Groupe de Recherche sur les Maladies Infectieuses du Porc, Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, 3200 Sicotte, CP 5000, Saint-Hyacinthe, Québec, CanadaJ2S 7C61
Author for correspondence: J. Daniel Dubreuil. Tel: +1 450 773 8521 ext. 8433. Fax: +1 450 778 8108. e-mail: daniel.dubreuil{at}umontreal.ca
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ABSTRACT |
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Keywords: enterotoxin b, mutagenesis, binding, ETEC
Abbreviations: CD, circular dichroism; SFT, sulfatide
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INTRODUCTION |
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In contrast to LT and STa toxins, there is no evidence that STb is associated with an intracellular elevation of cyclic nucleotide (Kennedy et al., 1984 ; Peterson & Whipp, 1995
; Weikel & Guerrant, 1985
). Instead, in vitro studies have shown that the intestinal secretion pathway involves the activation of a GTP-binding regulatory protein (G
i3 protein) sensitive to pertussis toxin. This action results in an intracellular calcium elevation through a receptor-dependent ligand-gated Ca2+ channel (Dreyfus et al., 1993
). The ensuing high level of intracellular Ca2+ is presumably involved in activation of a calmodulin-dependent protein kinase II (CaMK II) which could open an undetermined ionic channel (Fujii et al., 1997
). In addition, intracellular concentration of Ca2+ may regulate phospholipases A2 and C, which catalyse the release of arachidonic acid from the membrane phospholipids, and ultimately the formation of prostaglandin E2 (PGE2) known as a secretory agent (Fujii et al., 1995
; Hitotsubashi et al., 1992
; Peterson & Whipp, 1995
). Recently, our laboratory demonstrated in vivo the functionality of sulfatide (SFT) as a receptor for STb (Rousset et al., 1998
). We determined its affinity constant (Kd) between 2 and 6 µM (Beausoleil & Dubreuil, 2001
). Unsolved remain the steps between the attachment of STb to its receptor and the activation of the G protein presumably involved in signal transduction.
STb toxin synthesized as a 71 aa precursor (Fig. 1) undergoes cleavage of the N-terminus signal sequence (residues 123) (Kupersztoch et al., 1990
). This process results in a 48 aa polypeptide of 5200 Da, pI 9·6 (Handl et al., 1993
). Maturation also involves the formation of two disulfide bridges: between C10 and C48 and between C21 and C36. Both disulfide bridges are important for stabilization of the molecules tertiary structure (Sukumar et al., 1995
) and are indispensable for enterotoxicity (Arriaga et al., 1995
; Okamoto et al., 1995
). NMR studies of STb have established a structure having two antiparallel
-helix motifs separated by a loop. An amphipathic helix between residues 10 and 22 exposes several polar side chains to the solvent. Facing this is a hydrophobic helix extending from residue 38 to 44. The glycine-rich loop presents some amino acids that previous mutagenesis studies have demonstrated as being important in enterotoxicity (Sukumar et al., 1995
).
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METHODS |
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Circular dichroism analysis.
Circular dichroism (CD) measurements were carried out on a Jasco J-710 spectropolarimeter. Far-UV (300185 nm) runs were conducted in a 0·1 cm cell maintained at room temperature. Concentrations of STb and mutant STb were adjusted to 0·75 mg ml-1 in phosphate buffer 0·02 M (pH 7·2). The data collected were computer-averaged for ten scans, baseline subtracted, and smoothed.
Microtitre plate binding assay.
U-bottom polystyrene microtitre plates (Serocluster, Costar) were coated by overnight evaporation of 50 µl methanol-diluted SFT (500 ng) (Sigma). Free sites were blocked with 200 µl per well phosphate-buffer (PBS)-casein (0·3%, w/v) pH 7·4 for 2 h at room temperature. Purified STb toxin (500 ng) or mutant STb in 50 µl PBS (0·1 M phosphate containing 150 mM NaCl) pH 7·0 were added per well in triplicate for 1 h at 37 °C. After three washes with PBS-casein, each well was filled with 50 µl rabbit monospecific anti-STb antibodies diluted 1:1000 in PBS-casein for 1 h at 37 °C. The plates were washed three times with PBS-casein. Goat anti-rabbit IgG conjugated to horseradish peroxidase (Jackson Immunoresearch) was diluted 1:3000 in PBS-casein and 50 µl was applied to each well before incubation at 37 °C for 1 h. Following three washes with PBS-casein, 50 µl chromogenic substrate ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)] (Sigma) (0·4 mM) dissolved in citrate buffer pH 4·0 containing 0·5 mM H2O2 was added. The absorbance was measured at 410 nm using an automatic ELISA reader (model MR5000, Dynatech Laboratories).
