Department of Veterinary Science and Microbiology, The University of Arizona, 1117 East Lowell Street, Tucson, AZ 85721, USA1
Author for correspondence: Stephen J. Billington. Tel:+1 520 621 2745. Fax:+1 520 621 6366. e-mail: sbilling{at}u.arizona.edu
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ABSTRACT |
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Keywords: cholesterol-dependent cytolysin, virulence factor, amino acid substitution
Abbreviations: BME, ß-mercaptoethanol; CDC, cholesterol-dependent cytolysin; ILY, intermedilysin; LLO, listeriolysin O; PFO, perfringolysin O; PLO, pyolysin; PLY, pneumolysin; SRBC, sheep red blood cell
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INTRODUCTION |
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A. pyogenes secretes a haemolytic exotoxin, pyolysin (PLO) (Billington et al., 1997 ; Ding & Lämmler, 1996
), which is an important virulence factor, as a knockout mutation in the plo gene resulted in reduced virulence of the mutant in a mouse model (Jost et al., 1999
). Recombinant PLO is also an effective vaccine in the prevention of experimental infections in mice (Jost et al., 1999
), although its ability to protect domestic animals from natural infection is still to be tested. PLO is a novel member of a group of toxins historically known as the thiol-activated cytolysins. As all members of this family are not subject to thiol-activation, including PLO (Billington et al., 1997
; Nagamune et al., 1996
), a number of alternative designations have been proposed (Billington et al., 2000
; Gilbert et al., 1999
; Nagamune, 1997
; Shatursky et al., 1999
), with cholesterol-dependent cytolysins (CDCs) (Shatursky et al., 1999
) being the most widely accepted. This group of toxins, which includes listeriolysin O (LLO), perfringolysin O (PFO), pneumolysin (PLY) and streptolysin O, is characterized by an undecapeptide sequence (ECTGLAWEWWR) located near the C terminus of the protein (Fig. 1a
). Oxidation of the cysteine residue within this sequence is responsible for the thiol-activated nature of many CDCs. Both PLO and intermedilysin (ILY) of Streptococcus intermedius have variant undecapeptide sequences, in which the conserved cysteine has been substituted with an alanine residue, resulting in oxygen-resistant toxins (Billington et al., 1997
; Nagamune et al., 1996
; Nagamune, 1997
) (Fig. 1a
). Many advances have been made in recent years as to the function of the four domains within the CDC tertiary structures and their roles in pore-formation (Tweten et al., 2001
). However, while it has been evident for some time that the undecapeptide is critical for CDC activity (Boulnois et al., 1991
; de los Toyos et al., 1996
; Jacobs et al., 1999
; Korchev et al., 1998
; Michel et al., 1990
; Pinkney et al., 1989
; Saunders et al., 1989
; Sekino-Suzuki et al., 1996
), the precise function of this region of the molecule is unclear.
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Both PLO and the human-specific cytolysin ILY have variant undecapeptide sequences (Billington et al., 1997 ; Nagamune, 1997
) (Fig. 1a
). It is not known if this sequence divergence represents an attempt to regulate cytolytic activity or whether, in the case of PLO which is the most divergent member of the CDC family, the sequence divergence compensates for structural differences in other regions of the protein between PLO and the other CDCs. In this study, we examine the activity of various site-specific mutants in the undecapeptide of PLO and determine that the sequence variation in the PLO undecapeptide is required for full cytolytic activity.
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METHODS |
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DNA techniques.
Procedures for E. coli transformation and plasmid extraction, DNA restriction, ligation, agarose gel electrophoresis and Southern blotting were performed as described by Ausubel et al. (1994) . Plasmids pJGS59, encoding a 543 aa protein (His-PLO) composed of 507 aa of the mature PLO with an N-terminal extension of 36 aa encoded by pTrcHis B, including a hexahistidine sequence, and pJGS89, a site-directed mutant of pJGS59 that encodes a mutant version of His-PLO with an alanine to cysteine change at position 492 (His-PLO.C492), have been described previously (Billington et al., 1997
). pJGS59 and pJGS89 were used as templates for the construction of site-directed mutants, using the mutagenic primers indicated in Table 1
and the Transformer Site Directed Mutagenesis Kit (Clontech), according to the manufacturers instructions. Two selection primers were used. Primer 5'-GACTTGGTTGAGGCCTCACCAGTCAC-3' was designed to change the ScaI site of pTrcHis B to StuI and was used in conjunction with forward primers listed in Table 1
. A second selection primer, 5'-GAGTGCACCATGGGCGGTGTGAAAT-3', was designed to change the NdeI site of pTrcHis B to NcoI and was used in conjunction with the reverse primers listed in Table 1
. Introduction of appropriate mutations into either pJGS59 or pJGS89 was confirmed by automated DNA sequencing on a 373A DNA sequencer (Applied Biosystems) at the University of Arizonas DNA sequencing facility.
