From the Department of Pathology and Microbiology,
University of Bristol, School of Medical Sciences, Bristol BS8 1TD,
United Kingdom, the § NHLBI, National Institutes of Health,
Bethesda, Maryland 20892, and the ¶ Combined Program in Paediatric
Gastroenterology and Nutrition, Children's Hospital,
Boston, Massachusetts 02115
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ABSTRACT |
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Cholera toxin (Ctx) and E. coli
heat-labile enterotoxin (Etx) are structurally and functionally similar
AB5 toxins with over 80% sequence identity. When their
action in polarized human epithelial (T84) cells was monitored by
measuring toxin-induced Cl The severe, and at times fatal, diarrheal disease caused by
Vibrio cholerae is due to the potent action of cholera toxin
(Ctx)1 (for a review, see
Ref. 1). A structurally and functionally similar toxin, heat-labile
enterotoxin (Etx), is produced by certain strains of enterotoxigenic
Escherichia coli that are responsible for causing the
generally milder "traveler's" diarrhea of humans and scouring in
farm animals (2, 3). Both Ctx and Etx are hetero-oligomeric proteins
comprised of a single A-subunit (Mr 28,000) and
five B-subunits (Mr 12,000 each) (4-6). The
A-subunit contains two distinct structural domains linked by a
disulfide bridge: an A1-fragment (residues 1-192) that displays
ADP-ribosyl transferase activity and an A2-fragment (residues 193-240)
that mediates interaction with the B-subunit pentamer (4, 6). The
B-subunits of Ctx and Etx (CtxB and EtxB, respectively) bind to cell
surface receptors, principally GM1-ganglioside, found ubiquitously on the plasma membranes of eukaryotic cells (7). Although
Ctx and Etx exhibit a remarkable degree of structural homology, with
81.6% sequence identity between CtxA and EtxA and 82.5% sequence
identity between CtxB and EtxB (8-10), there are a number of subtle
physicochemical and functional differences between the two toxins. For
example, although the B-subunit pentamers of both toxins are resistant
to environmental conditions that normally lead to protein denaturation,
the pH stability of EtxB is almost 2 orders of magnitude greater than
that of CtxB (11). Studies have also shown that the receptor binding
specificities CtxB and EtxB are slightly different; whereas both bind
with high affinity to ganglioside GM1, and to a lesser
extent to GD1b, EtxB also binds significantly to
glycoprotein receptors and polyglycosylceramides, and with lower
affinity to GM2, asialo-GM1, and paragloboside (12-17). Sequence differences between CtxA and EtxA are spread throughout the polypeptides with the lowest identity surrounding the
A1/A2 cleavage site and near the C terminus of the A2-fragment (10).
The influence on toxin function of most of the primary amino acid
sequence divergence has not yet been fully explored.
The action of cholera toxin and related enterotoxins on eukaryotic
cells depends on a complex sequence of events that eventually leads to
alterations in ion fluxes and a concomitant loss of water, characteristic of cholera and related diarrheal diseases. The use of
polarized human colonic epithelial T84 cells has greatly facilitated
studies of Ctx and Etx, since toxicity can be readily monitored as the
induction of electrogenic Cl For Ctx and Etx to exhibit full toxicity, the A-subunits must undergo
proteolytic cleavage or "nicking" at Arg-192 to give separate A1-
and A2-fragments (31). In the case of cholera toxin, extracellular
proteases, such as HA protease produced by V. cholerae, can
efficiently nick and activate the A-subunit (32). By contrast, Etx from
enterotoxinogenic E. coli, as well as recombinant Ctx produced in E. coli, are normally isolated with their
A-subunits intact; trypsin or other gut-associated proteases have been
postulated to accomplish toxin activation in such cases (33). Recent
studies on T84 cells have shown that a serine protease, which
efficiently nicks and activates both CtxA and EtxA, is present on
either the apical surface or in apically-derived transport vesicles
(31). In this respect, no difference was found in toxicity of
commercial (nicked) preparations of Ctx and recombinant Ctx that was
either unnicked or nicked with trypsin.
The time course and magnitude of electrogenic Cl Materials
All reagents were purchased from Sigma or BDH unless otherwise
stated. Monoclonal antibodies CT-17, which recognizes CtxA was the gift
of Professor J. Holmgren (University of Gothenburg, Gothenburg,
Sweden), and 118-8, which recognizes the CtxB and EtxB, was provided by
Dr. H. Person (University of Umeå, Umeå, Sweden). Recombinant human
ADP-ribosylation factor-6 (rhARF 6) was provided by Dr. W. Patton
(NHLBI, National Institutes of Health, Bethesda, MD)
ctx and etx Operons
The ctxAB operon encoding the A- and B-subunits of
cholera toxin was amplified by PCR from the template plasmid,
pJBK33 (34) using the following primers;
5'-TTGGGCCCGATATCTTTCTGTTAAACAAAG-3' (CROL1) and
5'-GCTCTAGACTAGTTTGCCATACTAATTGCGGC-3' (CROL2) and ExpandTM high fidelity polymerase (Boehringer Mannheim).
The resulting fragment was cleaved with ApaI and
SpeI and then ligated into pBluescript II KS+ (Stratagene)
that had been cut with the same restriction enzymes to yield pRC1 (Fig.
1).2 To facilitate
controlled, high level expression of CtxAB, the EcoRV-SpeI fragment of pRC1 was subcloned into
pTTQ18 (35) at the SmaI and XbaI sites to yield
pRC9. Plasmid pTRH29 was described previously and is a pBluescript
derivative containing the etxAB operon of E. coli
enterotoxin flanked by EcoRV and SpeI restriction sites (36). A pTTQ18 derivative, harboring the
EcoRV-SpeI fragment from pTRH29 and designated
pAM29, has been described (37).
