Department of Microbiology, Moyne Institute of Preventive Medicine1, National Pharmaceutical Biotechnology Centre, BioResearch Ireland2 and Bioresources Unit3, Trinity College, Dublin 2, Ireland
Author for correspondence: Peter Owen. Tel: +353 1 6081188. Fax: +353 1 6799294. e-mail: powen{at}tcd.ie
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ABSTRACT |
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Keywords: IgG-binding protein, phagocytosis, autoaggregation, ligand-binding domains, strangles
Abbreviations: Em, erythromycin; Fg, fibrinogen; FgBP, fibrinogen-binding protein; HRP, horseradish peroxidase; i.p., intraperitoneal(ly); Km, kanamycin
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INTRODUCTION |
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The Lancefield group C streptococcus Streptococcus equi subsp. equi is the causative agent of strangles, which is a highly contagious disease of the upper respiratory tract of the family Equidae and one of the most frequently reported equine diseases world-wide. The disease is initially characterized by nasal discharge and fever, followed by abscess formation in local lymph nodes. Morbidity rates of up to 100% have been reported, and in up to 10% of cases mortality can occur as a result of disseminated abscessation (bastard strangles; Timoney, 1993 ).
S. equi has the potential to express at least five wall-associated proteins, viz. two fibronectin-binding proteins (FNE and SFS; Lindmark & Guss, 1999 ; Lindmark et al., 2001
), an
2-macroglobulin/albumin/IgG-binding protein (ZAG; Lindmark et al., 1999
) and two Fg-binding proteins termed FgBP or SeM and SzPse (Meehan et al., 1998
; Timoney et al., 1997
). Of these, FgBP is by far the most dominant wall-associated protein expressed by virulent S. equi (Meehan et al., 1998
). FgBP is a highly immunogenic protein that behaves as a multimer (220000 Da) during SDS-PAGE, reacts with convalescent horse serum, and is protective in a small animal model against lethal S. equi challenge. The sequence of the corresponding gene (fbp) has been determined and shown to encode a protein of 534 amino acids (58344 Da), which possesses some structural and sequence similarities to other streptococcal cell wall proteins. Based on experimentation and computer predictions, these have been shown to include a 36-residue signal sequence, a cell wall/membrane anchoring domain and two blocks (A and B) of degenerate repeated sequences (Meehan et al., 1998
, 2000a
). In addition, computer-assisted secondary structure analysis predicts that FgBP, like M proteins, possesses a high proportion (over 60%) of
-helical coiled-coil (dimer) structure (Meehan et al., 1998
). However, the protein shows little significant sequence similarity to other M(-like) proteins, except for the Fg/IgG-binding DemA protein from Streptococcus dysgalactiae, where some similarities in the A-repeat and C-terminal regions have been noted (Vasi et al., 2000
).
The region within FgBP required for maximum binding of Fg has been located, using a panel of 20 recombinant hexahistidyl (His6) -tagged FgBP truncates possessing overlapping N- and C-terminal deletions of sequence (FgBP117) and specific internal deletions of the A repeat and/or B repeats (FgBP[A-], FgBP[B-] and FgBP[A-B-]). This has been shown to extend over a large aspect (the N-terminal half) of the mature protein. Maximum ligand binding is not dependent on the presence of the A and/or B repeats, but is critically dependent on residues within the first 19 N-terminal amino acids and also on an extended region of (stabilizing) coiled-coil structure (Meehan et al., 1998 , 2000a
, b
). This contrasts with the situation for M1 and M5 proteins of group A streptococci where the centrally located B repeats appear to be critical for Fg binding (Ringdahl et al., 2000
).
Previous studies have shown that S. equi cells are resistant to non-immune phagocytosis, and there is some indirect evidence that FgBP may be involved (Boschwitz & Timoney, 1994a , b
; Chanter et al., 1994
). Thus, Timoney and co-workers showed that an isolate of S. equi expressing high levels of M protein (very likely FgBP) bound four times more complement C3b and survived 100-fold better than an unrelated isolate which apparently expressed lower levels of the antigen. In addition, specific antiserum inhibited survival of S. equi in whole blood, whereas the presence of fibrinogen enhanced the ability of S. equi to resist killing by equine neutrophils (Boschwitz & Timoney, 1994a
, b
; Chanter et al., 1994
).