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Haemolytic assays.
Microtitration haemolytic assays were performed essentially as described previously (Billington et al., 1997 ), except that dilutions of toxins were performed in 1 mg BSA ml-1 to aid in stabilization of the toxin. One haemolytic unit (HU) was defined as the amount of haemolysin (in ng) required to release 50% of the haemoglobin from 200 µl of 0·25% sheep red blood cells (SRBCs) in 1 h at 37 °C, and specific haemolytic activity was expressed as HU (µg protein)-1. The specific haemolytic activity of all His-PLO mutant proteins was expressed as a percentage of His-PLO haemolytic activity, which was designated as 100%. The effects of reducing compounds on haemolytic activity was assayed by incubating 10 µg toxin preparations ml-1 with final concentrations of either 2·5 mM DTT or 2% (v/v) ß-mercaptoethanol (BME) for 10 min at room temperature, prior to performance of the haemolytic assay.
Cholesterol-binding assays.
The relative cholesterol-binding affinity of His-PLO and mutant proteins was assayed by a modification of an ELISA technique developed by Shimada et al. (1999) . Microtitre plate wells were coated with 1 µmol of cholesterol, overnight at 37 °C. Following two washes in 0·1 M PBS (pH 7·2) containing 0·3% (v/v) Tween 20 (PBS-T), the wells were blocked for 1 h with 3% (w/v) BSA in PBS at 4 °C. The wells were washed twice with PBS-T and incubated for 1 h at 37 °C with dilutions of toxin in 1 mg BSA ml-1 (1 µg to 1 ng toxin or, where necessary, 10 µg to 10 ng toxin). Following nine washes with PBS-T, bound toxin was reacted with a 1/500 dilution of polyclonal goat anti-PLO serum (Billington et al., 1997
) at 37 °C for 1 h. Primary antibody was detected using a horseradish-peroxidase-conjugated rabbit-anti-goat IgG (H+L) peroxidase (KPL) and o-phenylenediamine as substrate (Sigma).
The cholesterol-binding activity was calculated as the amount of haemolysin (in ng) required to give an ELISA OD420 value 50% that given by 1 µg His-PLO. The binding activity of all His-PLO mutant proteins was expressed as a percentage of His-PLO binding activity, which was designated as 100%. An ELISA was also performed on known amounts of each mutant toxin to confirm that His-PLO and each mutant reacted equally with the anti-PLO serum.
Membrane-binding assays.
These were performed essentially as described by de los Toyos et al. (1996) . His-PLO and mutant toxins were diluted in 1 mg BSA ml-1 to a concentration of 2·5 µg ml-1. Toxin (0·5 ml) was added to an equal volume of 10% SRBC and incubated on ice for 20 min. SRBCs were pelleted by centrifugation at 14000 g for 5 min at 4 °C. Cell pellets were lysed by boiling in sample buffer and were then subjected to SDS-PAGE and Western blot analysis with the anti-PLO serum to detect bound toxin.
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RESULTS |
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The variation in the PLO undecapeptide is required for full cytolytic activity
Since there are other changes in the PLO undecapeptide that may compensate for the proline and arginine changes, a consensus undecapeptide was introduced into His-PLO. This protein was constructed in two forms, one containing a purely consensus CDC undecapeptide, His-PLOCON, which includes the conserved cysteine residue and another, His-PLOCON+A, containing all the consensus residues but retaining the alanine of PLO at position 492 (Fig. 1b). Purified His-PLOCON (Table 3
) and His-PLOCON+A (Table 2
) both had very low haemolytic activities of 0·1%. This result confirmed that the substituted residues in the PLO undecapeptide were required for full cytolytic activity of this protein. Since His-PLOCON contained the conserved cysteine residue responsible for the thiol-activation of many CDCs, haemolytic assays were performed with purified protein pre-treated with either DTT or BME. In the presence of these reducing agents, the haemolytic activity of His-PLOCON was increased, approximately 10-fold over no treatment, to 1·01·1% of His-PLO activity (Table 3
). While this activity is still very low, it represents a significant increase in activity over unreduced protein and indicates that His-PLOCON is subject to oxygen inactivation.