Construction of Mutant and Hybrid Toxin Operons
Mutant and hybrid operons were constructed by ligating two
separate fragments derived by PCR amplification of upstream and downstream segments of the ctxAB or etxAB operons.
Construction of a Mutant ctx Operon Encoding
CtxA(RDEL)CtxB--
pJBK33 was used as the template for
amplification of a 5'-fragment encoding CtxA using primers CROL1 and
5'-TCATCCCGAATTCTATTATGTG-3', and a 3'-fragment encoding CtxB using
primers 5'-ATAGAATTCGGGATGAA-3'and CROL2. The 3'-fragment was cloned
into pBluescript at the EcoRI-SpeI sites,
yielding pATA8. This plasmid was subsequently cut with ApaI
and EcoRI, and the 5' PCR fragment inserted to reconstruct the ctxAB operon, yielding plasmid pATA14 (Fig.
1).2 The EcoRI
site in pATA14 at the junction between the genes encoding the A- and
B-subunit resulted in replacement of Lys-237 by Arg in CtxA. The
EcoRV-SpeI fragment of pATA14 was subcloned into pTTQ18, yielding pCDR3.
Construction of a Hybrid Operon Encoding
EtxA(1-224)CtxA(225-240)CtxB--
pTRH29
was used as the template for amplification of a 5'-fragment from the
etxA gene using the universal reverse primer (Stratagene) 5'-AACAGCTATGACCATG-3' and oligonucleotide 5'-CCTCTGACTGGTACCCTGAA-3'. The DNA fragment was cleaved with EcoRV and KpnI
and inserted into these sites in pBluescript to yield pRC7. pRC1 was
used as the template for amplification of a 3'-fragment of the
ctxAB operon from codon 226 of ctxA using the
oligonucleotide 5'-TTTCAGGGTACCAATCTG-3' and the Construction of a Hybrid Operon Encoding
CtxA(1-225)EtxA(226-240)EtxB--
The
hybrid operon was constructed in a similar manner to that in pRC18. The
5'-fragment from the ctxA gene was amplified from pRC1 using
the universal reverse primer and oligonucleotide
5'-CAGATTGGTACCCTGAAA-3', and the 3'-fragment of the etxAB
operon from codon 226 of etxA was amplified from pTRH29
using the oligonucleotide 5'-TTTTCAGGGTACCAGTCAGA-3' and the Construction of a Hybrid Operon Encoding
CtxA(RDEL)EtxB--
The EcoRV-EcoRI
fragment from pTRH29 was substituted for the same fragment derived from
pATA14, yielding plasmid pCDR1 (Fig. 1). The EcoRV-
SpeI fragment of pCDR1 was subcloned into pTTQ18, yielding pCDR2.
The DNA sequences of all of the cloned operons and constructs were
verified by dideoxy nucleotide sequencing.
Toxin Purification
V. cholerae 0395NT (9) with a chromosomal
ctxAB deletion and harboring pTTQ-plasmid derivatives
encoding wild-type, mutant, or hybrid toxins were cultured overnight at
37 °C on Luria-Bertani agar plates supplemented with 30 µg/ml
streptomycin, 50 µg/ml kanamycin, and 200 µg/ml ampicillin.
Bacteria were inoculated into NZCYM medium (1% (w/v) NZ amine (ICN
Biochemicals), 0.5% (w/v) NaCl, 0.1% (w/v) casamino acids (Difco),
0.5% (w/v) yeast extract (Betalab), 0.2% (w/v)
MgSO4·7H2O) plus 200 µg/ml ampicillin and
grown at 37 °C in a rotary shaker. When cultures reached an absorbance of 0.6 ( Toxin Assays
Electrophysiology--
T84 cells (from passages 75-100)
obtained from ATCC were grown and passaged as described (18, 41).
Toxins were diluted in prewarmed Hanks' balanced salt solution (HBSS)
containing (per liter): 0.185 g of CaCl2, 0.098 g of
MgSO4, 0.4 g of KCl, 0.06 g of
KH2PO4, 8 g of NaCl, 0.048 g of
Na2HPO4, 1.0 g of glucose, and 10 mM HEPES at pH 7.4, and applied to the apical surface of confluent T84 cell monolayers in Transwell inserts (Costar, Cambridge, MA), followed by incubation at 37 °C. Alternatively, T84 cells were
placed at 4 °C; the toxins diluted in ice-cold HBSS were then
applied apically for 30 min at 4 °C. Cells were washed to remove
unbound toxin, fresh HBSS was added, and the cells were incubated at
37 °C. Measurements of short circuit current
(Isc) and resistance (R) were
performed as reported elsewhere (18, 41).
Toxin-catalyzed ADP-ribosylation of Agmatine--
To assay
NAD:agmatine ADP-ribosylation (30), the reaction was initiated by the
addition of 1 µg of toxin to a mixture (total volume 0.3 ml)
containing 10 mM agmatine, 50 mM potassium
phosphate (pH 7.5), 100 µM
[adenine-U-14C]NAD (90,000 cpm), 20 mM dithiothreitol, 5 mM MgCl2, 100 µM GTP, ovalbumin (0.1 mg/ml), 3 mM
dimyristoylphosphatidylcholine, and 0.2% cholate, with or without 1 µg of rhARF 6. After incubation for 1 h at 30 °C, duplicate
0.1-ml samples were transferred to columns (0.4 × 5 cm) of AG1-X2
(Bio-Rad) followed by five washes with 1.0 ml of water, which were
collected for radioassay in a liquid scintillation counter (30).
