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Previous Article | Next Article 
Infection and Immunity, January 2001, p. 579-583, Vol. 69, No. 1
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.1.579-583.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Mouse Toxicity and Cytokine Release by Verotoxin
1 B Subunit Mutants
Vince M.
Wolski,1,2
Anna M.
Soltyk,1 and
James L.
Brunton1,2,3,*
Samuel Lunenfeld Research Institute, Mount
Sinai Hospital,1 The Department of
Laboratory Medicine and Pathobiology, University of
Toronto,2 and The Toronto General
Hospital,3 Toronto, Ontario, Canada
Received 14 July 2000/Returned for modification 5 September
2000/Accepted 26 September 2000
 |
ABSTRACT |
The crystal structure of the verotoxin 1 (VT1) B subunit complexed
with a globotriaosylceramide (Gb3) analogue showed the presence of three receptor binding sites per monomer. We wished to
study the effects of altering the three sites, singly or in combination, on animal toxicity and cytokine induction in vitro. We
found that while the site 1 and 2 mutants were modestly (two- to
sevenfold) reduced in their ability to cause disease in BALB/c mice,
the site 3 mutant, W34A, was as toxic as VT1. However, all the
double-mutant proteins, irrespective of which two sites were mutated,
exhibited approximately a 100-fold reduction in their 50% lethal doses
for mice. These results suggest that multivalent receptor binding is
important in vivo and that all three binding sites make a similar
contribution to the latter process. The triple-mutant holotoxin, F30A
G62T W34A, administered intraperitoneally without adjuvant, stimulated
a strong antibody response in BALB/c mice, and the immune sera
neutralized the activity of VT1 in vitro. Induction of tumor neurosis
factor alpha release from differentiated human monocytes (THP-1 cells)
was relatively impaired for site 1 and site 2 but not site 3 mutants,
suggesting an auxiliary role for the latter site in mediation of
cytokine release in vitro. Cytotoxicity assays on undifferentiated
THP-1 cells have also demonstrated the importance of sites 1 and 2 and
the relatively small role played by site 3 in causing cell death. These
data suggest an association between the cytotoxicity of the protein and
its ability to induce cytokine release.
| |
TEXT |
Verotoxin-producing
Escherichia coli (VTEC) strains are recognized as the
etiological agents of a number of human diseases, including hemorrhagic
colitis (19, 26) and the hemolytic uremic syndrome (HUS)
(10). Much evidence suggests that verotoxins (VTs; also
known as Shiga-like toxins) produced by VTEC are directly involved in
the genesis of the vasculopathies of both of these conditions
(24, 25). VTs belong to the A:B5 family of bacterial toxins and consist of an enzymatic A subunit (32 kDa) and a
homopentamer of B subunits (7.5 kDa each) (8, 29). In the
case of VTs, the A subunit possesses N-glycosidase activity
which catalyzes the depurination of the 28S rRNA component of the
eukaryotic ribosome, leading to the inhibition of protein synthesis
(7). The B subunit pentamer mediates binding to the toxin
receptor, the globo-series glycosphingolipid globotriaosylceramide
(Gb3) (14, 35).
The structure of the cocrystal of the VT1 B pentamer and the Pk
trisaccharide, a Gb3 analogue, revealed three Pk binding
sites on each VT1 B monomer (12). Site 1 involved a
hydrophobic stacking interaction between F30 and Gal-
of the Pk
trisaccharide and hydrogen bonds with a number of residues, including
D17, E28, and T21. Site 2 involved hydrogen bonds with R33, N32, and
G62 and hydrophobic interactions with F30 and A56. Site 3 involved a
hydrophobic stacking of W34 on Gal- of the Pk moiety and hydrogen bonding with D18 of the adjacent monomer. Computer modeling studies predicted the presence of two Gb3 binding sites on the VT1
B subunit: site 1, a cleft defined by the solvent-exposed face of F30
and an aspartate loop (D16 to D18), and site 2, a crevice formed by the
back side of the same F30, a glycine loop (G60 to G62) and residues N32
and R33 (17, 18). Site 1 was very similar to that
demonstrated by crystallography, while the details of site 2 differed
significantly, although it was located in the same area. The third site
identified in the cocrystal, termed site 3, had not been predicted by
modeling. Studies of VT1 B mutants in our laboratory have demonstrated
that sites 1 and 2 function as the principal sites responsible for
high-avidity binding to Gb3 in the membrane context and
that both are important in mediating cell toxicity (3, 6;
A. M. Soltyk, C. R. MacKenzie, W. M. Wolski, T. Hirama,
and J. L. Brunton, submitted for publication). In contrast, site 3 was shown to contribute significantly to receptor binding but appeared
to play a relatively minor role in inducing cytotoxicity in vitro
(3; Soltyk et al., submitted).