In this paper we show for the first time that FgBP binds IgG as well as Fg. In addition, we describe the construction of an fbp insertion mutant of S. equi subsp. equi and demonstrate convincingly that FgBP plays a role in resistance to phagocytosis, and in virulence.
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METHODS |
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Construction of S. equi fbp mutants.
An insertion mutation in the fbp gene was constructed by replacement of the central 407 bp of the fbp sequence with the Km2 interposon (Fig. 1b
; Perez-Casal et al., 1991
; Prentki & Krisch, 1984
), and involved using the broad-host-range thermosensitive plasmid pG+host9 to mutagenize the wild-type chromosomal copy of fbp via a double-crossover integration event. pG+host9 cannot replicate at temperatures above 35 °C (Maguin et al., 1996
). Primers F1 and R1 were used to amplify a DNA fragment corresponding to the first 460 bp of fbp (encoding amino acids 1153 of unprocessed FgBP) together with 323 bp of upstream sequence; primers F2 and R2 were used to amplify a DNA fragment corresponding to the final 739 bp of fbp (encoding amino acids 290534 of unprocessed FgBP) plus 62 bp of downstream sequence. One hundred microlitre PCR reactions were performed in pfu polymerase buffer (Promega) containing 3 U pfu polymerase, 250 ng of forward and reverse primers, 250 µM dNTPs and 500 ng S. equi genomic DNA. Amplification conditions consisted of 30 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 2·5 min, followed by a final extension at 72 °C for 10 min. Gel-purified PCR fragments were cleaved as appropriate with EcoRI, HindIII and ApaI. The cleaved products were then ligated and cloned into pBluescript cleaved with EcoRI and ApaI, to generate the plasmid pFBP1. pFBP1 was then cleaved with HindIII, filled in with DNA polymerase I large (Klenow) fragment, and ligated with the purified
Km2 obtained from SmaI-digested pBR322
Km2. The resultant plasmid (pFBP2) was cut with EcoRI and ApaI, and the 3784 bp fbp::
Km2 DNA fragment (Fig. 1b
) was gel-purified and then ligated to plasmid pG+host9 (Emr) digested with EcoRI and ApaI. The resultant plasmid (pFBP3) was purified, electroporated into S. equi TW, and Kmr Emr transformants were grown at 30 °C in order to select derivatives carrying the replicating plasmid. A double-crossover event between homologous sequences on pFBP3 and the S. equi chromosome was facilitated by a temperature shift to 39 °C, resulting in the loss of pG+host9, integration of
Km2 into chromosomal fbp and a Kmr Ems phenotype. For this procedure, an overnight culture of S. equi TW(pFBP3) grown at 30 °C was diluted 1:100 into fresh THYE broth, grown for 24 h at 39 °C (subculturing once after 7 h), and finally plated onto THYE agar containing Km. Out of 150 Kmr transformants of S. equi, six were found to be Kmr Ems. PCR experiments using several primer sets covering different regions of fbp and Southern hybridization of restriction enzyme digested genomic DNA using probes specific for fbp, pG+host9 and the
Km2 element confirmed that these six transformants did not possess pG+host9 and were the result of integration of the
Km2 element into the fbp gene (data not shown). One of these S. equi fbp::
Km2 mutants was selected for further study.
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To construct a complemented derivative of S. equi fbp::Km2, a DNA fragment encompassing the entire fbp gene together with 323 bp of upstream and 136 bp of downstream sequence was amplified using primers F1 and R3, and S. equi genomic DNA as a template. Amplification conditions consisted of 30 cycles of denaturation at 95 °C for 1 min, annealing at 60 °C for 30 s and extension at 72 °C for 4 min, followed by a final extension at 72 °C for 5 min. The gel-purified product was cleaved with EcoRI and BamHI and cloned into pVA838 cleaved with EcoRI and BamHI, to generate pFBP4. pFBP4 was transformed into S. equi fbp::
Km2 by electroporation and complemented derivatives possessing a Kmr Emr phenotype were selected.
Phagocytosis assay.