Assessment of Holotoxin Stability
The stability of A- and B-subunit interaction in hybrid toxins
was determined using an adaptation of a GM1-based
enzyme-linked immunosorbent assay (42). 96-well microtiter plates were
coated with GM1-ganglioside (1.5 µg/ml) for 24 h at
4 °C and then washed with PBS. The plates were incubated with 1%
(w/v) BSA in PBS for 30 min at 37 °C and then washed with PBS. 0.1 ml of toxin (1.5 µg/ml) in PBS with 0.1% (w/v) BSA was then added to
each well. After 1 h at room temperature, the plates were washed
three times with PBS containing 0.05% (v/v) Tween 20, and once with
PBS, and then the effect of various treatments on the stability of
GM1-bound toxin was assessed. PBS alone, PBS containing
0.5% SDS, or McIlvaine buffer, pH 5.5, containing 0.5% SDS (0.15 ml)
was added for 30 min at room temperature. Plates were then washed with
PBS containing 0.05% (v/v) Tween 20, followed by the addition of
either an anti-CtxA monoclonal antibody (CT-17) or an anti-EtxB
monoclonal antibody (118-8) (43) in PBS containing 0.1% BSA with
0.05% (v/v) Tween 20. After 1 h at room temperature, plates were
washed with PBS containing 0.05% Tween 20, followed by the addition of
goat anti-mouse IgG-horseradish peroxidase conjugate for 1 h at
37 °C. The plates were then washed with PBS containing 0.05% (v/v)
Tween 20 and developed with 0.1 ml/well 1 mg/ml
o-phenylenediamine in 0.1 M citrate buffer, pH
4.5, containing 0.4 µl/ml 30% H2O2.
Absorbance was measured at 450 nm in a microtiter plate reader (Anthos).
Ctx and Etx Exhibit Differential Toxicity in T84
Monolayers--
Previously, when samples of periplasmic fractions from
E. coli that contained equivalent amounts of CtxAB or EtxAB
were applied to the apical surfaces of polarized T84 cells, the time
course of Cl
Comparison of the toxicity of 100 nM amounts of the
purified nicked and unnicked preparations of EtxAB revealed that they were equally active in inducing electrogenic Cl Is the K(R)DEL Motif Responsible for Differential
Toxicity?--
The Lys-Asp-Glu-Leu-COOH (KDEL) motif at the C terminus
of the A2-fragment of cholera toxin has been demonstrated to contribute to the efficiency of toxin action in T84 cells (22). In EtxAB, the same
motif contains a conservative amino acid replacement of Arg for Lys
(Fig. 4). Although the RDEL motif is
known to serve as a functional ER retention signal (44, 45) and
enhances EtxAB activity in T84 cells (22), the possibility remained
that the differential toxicity of cholera toxin and E. coli
enterotoxin might be attributable to the subtle alteration in this
C-terminal motif. To investigate this, the KDEL motif of cholera toxin
was replaced by an RDEL sequence, and resultant toxin,
CtxA(RDEL)CtxB, was expressed and purified as described
above (Fig. 5, lane 3). When the effects of 100 nM
CtxA(RDEL)CtxB and wild-type CtxAB on T84 cells were
compared, both toxins elicited similar anamnestic responses, with lag
periods of ~30 min (Fig. 6). We
therefore conclude that the different ER retention signals of cholera
toxin and E. coli enterotoxin are not the cause of their
differential toxicity. These findings are consistent with those of
Kreitman and Pastan (46), who found that KDEL or RDEL attached to the C
terminus of Pseudomonas exotoxin A were equally effective in
enhancing toxicity (46).
Construction of Hybrid Ctx/Etx Toxins to Determine the Basis for
Differential Toxicity--
To define whether the A- or B-subunits of
the two toxins are responsible for their different activities in T84
cells, a set of hybrid toxins was constructed. A schematic
representation of the holotoxin structure of EtxAB is shown in Fig.
4i. It illustrates that the A2-fragment can be considered as
two discrete structural segments primarily responsible for interacting
with the A1- and the B-subunit pentamer; a long
Construction of the hybrid toxins, with a fusion joint at codon 225 of
the A-subunit gene, involved a series of PCR amplifications of the
ctxAB and etxAB operons. The 5'-portion of the
ctxA and etxA genes (to codon 226) were amplified
and cloned separately as EcoRV/KpnI fragments,
while the downstream 3'-portions (from codon 226 of ctxA and
etxA, together with the entire ctxB and etxB gene, respectively) were cloned as
KpnI/SpeI fragments. Consequently, the ligation
of the 5' and 3' portions at the KpnI site (Fig. 1) enabled
the generation of two chimeric operons, one comprising codons
specifying the signal peptide plus residues 1-225 of ctxA, 226-240 of etxA, and the entire etxB gene (to
yield a hybrid toxin designated
CtxA (1-225)EtxA(226-240)EtxB), and the
other codons specifying the signal peptide and 1-224 of
etxA, 225-240 of ctxA, and the entire
ctxB gene (to yield a hybrid toxin designated
EtxA(1-224)CtxA(225-240)CtxB).
The generation of these chimeric operons resulted in the introduction
of a KpnI site, spanning codons 224-226 of the A-subunit genes; this had no effect on the encoded amino acid sequence in CtxA,
but changed codon 225 of EtxA to that normally found in CtxA,
i.e. from an Asp to Gly, accounting for the designation of
EtxA(1-224)CtxA(225-240)CtxB.
The two hybrid toxins were expressed and purified (Fig. 5,
lanes 4 and 5) using conditions
identical to those employed for the wild-type and RDEL mutant toxins,
above, then assayed for their capacity to induce electrogenic
Cl
Taken together, the findings implicate either the C-terminal portion of
the A2-fragment (from residues 226-240) or the B-subunits of the two
toxins, as important determinants of the difference in toxin activity.
Given that the B-subunits of E. coli enterotoxin are more
promiscuous in receptor binding than the B-subunits of cholera toxin,
one possible explanation for the lower potency of EtxAB was that
differential binding to receptors might influence the efficiency of
toxin delivery into T84 cells.