It has been proposed that two synergistic signals may play a role in
the pathogenesis of VT-mediated disease: systemic VTs and elevated
levels of proinflammatory cytokines, such as tumor neurosis factor
alpha (TNF-
) and interleukin-1 released in response to the toxins
(31). The latter have been shown to sensitize endothelial
cells to VTs by upregulating the cell membrane expression of
Gb3 (33, 34). In addition, clinical studies
have shown that HUS patients have elevated levels of these
proinflammatory cytokines in plasma as well as in urine (11,
15). Recently, it was demonstrated that, in contrast to the VT1
B subunit and anti-Gb3 monoclonal antibody, only the VT1
holotoxin was capable of inducing cytokine release from THP-1 cells, a
human monocytic cell line (28). Subsequently, Yamasaki et
al. showed that cytokine release induction was dependent on the
enzymatically active A subunit (37).
Given the central role that VTs are believed to play in the pathogenic
processes of both hemorrhagic colitis and HUS, immunization of humans
against these virulence factors could protect against the systemic
complications of VTEC infection, as has already been shown for edema
disease of swine (9). Although the B subunit of VT1
generates strong antibody responses (5, 27), holotoxin toxoids may be superior to vaccines incorporating only the B subunits of VTs, as they provide protection against both homologous and heterologus toxins (4).
In this study, we investigated the roles played by the three glycolipid
binding sites of the VT1 B subunit in inducing disease in the animal
host and stimulating cytokine release from monocytes in vitro. We also
assessed the immunogenicity of the triple-mutant protein, F30A G62T
W34A, and the ability of the induced humoral response to neutralize the
activity of VT1.
Toxicity of VT1 mutants in BALB/c mice.
Wild-type and mutant
holotoxins used in this study were purified from periplasmic extracts
of E. coli JM101 transformed with the plasmid pJLB128
(22) encoding the VT1 holotoxin and from derivative
plasmids carrying mutations in the B cistron of VT1 that were designed
to specifically disrupt the three Gb3 binding sites. The
mutant holotoxins were F30A (site 1 [6]), G62T, G62A,
and A56Y (site 2 [3; Soltyk et al., submitted]), and W34A (site 3 [3]); the double mutants were F30A W34A,
G62T W34A, and F30A G62T; and the triple mutant was F30A G62T W34A (Soltyk et al., submitted). Crystal structures of several mutants complexed with the Pk trisaccharide analogue have shown that the G62T
mutant can interact with Pk at site 1 but not site 2 while the F30A
mutant does not interact with Pk at site 1 (13). The holotoxins were purified as previously described (20). The
purified holotoxins were resuspended in phosphate-buffered saline,
purged of endotoxin with polymyxin B resin (Bio-Rad Laboratories,
Hercules, Calif.), and tested for residual endotoxin contamination by
the Limulus amebocyte lysate assay (Associates of Cape Cod,
Inc., Falmouth, Mass.). The toxin preparations were found to contain, on average, less than 1 pg of endotoxin per 1 µg of a given
holotoxin. Groups of five 4- to 6-week-old female mice of the inbred
BALB/c strain (Charles River Laboratories, Wilmington, Mass.) were
injected intraperitoneally with serial dilutions of the wild-type and
mutant holotoxins and observed for the development of hind limb
paralysis and death. To check for the lethality of the residual
endotoxin contamination of the protein samples, five mice were injected with boiled toxins (100°C, 1 h) at the highest toxin dose used in the
50% lethal dose (LD50) studies. Control mice for the
LD50 studies were injected with phosphate-buffered saline
by the protocol outlined above. The experimental procedures carried out
on the mice were in accordance with the principles of the Animal Care Committee of Mount Sinai Hospital, Toronto, Canada. The
LD50s (Table 1) were
calculated according to the method described by Reed and Muench
(23). For F30A and A56Y, the LD50s fell
between 1 and 2 µg (at 1 µg and lower doses, 100% of the mice
survived; at 2 µg and higher doses, 100% of the mice died). The
LD50s for F30A W34A, G62T W34A, F30A G62T, and F30A G62T
W34A fell between 50 to 100 µg (for all these toxins at 50 µg and
lower doses, 100% of the mice survived; at 100 µg and higher doses,
100% of the mice died). Ranges of LD50s are provided for
the toxins above, since the determination of precise
LD50s would involve sacrificing more animals at doses
between the twofold doses, which yielded an all-or-none response.
Control mice injected with boiled toxins showed 100% survival.