Triplicates of methanol with and without SFT served as negative controls to evaluate the background after the blocking step and the reaction of anti-STb antiserum with SFT, respectively. For analysis, the mean absorbance obtained for these wells was subtracted from the mean absorbance for STb or each mutant tested. The value for STb was considered as 100% binding and all the data were calculated relative to this value. The final value represents the mean of at least two assays.
Ligated rat jejunal loop assay.
Enterotoxicity studies were conducted using a ligated rat jejunal loop assay as described previously (Dubreuil et al., 1991 ). Briefly, 6- to 8-week-old white male rats (Sprague Dawley CD; Charles River Ltd) were fasted for 48 h and anaesthetized. The small intestine was exteriorized following a midline abdominal incision and rinsed for 5 min with 8 ml 0·85% saline solution containing 300 µg soybean trypsin inhibitor (Boehringer Mannheim) ml-1. A series of seven 5-cm-long ligated segments (loops) was made in the jejunum. The posterior 30% of the small intestine starting at the caecum was not used. Each loop received 5 µg test material in 500 µl 20 mM Tris/HCl buffer (pH 6·8) containing 300 µg trypsin inhibitor ml-1. Loops receiving only the buffer were considered as the negative controls. The abdominal incision was closed. Three hours later, the rats were killed and the amount of fluid in each loop was measured. Results are expressed as the ratio of millilitres fluid accumulated per centimetre ligated jejunal segment. Each mutant was tested twice in each rat, varying the position among the loops, and at least six rats were used for each mutant. To minimize the effect of sample position, two mutants and two STb-positive controls were alternated in each rat. Samples were always separated by buffer (negative control).
For analysis, the mean of three negative controls was subtracted from the values of two positive controls (STb alone) and from two samples of each mutant tested, in the same rat. Finally, from these results the mean and standard deviation were calculated, omitting the highest and the lowest values. The value for STb was considered as 100% biological activity and all the data were transformed relative to this value.
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RESULTS |
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Analysis of STb mutants by CD
Properly oxidized disulfide bonds have been shown to be important for full STb enterotoxicity (Arriaga et al., 1995 ). In our study, we observed that STb attachment to SFT is also dependent on the presence of oxidized disulfide bonds. Upon ß-mercaptoethanol treatment, the binding to SFT decreased to 10% (data not shown). Because important conformational changes induced by point mutations in STb could prevent the proper oxidation of cysteines in disulfide bridges, we analysed each mutant by measuring their CD spectra to confirm that all mutants retained their full structural integrity and in particular the presence of both disulfide bonds. Thus, the main chain structure of the STb enterotoxin, representing the
-helixloop
-helix pattern, appears not likely to be greatly affected by our site-directed mutagenesis. The CD spectra were similar to native STb, with the exception of mutant M42S, which seems slightly different (data not shown).
Hydrophobicity profiles of STb mutants
As shown in Fig. 2, STb mutants were purified to homogeneity as demonstrated by the single elution peak using a C8 reverse-phase column. These chromatograms represent the hydrophobicity profiles of the toxin mutants generated. As expected, mutations changing a non-polar residue for a polar one in the hydrophobic
-helix (F37, I41 and M42) diminished their retention time (tR) compared to native STb toxin. Almost all toxin mutants in the loop (C21C36) were produced using the alanine-scanning procedure (Cunningham & Wells, 1989
). In this case, the tR of the mutants was increased, indicating a predicted loss of polarity.
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Importance of the hydrophobic -helix in attachment to SFT
To reduce the hydrophobicity features of the C-terminal -helix, some highly non-polar residues pointing to the solvent (F37, I41 and M42) were targeted. None of them, even when mutated in combination, changed for negatively charged, positively charged or polar residues, showed a binding activity greater than 10% of native STb, with the exception of mutant M42R, which expressed the same attachment to SFT as STb. Among all mutants, the double mutation I41E-M42R displayed the lowest affinity for the receptor (3%).