To address this question, a third hybrid toxin was constructed. The
EcoRV-EcoRI fragment from plasmid pATA14 was
substituted for the same fragment in pRC19 to generate a hybrid
composed of codons specifying the signal peptide and residues 1-236 of
ctxA, 237-240 of etxA, and the entire
etxB gene (Fig. 1). The resultant toxin comprising the
entire A-subunit of cholera toxin with a C-terminal RDEL sequence and
the entire B-subunit of E. coli enterotoxin, was
designated CtxA(RDEL)EtxB. Since the presence of a KDEL or
RDEL sequence at the C terminus of the A-subunit had no effect on the
relative potency of the toxin in T84 cells (see above), the
CtxA(RDEL)EtxB hybrid allowed us to determine whether the
basis of differential toxicity is due to the C-terminal portion of the
A2-fragment (from residue 226) or to the B-subunit.
The A2-fragment Is the Determinant of Differential
Toxicity--
Purified CtxAB,
CtxA(1-225)EtxA(226-240)EtxB, and
CtxA(RDEL)EtxB (Fig. 5, lanes 2,
5, and 8) were tested on T84 cells at various
concentrations. In all cases the
CtxA(1-225)EtxA(226-240)EtxB
hybrid was less effective than CtxAB and CtxA(RDEL)EtxB in
triggering electrogenic Cl
The sequences of CtxA(RDEL)EtxB and
CtxA(1-225)EtxA(226-240)EtxB only
differ by 4 amino acids (Fig. 4ii): Asp-229, Ile-230,
Thr-232, and His-233 (in CtxA) and Glu-229, Val-230, Ile-232, and
Tyr-233 (in EtxA). The residues at these positions in
CtxA(RDEL)EtxB are those normally found in the A-subunit of
cholera toxin, whereas those present in
CtxA(1-225)EtxA(226-240)EtxB are
normally found in the A-subunit of E. coli enterotoxin. We
conclude that one or more of these residues influence toxin action in
T84 cells. Given that the amino acid differences are in the A2-fragment
that winds through the central pore of the B-pentamer (see Fig.
4i), a number of possible explanations for the difference in
toxicity can be considered.
The A2-fragment from residues 225 to 240 in cholera and E. coli enterotoxin is the most structurally different region of the two toxins, as revealed by x-ray crystallography (4, 6). In E. coli enterotoxin, this segment comprises an extended chain (residues 227-231), a small
To test the possibility that the four amino acid differences between
CtxA(RDEL)EtxB and
CtxA(1-225)EtxA(226-240)EtxB had resulted in
a change in holotoxin stability, each hybrid was subjected to a range
of in vitro conditions that might normally be expected to
cause protein denaturation. The toxins were bound to
GM1-ganglioside-coated microtiter plates, treated with
denaturant, washed, and probed with monoclonal antibodies specific for
CtxA or EtxB as described under "Experimental Procedures." The
hybrids were found to be remarkably resistant to denaturants,
maintaining their holotoxin structure in the presence of 8 M urea and at pHs as low as 4.0. These findings are not
unexpected, given the exceptional stability of the cholera toxin and
E. coli enterotoxin B-pentamers (11). When the hybrids were
incubated in the presence of 0.5% (w/v) SDS at pH 7.2 for 30 min, the
A-subunits were released from GM1-bound B-subunits, and
this was greater at pH 5.5 (Fig. 9). Of
particular interest was the finding that CtxA(RDEL)EtxB
(see H1 in Fig. 9) was more resistant than
CtxA(1-225) EtxA(226-240)EtxB
(see H2 in Fig. 9) to A/B-subunit dissociation, under these conditions. Given that the toxins are likely to encounter a lower pH in
endosomal compartments, and the A-subunits may well be subjected to
conditions that promote unfolding during translocation, these in
vitro conditions provide an important insight into holotoxin stability. Since CtxA(RDEL)EtxB is the more stable of the
two toxins under these in vitro conditions, as well as being
more active in inducing electrogenic Cl
An alternative explanation for the difference in cholera toxin and
E. coli enterotoxin activity in T84 cells may be that the structural differences in the A2-fragments, highlighted above, cause a
subtle but significant change in conformation of the K(R)DEL sequence.
Since it is known that this sequence plays a role in the efficiency of
toxin action in T84 cells, which presumably stems from its ability to
engage the KDEL receptor involved in retrograde transport to the ER, it
remains possible that the function of the KDEL sequence will be
influenced by its position at the opening of the central pore of the
B-pentamer. The absence of structural data on the RDEL sequence of
E. coli enterotoxin does not allow a comparison to be made
with that of the KDEL sequence of cholera toxin. It will clearly be
important to determine whether the conformation of RDEL differs in
CtxA(RDEL)EtxB and
CtxA(1-225)EtxA(226-240)EtxB. Crystallization
of these two hybrids is in progress.
Finally, it has been proposed that the amphipathic nature of the
A2-fragment may endow it with an ability to insert into eukaryotic cell
membranes, presumably the ER membrane during A-subunit translocation (24), the occurrence of which may depend on unfolding and release of
the A-subunit from the B-pentamer. Thus, the nature of interactions between the A2-fragment and the B-pentamer may affect this step in the
toxicity pathway.
Concluding Remarks--
This study provides definitive evidence
that the difference in activity of cholera toxin and E. coli
heat-labile enterotoxin in T84 cells is due to structural elements in
the C terminus of the A2-fragment. Preliminary x-ray crystallographic
data on the CtxA(RDEL)EtxB hybrid have confirmed that the
A2-tail penetrating the pore of the EtxB pentamer resembles that found
in CtxAB rather than EtxAB.4
This would be consistent with the view that the structural differences in the A2-fragments in CtxA(RDEL)EtxB versus
EtxAB would alter the stability of A/B-subunit interactions and/or
change the spatial location of the K(R)DEL motif. These studies also
highlight the importance of a region in the toxin molecule that has
hitherto been generally overlooked with respect to a role in toxin
action. In this regard, the difference in toxicities of Ctx and Etx is
likely to be of considerable significance in the pathobiology of
cholera and traveler's diarrhea.
ion secretion, Ctx was found
to be the more potent of the two toxins. Here, we examine the
structural basis for this difference in toxicity by engineering a set
of mutant and hybrid toxins and testing their activity in T84 cells.