Work in our laboratory has shown the importance of high-affinity
Gb3 binding sites 1 and 2 and the relatively small
contribution of low-affinity Gb3 binding site 3 in
mediating cell death in vitro (Table 1) (3, 6; Soltyk et
al., submitted). Thus, the high animal toxicity of the site 3 mutant,
W34A, both with respect to its LD50 and time elapsed to the
onset of disease symptoms and death (the latter being identical to that
of VT1), is consistent with the in vitro findings. These results show
that the presence of functional high-affinity sites 1 and 2 is
sufficient for the toxin to mediate VT1-like levels of toxicity in
mice. Surprisingly, however, site 1 (F30A) and site 2 (G62A and
G62T) mutants also exhibited relatively high toxicity levels in vivo,
even though their 50% cytotoxic doses (CD50s) were 5 to 6 logs higher than those for VT1 (Table 1). The A56Y site 2 mutation is
believed to block site 2 less effectively than the G62 mutations,
resulting in a relatively more toxic protein both in vivo and in vitro
(Table 1).
All the double-mutant proteins (F30A W34A, G62T W34A, and F30A G62T)
exhibited a significant decrease in toxicity in comparison to their
constituent single mutants. While the mutation of any one site resulted
in no reduction (W34A) or reductions of <1 order of magnitude in
mouse lethality (F30A, A56Y, G62A, and G62T), a mutation of any two
sites caused an approximately 100-fold decrease in toxicity relative to
that of VT1. This is particularly striking for the F30A W34A and G62T
W34A mutants, which in vitro exhibited no statistically significant
(P > 0.05) reductions in cytotoxicity relative to the
single mutants F30A and G62T, respectively (Table 1). The reductions in
animal toxicity caused by adding the W34A mutation to a preexisting
site 1 or 2 mutation are comparable to the reductions in binding to
Gb3-containing liposomes demonstrated by surface plasmon
resonance (Soltyk et al., submitted). A plausible explanation for these
observations is that maintaining multivalency of receptor-toxin
interactions is critical to achieve high-avidity binding in the
membrane context which is required for cellular internalization. Thus,
with any two sites disrupted, the one remaining Gb3 binding
site per B subunit is insufficient to support high levels of receptor
engagement. This results in a protein possessing only residual
lethality similar to that of the triple-mutant toxin, in which all
three sites are disrupted. The requirement for multivalent binding in
vivo is consistent with the results of VT1 mutants binding to
Gb3 in liposomes (Soltyk et al., submitted).
Another observation emerging from our studies is that the mutant toxins
are much more toxic in vivo than in vitro relative to the toxicity of
VT1. One reason for this may be the complexity of an animal system
versus a simple cell monolayer. Specifically, host response to the
toxins by cytokine release, resulting ultimately in target-cell
sensitization by means of increased Gb3 expression on the
cells of susceptible tissues, could in part be responsible for this
phenomenon (15, 31, 32). Another plausible explanation for
this increased toxicity of the mutants of VT1 relative to that of the
wild-type protein could be that, in mice, there exists a
Gb3-independent mechanism facilitating the intoxication of
renal tubular cells, the site of pathology in the murine model of
VT-mediated disease (30, 36). The latter might involve
tubular reabsorption of filtered proteins, including VTs, by a
nonspecific, receptor-independent process of pinocytosis
(16).
For all the mutants except for W34A, the time elapsed to the onset of
disease development and death was delayed relative to that of VT1
(Table 1). The mechanism underlying this phenomenon may consist of two
components. First, the reduced level of receptor recognition exhibited
by the mutants (3; Soltyk et al., submitted) may lead to
slow, reduced uptake in the susceptible tissues. This was in fact
observed for the F30A mutant in the rabbit model of verotoxemia
(1). The serum half-life of the mutant was close to
50-fold longer than that of VT1, and the mutant also failed to
preferentially localize to the Gb3-rich tissues targeted by the wild-type protein. Also, mutants exhibiting reduced Gb3
recognition are internalized by Vero cells to a lesser extent than the
wild-type protein (Soltyk et al., submitted). The second component of
the mechanism responsible for the observed delay may be that the mutant holotoxins are less efficient than the wild-type protein at stimulating cytokine release. We have shown that this is indeed the case in vitro
(Fig. 1). This reduced cytokine release
could lead to less target cell sensitization, and thus it would take
longer for the disease symptoms to develop.
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FIG. 1.
Net TNF- release induced by purified VT1 and its
mutants from a human monocytic cell line. TPA-differentiated THP-1
cells were incubated in media containing the indicated concentrations
of VT1 and its mutants for 12 h at 37°C. The culture
supernatants were then assayed for the presence of TNF- by ELISA.