Relative biological activity
The rat loop assay is a standard method that has proven to be useful for evaluating the toxicity of STb toxin. Among the 12 single mutants depicted in Fig. 3, the greatest decreases in toxicity were noted with three residues (F37K, I41S and M42R) located in the non-polar helix and two located in the loop (G24A and G31A). The biological activity detected in rat loop assay for these five mutants averaged 11, 11, 6, 11 and 1%, respectively. For the double mutation I41E-M42R, intestinal enterotoxicity (11%) was similar to the single mutations. Inside the STb loop, diminution of the toxicity was observed with three single mutants assigned to basic residues of the loop (K22A, K23A and R29A) for which the enterotoxicity decreased to about 65, 22 and 51%, respectively. The consequences of combining substitutions were also studied. The double mutants K22A-K23A and K23A-R29A showed enterotoxicities of about 17 and 7%, respectively. These values are lower than those for the corresponding single mutants. Small changes in toxicity were noted with D30A, for which the enterotoxicity was about 51%. For mutants G27P and G34A, the enterotoxicity was reduced to 20 and 13%, respectively.
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DISCUSSION |
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According to the highly basic pI (9·6) of STb and because of the anionic sulfate residue carried by SFT receptor, it is conceivable that some positively charged amino acid residues of STb could participate in bringing together and in binding to SFT. Previously, Fujii et al. (1994) demonstrated that the lysine residues K22 and K23 were important in the expression of the enterotoxic activity of STb. Dreyfus et al. (1992)
described the role of arginine R29 in toxicity. Therefore, these basic residues were tested for their affinity toward SFT using alanine replacement mutagenesis. The binding assay was done in a buffer containing 150 mM NaCl to more accurately mimic the physiological state. The dissociating salt should also help to neutralize the surface charges found on STb toxin. Because a significant decrease was observed in binding activity with mutants K22A, K23A and R29A, the individual amino acid residues K22, K23 and R29 evidently have an important and equivalent implication in the attachment to SFT. Yet, K22 and K23 seem to have an important role in binding as the double mutant K22A-K23A exhibited significantly less binding capacity to SFT than the double mutant K23A-R29A. Moreover, an additive effect in binding reduction was noted when K22 and K23 were co-mutated. The results obtained for the various mutants tested indicate that binding to SFT represents the initial event leading to the in vivo enterotoxicity observed. In fact, the reduction in toxicity was always either equal to or greater than the decrease in binding to SFT. This was expected, as the binding of a toxin to its receptor is the trigger step in pathogenesis leading to specific toxic consequences. Significantly, the lysine residue K23 seems to have an additional role in enterotoxicity as inferred by the dissociation observed for mutants K23A, K22A-K23A and K23A-R29A between the enterotoxic and binding activities. Discrepancies were noted between our results and those of Fujii et al. (1994)
, notably about the relevance of K22 for enterotoxicity. Their K22L mutant was shown to be less enterotoxic (10%) than the K22A mutant (70%) presented in this study. This divergence may be explained by the shorter side chain of the alanine of our K22A mutant compared to the longer leucine of Fujiis K22L mutant.
At position 30 inside the loop (C21C36), substitution of the aspartate for either alanine or valine reduced the enterotoxicity while the attachment to SFT seemed to be favoured. The suppression of the negative charge present on the aspartate side chain may eliminate a repulsive force that could occur in the vicinity of the SFT-binding site. Indeed, SFT bears an anionic sulfate group. Moreover, the substantial difference observed in attachment between mutants D30A and D30V could result from the preferential presentation of SFT due to the larger size of valine versus alanine or to the more hydrophobic property of valine compared to alanine. In contrast, this mutation appears to alter a subsequent step in enterotoxicity. The mutant D30V reported by Dreyfus et al. (1992) showed a similar enterotoxicity level (39%) to the D30A mutant examined in this study (49%).
Because of the flexibility conferred by four glycines in the loop (C21C36), we hypothesized that they would play a role in binding. Toxins such as -haemolysin of S. aureus are known to display this characteristic, which facilitates membrane insertion. To test this hypothesis, glycines G24, G27, G31 and G34 were selected for mutagenesis. The results showed a decrease in both attachment and enterotoxicity (except for mutant G34A, which showed a binding of 118%). This reduction may be due either to the elimination of the flexibility or to the low space occupancy of the glycine residue. As observed for K23, G31 and G34 seem to play an additional role in enterotoxicity other than simply the attachment. This conclusion is inferred by the dissociation between the enterotoxic and the binding activities observed for mutant G31A (1% and 41%, respectively) and mutant G34A (13% and 118%, respectively).