This revealed that the differential toxicity of Ctx and Etx was (i) not
due to differences in the A-subunit's C-terminal KDEL targeting motif
(which is RDEL in Etx), as a KDEL to RDEL substitution had no effect on
cholera toxin activity; (ii) not attributable to the enzymatically
active A1-fragment, as hybrid toxins in which the A1-fragment in Ctx was substituted for that of Etx (and vice versa) did not
alter relative toxicity; and (iii) not due to the B-subunit, as the replacement of the B-subunit in Ctx for that of Etx caused no alteration in toxicity, thus excluding the possibility that the broader
receptor specificity of EtxB is responsible for reduced activity.
Remarkably, the difference in toxicity could be mapped to a 10-amino
acid segment of the A2-fragment that penetrates the central pore of the
B-subunit pentamer. A comparison of the in vitro stability
of two hybrid toxins, differing only in this 10-amino acid segment,
revealed that the Ctx A2-segment conferred a greater stability to the
interaction between the A- and B-subunits than the corresponding
segment from Etx A2. This suggests that the reason for the relative
potency of Ctx compared with Etx stems from the increased ability of
the A2-fragment of Ctx to maintain holotoxin stability during uptake
and transport into intestinal epithelia.
INTRODUCTION
Top
Abstract
Introduction
References
secretion (18). Toxin action
is initiated by binding of the B-subunit moiety to cell surface
receptors. Recently, it was demonstrated that, following binding of Ctx
to T84 cells, Ctx-GM1 complexes cluster in caveolae-like
detergent insoluble subdomains of the plasma membrane (19).
Invagination and internalization of these membrane domains results in
the formation of smooth endocytic vesicles that enter vesicular
trafficking pathways leading to transport of the toxin to the
trans-Golgi network (TGN) (18, 20-25). The observation that brefeldin
A inhibits cholera toxin action (20, 21, 26), and the presence of a
KDEL sequence at that the C terminus of CtxA (RDEL in EtxA) have
suggested that the toxin is transported from the TGN to the endoplasmic
reticulum (ER). Indeed, mutations in the K(R)DEL sequence of CtxA and
EtxA reduce the efficiency of toxin-induced Cl
secretion
in T84 cells (22). It has been speculated that the A-subunit may detach
from the B-subunits in the TGN, since only the A-subunit
(A1/A2-fragments) has been detected in the ER (23-25), although
transport of the holotoxin from the Golgi to the ER, followed by rapid
dissociation and anterograde transport of the B-subunit back to the
Golgi, has not been excluded. Reduction of the disulfide bond between
the A1- and A2-fragments is thought to be catalyzed by protein
disulfide isomerase resident in the ER (27, 28), followed by
translocation of the A1-fragment across the ER membrane to the
cytosolic compartment. Because of its hydrophobicity, the A1-fragment
may remain associated with the cytosolic face of the ER membrane,
rather than being released free into the cytosol. The subsequent
trafficking and presumed delivery of the A1-fragment to the basolateral
membrane are less well understood, although the finding that the
A-subunit interacts with "so-called" ADP-ribosylation factors
(ARFs) (29), involved in vesicular transport, may facilitate its
anterograde targeting to the basolateral membrane. ARFs have also been
demonstrated directly to increase toxin activity in vitro
(30), a finding that may be important in determining the magnitude of
toxin action in vivo. The A1-fragments of Ctx and Etx
accomplish their toxic effects by ADP-ribosylating GS
, a
component of the trimeric GTP-binding protein that activates adenylate
cyclase. In T84 cells, toxin-induced elevations in cAMP levels lead to
electrogenic Cl
secretion: the primary ion transport
event responsible for secretory diarrhea in humans.
secretion in T84 cells demonstrated that Ctx (hereafter referred to as
CtxAB) was significantly more active than Etx (hereafter referred to as
EtxAB); CtxAB exhibited a shorter apparent lag period and generated a
higher short circuit current (22). In this paper, we describe the
structural basis for this difference in toxicity. The data reveal that
it is not due to structural or functional differences in the
ADP-ribosylating A1-fragment or in the receptor-binding B-subunits, but
to the A2-adapter fragment, which mediates A/B-subunit interactions.
These findings highlight the importance of a region in the toxin that
has hitherto been generally overlooked, and provides a possible
contributory explanation for the difference in severity of cholera and
traveler's diarrheal disease.
EXPERIMENTAL PROCEDURES
40 primer
(Stratagene) 5-TTTTCCCAGTCACGAC-3'. The fragment was then cleaved with
KpnI and SpeI and inserted into the corresponding sites of pBluescript, yielding pRC2. The hybrid operon was constructed using an intermediate vector pRCK18, a derivative of pK18 (38) in which
the polylinker sequence had been replaced by one with sites for
BamHI-XbaI-SpeI-ClaI-KpnI-NdeI-EcoRV-ApaI-PstI.3
The EcoRV-KpnI fragment from pRC7 was inserted
into pRCK18, followed by insertion of the
KpnI-SpeI fragment from pRC2. The resultant plasmid was cleaved with EcoRV and SpeI and the
hybrid operon cloned into the corresponding sites of pBluescript to
yield pRC18 (Fig. 1). The
EcoRV-SpeI fragment of pRC18 was subcloned into pTTQ18, yielding pRC20.