Actively growing cultures of S. equi derivatives (OD600 0·14) were diluted in THYE broth to approximately 10003000 c.f.u. ml-1. Aliquots (100 µl) were added to 800 µl of either heparinized horse blood or plasma (from horses with no history of strangles) and the suspensions were incubated at 37 °C for 3 h, with end-over-end rotation. One hundred microlitre aliquots of the resultant suspensions were then plated onto blood agar and incubated at 37 °C to determine viable counts, as described by Lancefield (1962)
.
Mouse challenge experiments.
All animal experiments were performed in compliance with EC directive 86/609/EC as implemented in Ireland under Statutory Instrument 17/94. Groups of Laca mice (12 weeks old) were challenged by intraperitoneal (i.p.) injection of 200 µl vols PBS containing either S. equi TW or S. equi fbp::Km2. Mice were monitored for 30 d post challenge and, for humane reasons, were killed if considered terminally ill with body temperatures below 32 °C. Survival times among the challenge groups were analysed statistically using the MannWhitney U-test.
Isolation of bacterial cell envelopes and purification of FgBP and recombinant FgBP truncates.
Bacterial cell envelopes were isolated from stationary-phase S. equi cells, using a method based on French pressure cell lysis, as described previously (Meehan et al., 1998 ). Native (wild-type) FgBP was purified from mutanolysin extracts of S. equi envelopes using horse Fg affinity chromatography, as previously described by Meehan et al. (1998)
. The construction of recombinant plasmids expressing different affinity-tagged FgBP truncates (FgBP117, FgBP[A-], FgBP[B-] and FgBP[A-B-]) has been described in detail elsewhere. Briefly, this involved ligation of appropriate PCR-amplified fbp fragments into the pQE30 plasmid vector. His6-tagged FgBP derivatives were then purified from the soluble fractions of transformed E. coli XL-1 Blue lysates by metal-chelate affinity chromatography in the presence of protease inhibitors (Meehan et al., 1998
, 2000a
, b
; see Fig. 1a
).
Biochemical procedures.
SDS-PAGE was performed using both 7·5% and 12·5% (w/v) polyacrylamide separating gels and a 4·5% (w/v) polyacrylamide stacking gel, as described by Laemmli (1970) . Samples were routinely heated for 3 min at 100 °C in sample buffer (Laemmli, 1970
) prior to electrophoresis. Proteins were detected by staining with Coomassie brilliant blue. Molecular masses were determined from the relative mobilities of 15 standard molecular mass marker proteins (BenchMark protein ladder; Gibco-BRL). Protein concentrations of purified FgBP truncates and of cell envelope preparations of S. equi strains were estimated by a modification (Dulley & Grieve, 1975
) of the Lowry method, using BSA as a standard. The protein concentrations of IgG solutions were based on details provided by the suppliers.
Immunochemical and affinity procedures.
Test tube precipitation experiments were performed using a modification of the immunoprecipitation technique described by Doherty et al. (1986) . Purified IgGs (100 µg), in 50 mM sodium phosphate (pH 7·4) containing 0·1 M NaCl, were preincubated for 1 h at 25 °C with Complete protease inhibitor cocktail (Roche) and then centrifuged (13000 g, 15 min, 20 °C). Purified FgBP truncate (20 µg) and Triton X-100 (final concn 2%, v/v) were added to the cleared supernatant, and the solutions were mixed and incubated for 4 h at 25 °C. The resultant affinity precipitates were harvested by centrifugation (13000 g, 15 min, 20 °C), washed twice in 200 µl 0·1 M NaCl containing 0·5% (v/v) Triton X-100, and finally resuspended in Laemmli sample buffer with and without 2-mercaptoethanol (Laemmli, 1970
). Affinity diffusion assays were conducted as described previously (Meehan et al., 1998
).