The data shown are the means ± standard deviation of three
independent experiments. The release mediated by A56Y at 10 µg/ml was
not statistically significant (P > 0.05). The
background release from cells incubated in medium only was 5.3 ± 0.58 pg/ml. Student's t test was used to determine statistical
significance.
|
|
Immunogenicity of the F30A G62T W34A holotoxin.
Five mice were
injected intraperitoneally with 50 µg of the biologically active
triple-mutant holotoxin, followed by boosters with the same dose 14 days later; 14 days after the booster injections, mice were bled by
cardiac puncture. Control mice were injected with phosphate-buffered
saline. Anti-F30A G62T W34A-specific antibodies and anti-VT1-specific
antibodies were detected using a solid-phase enzyme-linked
immunosorbent assay (ELISA) as previously described (2).
Briefly, each well of an Immulon 1 plate (Dynatech Laboratories, Chantilly, Va.) was coated with 400 ng of F30A G62T W34A and VT1 holotoxins dissolved in 100 µl of the carbonate-bicarbonate buffer (pH 9.6). The coated wells were subsequently blocked with 2% (wt/vol) bovine serum albumin (Sigma Chemical Co., Oakville, Ontario, Canada), incubated with serial dilutions of sera, peroxidase-conjugated goat
anti-mouse immunoglobulin G secondary antibody (Bio-Rad Laboratories), and developed with o-phenylenediamine dihydrochloride (Sigma
Chemical Co.) for 30 min. The chromogen produced was measured by
determining the optical density at 490 nm (OD490) on an
MR600 ELISA plate reader (Dynatech Laboratories). The antibody titer
was defined as the highest serial dilution of serum at which the
OD490 was 2 standard deviations above the mean
OD490 of the negative-control sera at a 1:100 dilution
(2). The ability of anti-F30A G62T W34A holotoxin antisera
to inhibit the cytotoxic effects of VT1 toward Vero cells (a kind gift
of C. Lingwood, The Hospital for Sick Children, Toronto, Canada) was
determined in an in vitro neutralization assay (10). Equal
volumes of serial twofold dilutions of sera and toxin at 0.03 ng/ml (10 times the measured CD50 for VT1) were preincubated for 1 h
and then added to the wells of a 96-well plate. The serum
neutralization titer was defined as the highest serial dilution of
serum at which 50% of the cells were killed by VT1. Antibody titers
were converted to logarithmic values (log5 x and
log2 x for ELISA and neutralizing titers,
respectively, where x equals the reciprocal of the serum
dilution) for calculation of geometric means and standard deviations.
Mice injected with phosphate-buffered saline produced no anti-toxin antibodies.
Our results show that the F30A G62T W34A holotoxin produced a strong
anti-toxin antibody response in mice, even without the use of any form
of adjuvant. Importantly, the immune sera reacted equally well with
both the triple-mutant and the wild-type holotoxins in a solid-phase
ELISA assay. The reciprocal geometric mean (± standard deviation)
titers of specific antibody to F30A G62T W34A and VT1 holotoxins were
8.29 ± 0.21 (serum dilution, 1:625,000) for both proteins. The
sera also neutralized the cytotoxic activity of VT1 on Vero cells. The
reciprocal geometric mean titer of neutralizing antibodies determined
against 10 times the CD50 of VT1 was 9.93 ± 0.42 (1:975)
(n = 5). The advantage of using holotoxin molecules as
vaccines is that, unlike B subunit vaccine formulations, they would be
expected to provide protection against both homologous and heterologous
toxins (VT2) in vivo (4). In addition, Bast et al. showed
that the antibody response to B subunit presented as holotoxin is major
histocompatibility complex independent (in the haplotypes tested),
while the response to the B subunit alone is major histocompatibility
complex dependent (2). Their data were compatible with the
thesis that the A subunit contained T-helper epitopes. However, even
the disruption of all three Gb3 binding sites of VT1 (F30A
G62T W34A) resulted in a holotoxin which, despite an 8-log reduction in
toxicity in vitro (Soltyk et al., submitted), mediated relatively high
levels of toxicity in vivo. Consequently, additional mutations, most
likely in the active site of the A subunit, would have to be made to
produce a safe natural toxoid.
Activity of B subunit mutant toxins on THP-1 cells.