View larger version (21K):
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Fig. 1.
Wild-type and hybrid toxins. Plasmid
pRC1 contains the entire ctxAB operon encoding wild-type
CtxAB; plasmid pTRH29 (36) contains the entire etxAB operon
encoding wild-type EtxAB; plasmid pATA14 encoding
CtxA(RDEL)CtxB was generated by PCR amplification of
upstream and downstream segments of the ctxAB operon
followed by their ligation at an engineered EcoRI site to
introduce a Lys-237 to Arg substitution; plasmid pRC18 encoding
EtxA(1-224)CtxA(225-240)CtxB was generated by
ligating an upstream segment of etxA to a downstream segment
of the ctxAB operon at an engineered KpnI site;
pRC19 encoding CtxA(1-225)EtxA(226-240)EtxB
was generated by ligating an upstream segment of ctxA to a
downstream segment of the etxAB operon at an engineered
KpnI site; and pCDR1 encoding CtxA(RDEL)EtxB was
constructed by substituting the EcoRV-EcoRI
fragment in pTRH29 for the corresponding fragment from pATA14. In all
cases, the vector pBluescript II KS is not depicted.
40
primer. These were cloned separately into pBluescript to yield pRC3 and
pRC6, respectively. The subsequent subcloning of these fragments to
generate a hybrid operon was carried out in a manner identical to that
described above for the construction of pRC18. The resultant plasmid
encoding CtxA(1-225)EtxA(226-240)EtxB was
designated pRC19 (Fig. 1). The EcoRV-SpeI
fragment of pRC19 was subcloned into pTTQ18, yielding pRC21.
= 600 nm),
isopropyl-1-thio-
-D-galactopyranoside was added to a
final concentration of 0.5 mM and cells were cultured for
an additional 12 h. A total of 3 liters of culture was centrifuged at 8,000 × g in a Sorvall RC-5B centrifuge at 4 °C.
Sodium hexametaphosphate (39) was added to the culture supernatant to a
final concentration of 0.25% (w/v), the pH adjusted to 4.5 with
concentrated HCl, and the mixture stirred for 2 h at room
temperature. Precipitated material was recovered by centrifugation at
8,000 × g in a Sorvall RC-5B centrifuge at 4 °C and
then dissolved in 150 ml of 0.1 M sodium phosphate, pH 8.0. The sample was dialyzed against two changes of 20 mM
Tris-HCl, pH 7.0, at 4 °C in the presence of 0.02% (w/v) sodium
azide and then centrifuged in a Sigma 4K10 centrifuge at 15,000 × g for 1 h at 4 °C. The supernatant was adjusted to
1.0 M (NH4)2SO4,
stirred for 1 h at room temperature, and then centrifuged at
15,000 × g for 1 h at 4 °C. The supernatant was filtered through a 0.2-µm filter (Millipore) and toxin purified by FPLC (Amersham Pharmacia Biotech) using a modification of the method
described by Amin and Hirst (42). Briefly, the sample was applied to a
hydrophobic interaction column (2 × 11 cm, 35-ml bed volume,
Source 15PHE, Amersham Pharmacia Biotech) and eluted with a linear
gradient (1.0 to 0 M) of
(NH4)2SO4 containing 20 mM Tris-HCl, pH 7.5. Fractions containing the toxin were
pooled, desalted by overnight dialysis against 20 mM Tris
base, 20 mM NaCl, pH 8.75, at 4 °C and then applied to a
Resource Q column (6 ml, Amersham Pharmacia Biotech). Samples were
eluted with a linear gradient of NaCl (20 mM to 1.0 M) in 20 mM Tris base, pH 8.75. Fractions
containing the toxin were added to a NAP-10 column (Amersham Pharmacia
Biotech) to replace the buffer with phosphate-buffered saline (PBS:
0.15 M NaCl, 10 mM sodium phosphate, pH 7.2)
and stored at -80 °C. Toxin concentration was determined by UV
absorbance at
= 280 nm using the theoretical molar extinction
coefficients for each holotoxin calculated according to Gill and von
Hippel (40).
RESULTS AND DISCUSSION
efflux elicited by the toxins were markedly
different; a higher rate of Cl
secretion was induced by
CtxAB than by EtxAB (22). This suggested that structural or functional
attributes in either the A- or B-subunits of cholera toxin conferred
enhanced toxicity. To investigate this further, highly purified CtxAB
and EtxAB were prepared as described under "Experimental
Procedures." Briefly, recombinant plasmids encoding CtxAB or EtxAB
were introduced into the non-toxinogenic V. cholerae O395NT
strain, and the toxins purified from the culture media. The CtxAB
preparation eluted as a single homogeneous peak on anion-exchange
chromatography. When analyzed by SDS-polyacrylamide gel
electrophoresis, the A-subunit of CtxAB migrated with an apparent Mr of 21 kDa, corresponding to the A1-fragment,
indicating that it had been proteolytically nicked by V. cholerae, as expected (Fig. 2,
lane 1). The EtxAB preparation contained a
mixture of both unnicked and nicked toxin, which eluted at slightly
different salt concentrations, enabling separation of the two
populations (Fig. 2, lanes 2 and 3).
When the toxicity of purified CtxAB and EtxAB (100 nM,
nicked) was tested on T84 cells the toxins exhibited clear differences
in time course and magnitude of Cl
secretion, with CtxAB
being more potent than EtxAB (Fig. 3). This confirmed the earlier observations on CtxAB and EtxAB derived from
periplasmic fractions of E. coli (22).
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Fig. 2.