Western immunoblotting was done as described by Caffrey et al. (1988) using the following reagents: 5% (w/v) dried skimmed milk as blocking reagent; mouse anti-recombinant FgBP1 (1:30000 dilution) as primary antibody; and horse radish peroxidase (HRP)-labelled affinity-purified goat anti-mouse IgG as localizing antibody. Fg-affinity blotting using HRP-labelled horse Fg in 2% (w/v) dried skimmed milk was performed as described previously (Meehan et al., 1998
, 2000a
). For IgG-affinity blotting, nitrocellulose blots were blocked with a solution of 2·5% (w/v) dried skimmed milk followed by incubation, as appropriate, with unlabelled horse IgG-Fab, unlabelled or HRP-labelled horse IgG-Fc, HRP-labelled Fc of human or rabbit origin, or HRP labelled Fab fragments of either human or rabbit origin (210 µg ml-1). Where appropriate HRP-labelled affinity-purified goat anti-horse IgG (H+L) was used as localizing antibody (Meehan et al., 1998
). For FgBP1-affinity dot blot experiments, IgGs were probed with HRP-labelled FgBP1 (10 µg ml-1). In whole-cell dot blots, S. equi suspensions (OD600 0·4) were subjected to doubling dilutions and 150 µl aliquots were transferred onto nitrocellulose using the Bio-Dot apparatus (Bio-Rad). Dried blots were then blocked and developed as described above for Western immunoblotting or equine Fg/IgG affinity electroblots. All peroxidase labelled probes were detected using 4-chloro-1-naphthol as developing reagent (Meehan et al., 1998
). For immunofluorescence microscopy (Henderson et al., 1997
), glutaraldehyde-fixed bacterial cells were probed with mouse anti-FgBP1 antiserum (1:400 dilution) followed by FITC-conjugated goat anti-mouse immunoglobulins. All experimentation involving Western and dot immunoblotting, immunoprecipitation and immunofluorescence microscopy was repeated two to four times to ensure reproducibility, and representative data are shown in Figs 25
.
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Immunochemical and affinity reagents.
Bovine IgG was from Chemicon. All other purified IgGs, together with horse Fg and HRP- and FITC-conjugated affinity-purified goat anti-mouse immunoglobulins (Fab-specific), were from Sigma. Goat anti-horse IgG (H+L) was obtained from ICN. Unlabelled horse IgG-Fc/Fab and HRP-labelled human and rabbit IgG-Fc/Fab fragments were obtained from Jackson ImmunoResearch Laboratories. Horse Fg, horse IgG-Fc and FgBP1 were labelled with HRP, as previously described (Meehan et al., 1998 ). Mouse anti-FgBP1 was generated by immunizing mice subcutaneously on days 0 and 28 with 50 µg purified FgBP1 emulsified in 200 µl MPL+S-TDCM Ribi adjuvant (active ingredients monophosphoryl lipid A and trehalose dimycolate; RIBI Immunochem Research). On day 35, mice were exsanguinated and serum was obtained.
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RESULTS |
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Most other IgG-binding proteins, such as protein A from Staphylococcus aureus, protein H from Streptococcus pyogenes, and protein G from group C and G streptococci, are known to bind to the Fc region of IgG (Frick et al., 1992 ). To ascertain whether FgBP binds equine IgG by a similar non-immune mechanism, Western blots were performed using FgBP1 probed with either purified Fab or Fc fragments of horse/human/rabbit IgG. The results showed that FgBP1 bound strongly to Fc fragments of equine, human and rabbit origin. In contrast, FgBP1 showed only faint reactions with equine Fab fragments and showed no detectable binding to either human or rabbit Fab fragments (Fig. 2c
). These results were confirmed using affinity dot blots and ELISA tests (data not shown). Whether the faint reaction observed between equine IgG-Fab and FgBP1 is a consequence of immune or non-immune binding remains to be determined.