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Cholesterol-binding activities of His-PLO mutant proteins
Cholesterol is the major host-cell-surface receptor for CDCs and the undecapeptide region of these toxins has been implicated either in direct binding of cholesterol or in membrane insertion as a result of cholesterol binding. Each of the His-PLO mutant proteins was compared with His-PLO for its ability to bind to cholesterol in an ELISA-based assay. The ability of all mutant toxins, with the exception of His-PLO.C492, to bind cholesterol was in direct proportion to the haemolytic activity of the toxin (Tables 2 and 3
). For example, His-PLO.
R502 had a haemolytic activity of 27·5% and a cholesterol-binding activity of 26·2% compared to His-PLO, while His-PLO.F497 had 0·2 and 1·6% of His-PLO haemolytic and cholesterol-binding activities, respectively. Reduced His-PLO.C492, however, appeared to bind cholesterol as well as, if not better than, wild-type His-PLO (mean=166·2% His-PLO cholesterol-binding activity), despite having a haemolytic activity only 44·2% that of His-PLO (Table 3
).
Membrane binding of His-PLO mutant proteins
Cholesterol is the proposed major target cell receptor for CDCs, although it is not known if initial interactions occur through other host molecules. To test the ability of the mutant toxins to bind to host-cell membranes, SRBCs incubated with each toxin were analysed by Western blotting using anti-PLO serum (Fig. 2). Despite the variations in haemolytic activity (undetectable to 44·2% of His-PLO) and cholesterol binding (undetectable to 166·2% of His-PLO), all mutant toxins bound to SRBC membranes, and no obvious defects in binding compared to His-PLO could be determined from this assay.
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DISCUSSION |
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Of the changes in the undecapeptide sequence of PLO compared to the CDC consensus, the proline insertion appears to have the greatest effect, since removal of this amino acid resulted in a protein that was non-haemolytic, even at very high toxin concentrations. The importance of P499 may be to provide the correct spacing between important residues, such as the three undecapeptide tryptophan residues. However, the substitution of P499 with either the similarly bulky amino acid phenylalanine or the flexible amino acid glycine did not result in functional proteins, suggesting that spacing alone does not explain the importance of P499. Proline residues are important in the structural integrity of many proteins, as they can force peptide bends. In fact, the general conservation of proline residues within CDC primary sequences suggests that they may be involved in the maintenance of higher-order structures (Billington et al., 1997 ). The undecapeptide proline residue may be required to align the PLO undecapeptide to compensate for structural differences imposed on the PLO undecapeptide by other regions of the PLO molecule, which vary considerably in primary amino acid sequence from the structural backbone of other CDCs. Interestingly, ILY, which is not nearly as divergent in its primary sequence, also contains a proline at the same position in the undecapeptide as PLO (Fig. 1a
; Nagamune, 1997
). However, in ILY the terminal arginine is retained and the third tryptophan residue is missing, which may have implications for its binding to host cells. Indeed, ILY appears to show more specificity in host-cell type than other members of the CDC family (Nagamune, 1997
; Nagamune et al., 1996
).
Despite the differences in the PLO undecapeptide, it shares the three hydrophobic tryptophan residues which have been demonstrated to play a pivotal role in the cytolytic activity of many CDCs (Boulnois et al., 1991 ; Korchev et al., 1998
; Michel et al., 1990
; Nakamura et al., 1998
; Sekino-Suzuki et al., 1996
). However, the spacing between the first and second tryptophan is altered by insertion of P499. Mutations in each of the tryptophan residues resulted in vastly reduced cytolytic activity in LLO, PFO and PLY (Boulnois et al., 1991
; Korchev et al., 1998
; Michel et al., 1990
; Sekino-Suzuki et al., 1996
). Consistent with results from other CDCs, substitution of any of the tryptophan residues of PLO with phenylalanine resulted in a substantial reduction in haemolytic activity compared to the wild-type, suggesting that the undecapeptide plays a similar role in PLO as it does in other members of the family. The role of the undecapeptide sequence in the initial binding, tethering or membrane sampling of CDCs has yet to be proven. However, the position of the first undecapeptide tryptophan residue at the very tip of the undecapeptide loop (Rossjohn et al., 1997
) implies a prominent role in any initial interaction with the target cell membrane precipitated by domain 4. The marked effect of mutations at this position in PLO (Table 2
) and other CDCs (Boulnois et al., 1991
; Sekino-Suzuki et al., 1996
) supports the importance of this residue in CDC cytolytic activity.