Purification of homogeneous preparations of
"nicked" and "unnicked" toxins. CtxAB and EtxAB were
purified as described under "Experimental Procedures," and boiled
in SDS sample buffer containing the reducing agent dithiothreitol prior
to analysis by SDS-polyacrylamide gel electrophoresis. Lane
1, CtxAB (nicked); lane 2, EtxAB
(unnicked); lane 3, EtxAB (nicked). The migration
positions corresponding to intact (unnicked) A-subunits (A),
the nicked A1-fragment (A1), and B-subunit monomers
(B) are indicated. The position of the molecular weight
markers are indicated on the right-hand side of the
figure.
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Fig. 3.
Ctx and Etx exhibit differential toxicity in
T84 cells. Time course of electrogenic Cl secretion
induced by the addition of 100 nM nicked CtxAB (
),
nicked EtxAB (
), or unnicked EtxAB (
) to the apical surface of
T84 cell monolayers (with the data points representing the mean ± S.E., where n = 2 independent monolayers). Twelve
independent experiments gave similar results.
secretion
by T84 cells (Fig. 3). This is consistent with our previous finding
that T84 cells contain a serine protease that can nick and activate the
A-subunits of cholera toxin and E. coli enterotoxin, and it
further demonstrates that nicking per se is not the
rate-determining step defining the magnitude and time course of
Cl
secretion.
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Fig. 4.
Schematic representation of the positions
within the holotoxin structure used in the construction of mutant and
hybrid toxins. i, the A2-fragment within the EtxAB
holotoxin structure and the sites corresponding to A-subunit nicking
(Arg-192), hybrid fusion at either Asp-225 or Ile-236 and the RDEL
sequence are illustrated. ii, primary amino acid sequences
of the C-terminal domains of CtxA and EtxA from residue 187 to 240. Amino acid differences are in bold.
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Fig. 5.
SDS-polyacrylamide electrophoretic analysis
of recombinant toxins. Purified toxins from culture supernatants
of V. cholerae O395NT harboring the appropriate plasmids
were analyzed by SDS-polyacrylamide gel electrophoresis. In this
instance, the toxins were boiled in sample buffer lacking
dithiothreitol; as a consequence, the apparent molecular mass of both
nicked and unnicked A-subunits was approximately 30 kDa.
Lane 1, CtxB; lane 2,
CtxAB; lane 3, CtxA(RDEL)CtxB;
lane 4,
EtxA(1-224)CtxA(225-240)CtxB; lane
5,
CtxA(1-225)EtxA(226-240)EtxB;
lane 6, EtxAB; lane 7,
EtxB; lane 8, CtxA(RDEL)EtxB. The
migration positions of the A-subunit (A) and B-subunit
monomers (EtxB and CtxB) are shown on the
right-hand side of the figure. Note that EtxB has a slower
electrophoretic mobility than CtxB.
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Fig. 6.
The Arg for Lys substitution in the -K(R)DEL
motif is not responsible for the differential toxicity of Ctx and
Etx. Time course of electrogenic Cl secretion
induced by the addition of 100 nM of CtxAB (
) or
CtxA(RDEL)CtxB (
) to the apical surface of T84 cell
monolayers. (with the data points representing the mean ± S.E.,
where n = 2 independent monolayers). Five independent
experiments gave similar results.
-helix in A2-
(residues 197-224) abuts the A1-fragment, while the remainder (A2
residues 225-236) winds through the pore of the toroidal B-pentamer.
Because of the relatively large number of non-conservative differences
in amino acids in the A2-fragments of CtxA and EtxA (Fig.
4ii), hybrid toxins were designed with the fusion site
located at the end of the long
-helix of the A2-fragment,
corresponding to amino acid 225. By so doing, the interactions between
the A1- and A2-fragments and between the A2-fragment and B-subunits in
the hybrids would involve homologous toxin domains, which we considered
would favor toxin assembly and stability.
secretion by T84 cells. The hybrid toxin
CtxA(1-225)EtxA(226-240)EtxB was less potent
than wild-type CtxAB at inducing Cl
secretion (Fig.
7A). Indeed the delay in the
time course of Cl
secretion with the hybrid more closely
resembled that of wild-type EtxAB (Fig. 7B). By
contrast, the activity of the other hybrid, EtxA(1-224)CtxA(225-240)CtxB,
more closely resembled that of wild-type CtxAB (Fig. 7, compare
panels A and B). These findings
demonstrated that a potent electrogenic response could be elicited by
toxins in which the first 224 amino acids were derived from either CtxA
or EtxA. Since this region contains the entire ADP-ribosylating
A1-fragment (i.e. residues 1-192), we conclude that the
differential toxicity of cholera toxin and E. coli
enterotoxin cannot be due to differences in the enzymatic activities of
the A-subunits. This conclusion was further substantiated by analysis
of the intrinsic ADP-ribosyltransferase activity of both the wild-type
and hybrid toxins (Table I). Agmatine was
used as the substrate for ADP-ribosylation, and the ADP-ribosylating activity measured with or without recombinant hARF6. Comparison of
wild-type cholera toxin with the various toxin constructs
containing the CtxA1-fragment, revealed that they all had similar
ADP-ribosyltransferase activity. The activities of wild-type E. coli enterotoxin and the hybrid toxin containing the Etx
A1-fragment were found to be approximately 1.6-2.8-fold higher than
that exhibited by cholera toxin (Table I). Thus, the lower potency of
E. coli enterotoxin in T84 cells cannot be
attributed to a lower level of intrinsic enzymatic activity. Lee
et al. (47) also noted that the activity of E. coli enterotoxin, in the agmatine assay, was slightly higher than
that of cholera toxin (47), but the reasons for this remain unknown.
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Fig. 7.
The differential toxicity of Ctx and Etx is
not attributable to the enzymatic A1-fragment. Time course of
electrogenic Cl secretion induced by T84 cell monolayers
following the addition to apical surface of 600 nM CtxAB
(
) or CtxA(1-225)EtxA(226-240)EtxB (
)
(panel A) and EtxAB (
) or
EtxA(1-224)CtxA(225-240)CtxB (
)
(panel B). The data points represent the mean ± S.E.,
for two independent monolayers. Four independent experiments gave
similar results.