Among other SFT-binding proteins, the FasG subunit of the 987P fimbriae was shown to bind to its SFT receptor (Choi & Schifferli, 1999 ). Specifically, residues R116, K117 and K118 in alanine-scanning site-specific mutagenesis experiments have been shown to play a role in attachment to SFT. Thus, for STb and FasG, closely gathered basic residues appeared to interact with SFT, involving a salt-bridge interaction with the sulfate group of SFT (Choi & Schifferli, 1999
). Similar observations were made with properdin, another SFT-binding protein (Holt et al., 1990
). In contrast, the flavonol 3-sulfotransferase (ST) displays some similarities with the STb toxin. ST is an enzyme using 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as cosubstrate. Structurally, PAPS is closely related to SFT, both molecules having an acidic sulfate group linked to a sugar ring. Marsolais & Varin (1995)
did site-specific mutagenesis on ST throughout an amino acid stretch known to bind the cosubstrate PAPS, 276RKGXXGDWK(N/T)XFT288. They demonstrated that residues R276, K277, G281 and K284 were involved in cosubstrate binding by using mutants. Those residues could correspond to K22, K23, G27 and R29, found within STb loop 22KKGFLGVRDGTAGA35. Marsolais & Varin (1995)
also established that conservative replacements such as K284R have little effect on PAPS-binding activity.
To test the hypothesis concerning the implication of the hydrophobic -helix in enterotoxicity according to the STb NMR structure, three major hydrophobic residues facing the solvent (F37, I41 and M42) were mutated (Sukumar et al., 1995
). These mutants demonstrated weak enterotoxicity in the rat loop assay, providing compelling evidence for the role of selected non-polar residues in pathogenesis. Chao & Dreyfus (1998)
have shown that STb toxin promotes leakage of lipid vesicles, indicating local disruption or membrane insertion. Considering that amphipathic and hydrophobic helical structures are required for membrane insertion of the toxin, we postulated that F37, I41 and M42, belonging to the C-terminal hydrophobic
-helix, could be involved in the internalization pathway inferred by Chao & Dreyfus (1997)
.
Mutants F37K, I41S and M42S showed the same reduction in binding to SFT compared to the reduction in enterotoxicity. However, these results were difficult to reconcile with other results obtained in our laboratory. In fact, Rousset et al. (1998) have shown that the epitope recognized by STb is specifically a galactose residue linked to a sulfated group at position 3. This structure displays no hydrophobic region enabling an interaction with STb. In fact, galactose is comprised of alcohol groups and one hemiacetal group conferring a polar environment. From these observations, theoretically no direct hydrophobic interaction could occur between STb and SFT. To explain the involvement of hydrophobic amino acids in binding to SFT, a model was developed implying an oligomerization process between STb molecules. As demonstrated recently, STb enterotoxin forms hexamers and heptamers through hydrophobic bonds between C-terminal hydrophobic
-helix residues F37, I41 and M42 (Labrie et al., 2001
). Full STb structure integrity is necessary for the oligomerization process to take place. We suggest that there is basic binding activity between STb and SFT with a STbSTb inter-binding process, in part via the hydrophobic
-helix. In this case, the propensity of hydrophobic groups to associate due to their repulsion from the aqueous solvent compels STb molecules to interact. This could explain the decrease in attachment observed with polar mutants located in the hydrophobic
-helix. Oligomerization is observed for the
-toxin of S. aureus following a preliminary binding either with a high-affinity protein receptor, or, more commonly, with ubiquitous low-affinity acceptors (phosphatidylcholine and cholesterol). As for STb, the S. aureus
-toxin contains a glycine-rich flexible motif (Menestrina & Ferreras, 1997
).
The most prominent result of this study is the establishment of F37, I41 and M42 as important residues for binding and enterotoxicity activities. In addition, we have demonstrated the importance of charged amino acid residues present in the loop (K22, K23, R29 and D30) in the attachment to SFT and confirmed their role in enterotoxicity. Mutations of these residues strongly inhibited both binding and toxic activities. Moreover, these latter hydrophobic residues appear to be more important in both attachment to SFT and enterotoxicity than the previously identified charged amino acids situated in the loop. Taken together, the results indicate that hydrophobic amino acids are directly implicated in the oligomerization process that precedes the binding to SFT. Binding to SFT is due to interaction of SFT with the positively charged lysine and arginine residues of STb. Our study has also clearly established that the binding to SFT is a prerequisite step preceding fluid secretion, as a reduction in binding was concomitantly associated with an equivalent or greater decrease in toxicity.
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ACKNOWLEDGEMENTS |
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We thank M. Tousignant and C. Bérubé for their valuable collaboration with the rat loop assay; F. Dumas for the protein sequencing service and HPLC resourcefulness; and Dr Kornblatt for her helpful advice with CD measurements.
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REFERENCES |
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Received 1 March 2001;
revised 21 June 2001;
accepted 10 July 2001.
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