pro-peptide to mature HlyA was 40 or higher. Bovine serum albumin, used as a control instead of the Pro-peptide, had no effect, even at a concentration 1200 times higher than that of mature HlyA (data not shown). Incubation for shorter and longer times following dilution did not affect the recovery (data not shown). These findings indicate that the isolated pro-region facilitates the formation of denatured mature HlyA in vitro.
In this study, we investigated the possible folding ability of the
pro-region of El Tor cytolysin (HlyA). The guanidine HCl-denatured pro-HlyA recovered hemolytic activity following dilution of the denaturant; the mature HlyA lacking the pro-region was not activated (Fig. 3 and Table II). This indicates that the pro-region may support
the folding of HlyA. The pro-region-deleted HlyA, HlyAPro, was not
secreted and was degraded in the periplasmic space (Fig. 2). These
results suggest that the degradation of HlyA
Pro in vivo
may be due to its failure to fold in the periplasmic space.
Molecular chaperones (22, 23) are known to enhance the formation of the native conformation of other proteins. In the organelles (e.g. mitochondria and endoplasmic reticulum), molecular chaperones facilitate the folding of proteins exported across membranes. In bacterial cells, although several cytoplasmic molecular chaperones have been found and well characterized (22, 23), few chaperones have been reported in the periplasm (20). In the absence of chaperone, the HlyA pro-region is likely to function in a manner similar to molecular chaperones and be effective in the periplasm. Of interest, V. vulnificus cytolysin, which is evolutionarily close to El Tor cytolysin, does not have a pro-region (27). Why El Tor cytolysin has a special domain for folding remains unclear.
The HlyA pro-region has a domain highly homologous to Hsp90, a family of molecular chaperones that stimulates protein folding (15). This chaperone is widely distributed and found in bacteria from humans (24, 25, 26). The region in Hsp90, which is similar to the pro-region of El Tor cytolysin, is one of the most conserved regions among the Hsp90 family proteins (24, 25, 26). The pro-region was found to act like a chaperone not only intramolecularly but also intermolecularly. These results suggest that the HlyA pro-region and molecular chaperone Hsp90 may share a common mechanism for supporting protein folding. These findings raise the intriguing possibility that the HlyA pro-region may have structural and functional similarities to molecular chaperones.
Recently, the pro-regions of precursor proteins for a few serine
proteases, subtilisin (3), -lytic protease (4) and carboxypeptidase
Y (5), have been shown to have chaperone-like ability in
vitro. To distinguish them from molecular chaperones, these
pro-regions are called intramolecular chaperones. All these chaperone-like peptides are part of precursor proteins and the peptides
possessing chaperone-like function only for the folding of the
precursors (6, 7). All the intramolecular chaperones known so far,
including the HlyA pro-region, are found in the N terminus of the
precursor proteins.
The pro-regions of subtilisin and -lytic protease have been reported
to act not only as intramolecular chaperones but also like molecular
chaperones (3, 4, 7); that is, these pro-regions fold their target
peptides when these pro- and the mature regions are expressed in
trans. The pro-regions of these proteases have no
significant homology to the known molecular chaperones, including Hsp90. The HlyA pro-region has no significant homology with the pro-regions of other proteases either. HlyA pro-region may function in a manner different from the pro-regions of these proteases.
We thank Dr. Joel Moss (National Institutes of Health, Bethesda, MD) for critical review of the manuscript.