Localization of the IgG-binding domain
To localize the equine IgG-Fc binding domain within FgBP, use was made of a panel of 20 recombinant FgBP proteins containing defined N-terminal, C-terminal and internal deletions of sequence (Meehan et al., 2000a , b
; Fig. 1a
). Initial Western IgG-affinity blotting experiments showed that FgBP3 and FgBP4 bound equine IgG-Fc weakly or not at all and that the native protein and remaining truncates (Fig. 1a
) all bound detectable levels of equine IgG-Fc (data not shown). In order to place these observations on a more quantitative basis and to directly relate IgG-Fc binding with comparable Fg-affinity experiments (Meehan et al., 2000a
, b
), IgG-affinity dot blots were performed using undenatured FgBPs (Fig. 3
). These semi-quantitative experiments confirmed the general trend observed during Western affinity blotting and revealed the following in the absence of SDS: (a) wild-type FgBP, FgBP1 (which lacks the wall/membrane anchor domain), FgBP2 (missing 113 amino acids from its C-terminus), FgBP1213 (missing 19 and 34 residues, respectively, from the N-terminus) and FgBP[A-], FgBP[B-] and FgBP[A-B-] all bound similar levels of equine IgG-Fc to a first approximation; (b) FgBP3 (missing 182 amino acids from its C-terminus) bound 32-fold less IgG-Fc than FgBP1; (c) FgBP4 (missing 255 residues from the C-terminus) bound no detectable IgG-Fc; (d) N-terminal truncates FgBP14, 15 and 16 (which have 70, 102 and 148 residues, respectively, deleted from their N-termini) bound approximately fourfold less IgG-Fc than their FgBP2 control; and (e) FgBP17 (missing 182 N-terminal residues) bound approximately 32-fold less IgG-Fc than its FgBP2 control. Very similar results were obtained when blots were probed with human IgG-Fc; the major difference being that FgBP3 exhibited wild-type binding to human IgG-Fc (data not shown). These data suggest that a large central region of FgBP encompassing residues 185421 and including the A and B repeats is important for IgG-Fc binding, but that neither the A nor B repeats are essential for that binding.
Construction and characterization of S. equi fbp::Km2
Insertional inactivation of the fbp gene was accomplished by replacement of the central 406 bp with the Km2 element (Perez-Casal et al., 1991
; Prentki & Krisch, 1984
; Methods section and Fig. 1b
). The
-interposon is stably maintained in the chromosome of S. equi in the absence of selective pressure and contains strong terminators of transcription and translation. Theoretically, this construct could secrete a 12·9 kDa FgBP truncate lacking the C-terminal three-quarters of the native protein. However, several lines of evidence confirm that S. equi fbp::
Km2 no longer expresses any FgBP and that, under laboratory conditions, FgBP is likely to be the major Fg-binding protein expressed by wild-type S. equi. Firstly, SDS-PAGE and Western immuno/affinity blot analyses of mutanolysin-extracted bacterial cell envelopes showed that wild-type S. equi, but not S. equi fbp::
Km2, possessed the mature wall-associated 220 kDa FgBP which reacted with specific antiserum to FgBP and bound Fg. Secondly, no cross-reacting FgBP polypeptides or any Fg-binding protein could be detected in the envelopes of the fbp mutant strain (Fig. 4
) or in the soluble cytoplasmic extracts and culture supernatants of either the wild-type or an isogenic mutant (data not shown). Finally and importantly, immunofluorescence microscopy and whole-cell dot blot analysis performed with specific anti-FgBP1 serum, horse Fg or horse IgG-Fc revealed that S. equi fbp::
Km2 cells, in contrast to those of the wild-type, no longer express FgBP on their cell surface and do not bind detectable levels of either Fg or IgG-Fc (Fig. 5a
, b
). It should also be noted that both S. equi and its fbp::
Km2 derivative were encapsulated and grew at the same rate in broth culture (data not shown). However, a characteristic feature of the fbp mutant was its failure to autoaggregate in liquid media during stationary phase in a manner observed for wild-type cells (Fig. 5c
). This phenomenon was also evident but to a less dramatic extent in exponential phase (compare Fig. 5a
left and right panels).
Analogous experimentation conducted with the complemented derivative S. equi fbp::Km2(pFBP4) revealed that it expressed mature functional FgBP as anticipated, but at considerably lower levels than the wild-type strain (Fig. 4
; lanes 3, 6 and 9). This was confirmed by immunofluorescence microscopy, whole-cell dot blots and settling experiments (data not shown). The reason for the low expression is unclear, but was a consistent feature in attempts to create such constructs. pVA838 does not appear to affect parental levels of FgBP, since S. equi TW(pVA838) expressed wild-type levels of the protein as determined by immunofluorescence microscopy and whole-cell dot blot analysis (data not shown). Furthermore, the complemented mutant harboured replicating recombinant pVA838 with full length fbp as evidenced by: (a) Southern blot analysis of restriction enzyme digested-genomic DNA of S. equi fbp::
Km2(pFBP4) using pVA838- and fbp-specific probes; (b) PCR analysis of genomic DNA of S. equi fbp::
Km2(pFBP4); (c) restriction enzyme digestion analysis of pFBP4 purified from S. equi fbp::
Km2(pFBP4); and (d) retention of appropriate antibiotic resistance markers. Moreover, there was no evidence of any recombination between recombinant pVA838 and the chromosomal copy of fbp (data not shown). Others in the field have also experienced the problem of poor expression of surface proteins from complemented streptococcal derivatives (Kihlberg et al., 1999
).