ADP-ribosyltransferase activities of wild-type and hybrid toxins
secretion, even at fully
saturating concentrations of 600 nM (Fig.
8). Importantly, the time course and
magnitude of Cl
secretion elicited by CtxAB and
CtxA(RDEL)EtxB were nearly identical (Fig. 8). Since these
two toxins contain different B-subunit components, but are equally
effective in triggering Cl
secretion in T84 cells, the
differential toxicity of cholera toxin and E. coli
enterotoxin cannot be associated with their respective B-subunits. By
contrast, the data shown in Fig. 8 demonstrate that
CtxA(RDEL)EtxB and
CtxA(1-225)EtxA(226-240)EtxB, which only
differ in the C-terminal portion of the A-fragment, have toxicities
characteristic of cholera toxin and E. coli enterotoxin, respectively. We therefore conclude that the structural basis for
differential toxicity resides in the A2-fragment.
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Fig. 8.
The A2-fragment is the determinant of
differential toxicity. Time course of electrogenic
Cl secretion induced by the addition of 600 nM CtxAB (
), CtxA(RDEL)EtxB (
), or
CtxA(1-225)EtxA(226-240)EtxB
(
) to the apical surface of T84 cell monolayers (with the data
points representing the mean ± S.E., where n = 2). Five independent experiments gave similar results.
-helix (232-236), and the last four RDEL residues. The extended chain and
-helix are located within the
central pore of the B-pentamer, while the exact location and structure
of the RDEL sequence remain uncertain due to lack of definition in the
electron density. Many of the residues in this segment are involved in
intersubunit salt bridge, hydrophobic, and hydrogen bond interactions
with the B-subunits. These include Glu-229, Ile-232, and Tyr-233, which
interact with specific residues in the B-subunit pentamer (4). The
x-ray crystal structure of cholera toxin revealed that the A2-segment
from residue 227 enters the central pore of the B-pentamer as an
-helix rather than as an extended chain (6). The last four (KDEL)
residues were also visible in the electron density of cholera toxin and may reflect the more compact structure of the Ctx A2- than the Etx
A2-fragment. Given these structural differences, a possible explanation
for differential toxicity would be that amino acid changes between
cholera toxin and E. coli enterotoxin in the A2-fragment alter the stability of A/B-subunit interaction, which may in turn influence maintenance of the holotoxin structure during its transport along the endocytic pathway or potentiate liberation of the A-subunit at a stage when translocation occurs.
secretion in T84
cells, we hypothesize that the difference in toxicity of cholera toxin
and E. coli enterotoxin is due to the greater ability of
cholera toxin to maintain its holotoxin structure during transit
through the endocytic pathway and thus to deliver a greater proportion
of internalized toxin to the site of translocation and action.
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Fig. 9.
Comparison of the stability of hybrid toxins
which differ only in the C-terminal segment of the A2 polypeptide.
The maintenance of A-subunit/B-subunit interaction in toxins bound to
GM1 was determined for CtxA(RDEL)EtxB
(designated H1) and
CtxA(1-225)EtxA(226-240)EtxB (designated
H2) after incubation for 30 min at room temperature in PBS,
PBS containing 0.5% SDS, or McIlvaine buffer, pH 5.5, containing 0.5%
SDS. The amount of pentameric EtxB present following such treatment was
assessed by enzyme-linked immunosorbent assay using an anti-EtxB
monoclonal antibody (118-8), whereas the amount of A-subunit associated
with EtxB was assessed using an anti-CtxA monoclonal antibody (CT-17).
Each sample was analyzed in duplicate (with the data representing the
mean ± S.E., where n = 2). The figure is a
representative data set from three independent experiments.
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ACKNOWLEDGEMENTS |
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We thank Chris Rees and Linda Stevens for technical contributions and Drs Doug Millar, Richard Pitman, Martha Vaughan, and Neil Williams for critically reading the manuscript.
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FOOTNOTES |
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* This work was supported by grants from the Wellcome Trust (to T. R. H.), from the National Institutes of Health (to W. I. L. and the Harvard Digestive Center) and from the Università Cà Foscari di Venezia and the University of Bristol (to C. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom all correspondence and reprint requests should be
addressed: Dept. of Pathology and Microbiology, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, United
Kingdom. Tel.: 44-117-9287538; Fax: 44-117-9300543; E-mail: t.r.hirst{at}bristol.ac.uk.
The abbreviations used are:
Ctx, cholera toxin; Etx, E. coli heat-labile enterotoxin; CtxA, A-subunit of
cholera toxin; CtxB, B-subunit of cholera toxin; EtxA, A-subunit of
E. coli heat-labile enterotoxin; EtxB, B-subunit of E. coli heat-labile enterotoxin; ER, endoplasmic reticulum; TGN, trans-Golgi network; ARF, ADP-ribosylation factor; rhARF 6, recombinant
human ADP-ribosylation factor 6; PBS, phosphate-buffered saline; HBSS, Hanks' balanced salt solution; GM1, monosialoganglioside-GM1
(Gal1-3GalNAc
1-(Neu5Ac
2-3)-4Gal
1-4Glc
1-Cer); PCR, polymerase chain reaction; BSA, bovine serum albumin.
2 The ApaI site is immediately adjacent to the EcoRV site shown in Fig. 1.
3 Difficulties were encountered in the construction of the hybrid operons, possibly due to the toxicity of gene products encoded by fragments of the toxin genes; and necessitated the design and use of an intermediate vector.
4 B. Bovey, C. Rodighiero, W. G. J. Hol, and T. R. Hirst, unpublished observations.
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REFERENCES |
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