THP-1
cells, a human monocytic cell line (a kind gift of O. Rotstein, The
Toronto General Hospital, Toronto, Canada), were used to assess the
toxicity and cytokine release induction by VT1 and its mutants. For
undifferentiated THP-1-cell cytotoxicity assays, THP-1 cells grown in
RPMI 1640 (Gibco BRL, Bethesda, Md.) containing 10% fetal calf serum
(Sigma Chemical Co.) were seeded in 96-well plates, and serial 10-fold
dilutions of lipopolysaccharide-purged holotoxins were added to the
wells in triplicate. After 72 h of incubation, the assays were
developed using an MTT cell viability assay kit (Promega, Madison,
Wis.). Wells with no added toxin were used to calculate percent
survival. For TNF- release assays, THP-1 cells were differentiated
for 65 h in the presence of 60 nM
12-O-tetradecanoylphorbol-13-acetate (TPA) (Sigma Chemical Co.) in complete RPMI 1640 containing 10% fetal calf serum. Following differentiation (assessed by flow cytometry analysis of cell surface expression of CD11b and morphological changes), the cells were first
incubated for 24 h in complete RPMI 1640 containing no TPA and then for
12 h with serial dilutions of endotoxin-purged wild-type and
mutant holotoxins. The supernatants were collected and assayed for the
presence of immunoreactive TNF- by using a human TNF- ELISA kit
(Medicorp, Inc., Montreal, Quebec, Canada). Boiled (100°C, 1 h)
toxins served as controls for cytokine induction due to the residual
endotoxin contamination of the protein samples. The Student t test was used to determine statistical significance for
the toxicity and cytokine release assays using the functions contained within Primer of Biostatistics (McGraw-Hill). The CD50s for
undifferentiated THP-1 cells are presented in Table 1. The
CD50 for VT1 on TPA-differentiated THP-1 cells was 500 ± 150 (mean ± standard deviation) ng/ml (72-h incubation
period). Differentiated THP-1 cells released TNF- upon exposure to
the toxins. At 10 µg of toxin/ml, F30A, G62T, F30A W34A, D17E W34A,
and F30A G62T produced no net TNF- release (Fig. 1). The cytokine
release induced by the wild-type and W34A proteins at both the toxin
doses was not significantly different (P > 0.05); VT1 and
W34A also produced equivalent amounts of TNF- at 1 and 5 µg of
toxin per ml (data not shown). The induction mediated by A56Y at 10 µg/ml did not differ significantly from background release from cells
incubated in medium only (P > 0.05). The above
cytokine induction was toxin dependent, since the equivalent amounts of
boiled holotoxins produced no net TNF- production. Undifferentiated
THP-1 cells did not release TNF- upon exposure to VT1 (results not shown).
Our results indicate that while site 1 (F30A) and site 2 (G62A and
G62T) mutants were severely impaired in their ability to cause
THP-1-cell death, the site 3 mutant, W34A, exhibited relatively little
reduction in that respect (Table 1). These findings closely mirror the
results of cytotoxicity assays performed with these mutant proteins on
other cell lines, such as the Gb3-rich Vero cells, and thus
confirm the importance of the high-affinity Gb3 binding
sites 1 and 2 in toxicity mediation (3; Soltyk et al., submitted). Interestingly, the mutant proteins exhibited much smaller
reductions in their ability to induce TNF- release than they did in
their ability to mediate cell killing. However, in keeping with the
cytotoxicity assay results, site 1 (F30A) and site 2 (A56Y, G62A, and
G62T) mutants were relatively impaired in their ability to elicit
cytokine release whereas the site 3 mutant, W34A, was as effective an
inducer as VT1 at the doses tested. These results imply that toxin
uptake mediated by Gb3 binding through sites 1 and 2 is
important for both toxicity and cytokine mediation, with site 3 playing
an auxiliary role in those processes. Thus, these data suggest an
association between the cytotoxicity of the protein and its ability to
induce cytokines, an idea which was recently proposed by Yamasaki et
al., who showed that the ability of an A subunit VT1 mutant to induce
cytokines, at both the mRNA and protein levels was directly related to
the mutant's toxicity (37).
| |
ACKNOWLEDGMENTS |
This work was supported by grant FRN 13071 from the Medical
Research Council of Canada. V.M.W. was a recipient of the University of
Toronto Open Fellowship.
We thank D. J. Bast for many helpful discussions and for preparing
some holotoxins.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, The Toronto General Hospital, 200 Elizabeth St.,
Norman Urquhart Wing, 13th floor, Room 122, Toronto, Ontario M5G 2C4, Canada. Phone: (416) 340-3183. Fax: (416) 340-5047. E-mail:
james.brunton{at}uhn.on.ca.
Editor:
A. D. O'Brien
| |
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Infection and Immunity, January 2001, p. 579-583, Vol. 69, No. 1
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.1.579-583.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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