Contrary to results with PFO (Sekino-Suzuki et al., 1996 ; Shimada et al., 1999
), mutations in undecapeptide amino acids of His-PLO that resulted in decreased cytolytic activity, with the exception of the alanine to cysteine change in His-PLO.C492, also resulted in a concomitant reduction in cholesterol binding. There are several possible explanations for this observation. The undecapeptide of PLO may be intimately involved in cholesterol binding or mutations in undecapeptide residues may directly affect the cholesterol-binding site of PLO. Alternatively, mutations in the PLO undecapeptide may have a considerable effect on the structure of the PLO molecule, in particular domain 4, which likely contains the cholesterol-binding domain, such that it can no longer bind cholesterol. The variant nature of the PLO undecapeptide may be required not only to properly align the undecapeptide sequence but also to maintain the structure of domain 4 in general. Support for the latter comes from minor substitutions at the C terminus of CDCs (Owen et al., 1994
; Shimada et al., 1999
), which disrupt the ability of these toxins to bind to cholesterol and host-cell membranes. Aligned with this final suggestion is the possibility that mutations introduced in this study result in the misfolding of domain 4, or indeed the entire PLO protein structure, leading to the reduction in haemolytic and cholesterol-binding activities. This is not the case with the majority of mutations in this region of PFO (Sekino-Suzuki et al., 1996
), where reduction in haemolytic activity was observed in the absence of major structural changes. Despite the reduced ability to bind to cholesterol, each of the His-PLO mutant toxins was still able to bind to erythrocyte membranes. These results suggest that CDCs may also bind through additional host-cell molecules (Dubail et al., 2001
; Owen et al., 1994
). This suggestion is supported by experiments with a truncated LLO molecule composed of domains 1, 2 and 3 but lacking the cholesterol-binding domain 4, which indicated that this truncated molecule could still bind to host-cell membranes (Dubail et al., 2001
).
The thiol-activated nature of the original members of the CDC family is the result of oxidation of the cysteine residue at position 2 of the undecapeptide motif (Michel et al., 1990 ; Pinkney et al., 1989
; Saunders et al., 1989
). Generally, mutagenesis of this cysteine residue to alanine resulted in a toxin with a similar cytolytic activity, but which was not inactivated by oxygen (Michel et al., 1990
; Pinkney et al., 1989
; Saunders et al., 1989
). The identification of PLO and ILY confirmed speculation by Pinkney et al. (1989)
as to the presence of members of the CDC family that lack the cysteine residue and thus would be able to stably express their cytolytic activity. The significance of the thiol-activation of CDCs is unknown. It may be a fortuitous event in evolution, but the simplicity of the substitution to an oxygen-resistant form suggests some selection for the oxygen-sensitive form. It is possible that oxygen sensitivity may regulate toxin function following secretion by pathogenic bacteria in the host, although expression of an oxygen-stable form of LLO had no effect on the pathogenesis of Listeria monocytogenes in mice (Michel et al., 1990
). The His-PLO.C492 toxin, while reduced for haemolytic activity, appeared to bind cholesterol better than wild-type His-PLO. Perhaps in a molecule more amenable to its presence, the cysteine aids in the interaction of domain 4 with the primary host-cell receptor. Anecdotally, the His-PLO.C492 protein loses activity much more slowly than His-PLOCON, although both lose approximately 90% of activity to oxygen inactivation. This observation may support the initial assertion that the variant sequence of the PLO undecapeptide affects the stability of PLO to oxygen, even in the presence of the cysteine substitution (Billington et al., 1997
).
We conclude that the variant undecapeptide of PLO is required for full cytolytic activity of this toxin. It seems likely that the altered residues in the PLO undecapeptide may compensate for structural changes in other parts of PLO, with respect to other CDCs, as PLO has the most divergent amino acid sequence (Billington et al., 1997 ). It is also possible that the undecapeptide controls host-cell-membrane sampling. PLO is known to more-readily lyse erythrocytes from some animals species (e.g. rabbit or sheep) than other species (e.g. bovine) (Funk et al., 1996
). This suggestion has implications for the variant undecapeptide of ILY and its limited host range (Nagamune, 1997
; Nagamune et al., 1996
).
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ACKNOWLEDGEMENTS |
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Received 7 June 2002;
revised 9 August 2002;
accepted 13 September 2002.
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