FgBP is antiphagocytic
To assess whether FgBP plays a role in resistance of S. equi to phagocytosis, the ability of wild-type S. equi TW to survive and grow in non-immune horse blood was compared with that of its isogenic fbp::Km2 derivative (Table 2
). The experiment was conducted using blood from two different donor horses that had no history of strangles. As expected both strains grew well in horse plasma. In contrast, only wild-type S. equi survived and grew (equally well) in whole blood. The fbp::
Km2 derivative lacking FgBP was killed. As anticipated (Boschwitz & Timoney, 1994b
; Timoney et al., 1997
), the survival of wild-type S. equi was reduced substantially (9- to 45-fold) following the addition of (opsonizing) rabbit anti-FgBP antibodies (data not shown). From this it can be concluded that FgBP plays an important role in resistance of S. equi to killing by phagocytes (and/or other blood factors) in the absence of specific antibody.
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DISCUSSION |
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The binding of Fg and IgG to FgBP clearly requires distinct structural features. Binding of Fg is critically dependent on the N-terminal 19 aa and additionally requires a significant stretch of (stabilizing) downstream coiled-coil structure (Meehan et al., 2000a , b
). In contrast, IgG-Fc binding does not appear to require residues at the immediate N-terminus, but instead is dependent on a sizeable central domain of FgBP which encompasses both the A and B repeat regions. However, in common with the situation for Fg, it is evident that neither the A nor B repeats regions are essential for IgG-Fc binding. In other IgG-binding proteins, the actual location of and residues involved in IgG binding varies. Thus, IgG binding is located closer to the N-terminus in the case of protein H and to the C-terminus for protein G. Furthermore, the IgG-binding domains in protein A and protein G are located in repeat regions, whereas protein H and M1 protein appear to have two separate binding sites for IgG which are not present in repeat domains and which appear to bind to different human IgG subsets (
kerström et al., 1987
; Frick et al., 1994
; Navarre & Schneewind, 1999
; Raeder et al., 1998
; Uhlén et al., 1984
). Searches of databases for homologies to the amino acid sequence of the putative IgG-binding domain of FgBP reveals significant similarities only with (a) a new protective antigen (Spa) of group A streptococci which shows a curious and remarkable (98%) homology with S. equi FgBP over the C-terminal half of the molecule (Dale et al., 1999
; GenBank accession no. AF0876813), and (b) the central regions of the Fg/IgG-binding protein DemA from S. dysgalactiae. Perhaps significantly, efficient binding of bovine IgG to DemA appears to require this region of the protein (Vasi et al., 2000
).
Ligand-binding studies performed with S. equi and its fbp insertion mutant provide convincing evidence that FgBP is the dominant surface antigen responsible for the binding of both Fg and IgG by wild-type cells. This confirms and extends previous studies showing that FgBP in whole cells is accessible to both specific antiserum and Fg and that it is the major Fg-binding protein in mutanolysin-extracted bacterial cell envelopes (Meehan et al., 1998 ; Timoney et al., 1997
). S. equi has the potential to express other surface proteins capable of binding Fg (viz. SzPse; Timoney et al., 1997
) or IgG (ZAG; Jonsson et al., 1995
; Lindmark et al., 1999
). However, these are clearly relatively minor ligand-binding species under the conditions of laboratory growth, since S. equi fbp::
Km2 binds no detectable Fg and IgG-Fc.
Previous studies have provided evidence that the M protein of S. equi is antiphagocytic (see Introduction). Here, through use of an isogenic fbp derivative, we show conclusively and for the first time that FgBP contributes in a major way to the survival of S. equi in whole horse blood. The precise mechanism by which this occurs remains to be elucidated. There is evidence that reduction in C3b deposition and Fg binding contribute to the ability of S. equi to resist killing by equine neutrophils (Boschwitz & Timoney, 1994a , b
; Chanter et al., 1994
), although in our own hands the levels of enhanced survival in the presence of Fg are marginal (data not shown). The ability of FgBP to bind IgG-Fc suggests another possible mechanism for resistance to phagocytosis. Certainly, for the clinically important (M1) serotype of group A streptococcus it has been convincingly established that the IgG-binding protein H can block C3 deposition due to inhibition of the classical complement pathway, and that the presence of either protein H or the Fg-binding M1 protein on the cell surface is sufficient for survival in human blood (Berge et al., 1997
; Kihlberg et al., 1999
). In other cases, reduction in C3b deposition has been attributed to the ability of M proteins to bind complement regulators (see Introduction).
Some streptococcal M proteins can also mediate bacterial autoaggregation, and this property has been shown to be crucial for adherence and for resistance to phagocytosis (Frick et al., 2000 ). Such a mechanism cannot be ruled out for virulent S. equi. Comparative microscopic and sedimentation analysis shows that wild-type S. equi routinely grows as large sedimentable aggregates, whereas the fbp::
Km2 knockout mutant does not (see Fig. 5
; data not shown). FgBP does not possess the conserved 19 amino acid residue (AHP) sequence which has been implicated in the homophilic proteinprotein interactions responsible for formation of S. pyogenes aggregates (Frick et al., 2000
); however, it can form apparent multimers under certain conditions and the B-repeats of FgBP have been heavily implicated in this process (Meehan et al., 2000b
).
It is relevant to note that other streptococcal surface components, such as the hyaluronic acid capsule, have also been implicated in resistance to phagocytosis. In the case of S. equi, strains expressing lower amounts of capsule seem to be more susceptible to phagocytosis and to cause a less invasive disease than heavily capsulated strains (Anzai et al., 1999b ). The virulent strain of S. equi TW used in the current studies does express capsule under conditions of laboratory growth; however, it is clear from the experimental data that the level of capsule produced is insufficient to prevent efficient phagocytic killing in the absence of both FgBP and specific antibody.
The M-like protein (FgBP) of S. equi has been recognized for a number of years as a likely virulence determinant. It is structurally and functionally similar to the well-studied M protein virulence factors of group A streptococci, possesses anti-phagocytic properties (see above), and elicits strong serum and mucosal antibody responses in infected horses. However, direct evidence that FgBP contributes to virulence has been lacking until now. The results of the small animal experiments conducted with S. equi and S. equi fbp::Km2 provide compelling evidence that this is indeed the case. Supporting evidence that FgBP is important in S. equi infection of the target species comes from a recent study of horses that were outwardly healthy but were shown to be persistent carriers of S. equi. Streptococcal isolates from about 25% of these carriers expressed truncated forms of FgBP (lacking the N-terminal Fg-binding region) and were more sensitive to phagocytosis (Chanter et al., 2000
). Clearly, the balance of evidence presented here and elsewhere strongly suggests that the antiphagocytic properties of FgBP contribute to the virulence of S. equi. This is not to exclude other possible roles. Certainly, there is a multitude of studies suggesting that Fg- and/or IgG-binding M proteins of group A streptococci are involved in adhesion, invasion and colonization (Cleary & Retnoningrum, 1994
; Dombek et al., 1999
; Molinari & Chhatwal, 1999
; Navarre & Schneewind, 1999
). Furthermore, wall-anchored M protein has recently been implicated in the maturation of cysteine proteinase (Collin & Olsén, 2000
). In turn, cysteine proteinase has been shown to release, from the cell surface, biologically active fragments of M protein and protein HIgG complexes (Berge & Björck, 1995
; Berge et al., 1997
). It has been suggested that the deposition of the protein HIgG complexes onto body organs may cause localized inflammation and tissue damage, and thus contribute to severe complications of suppurative S. pyogenes infection. Interestingly, purpura haemorrhagica, a fatal sequel to strangles, has been proposed to be an immune-complex-mediated disease (Galán & Timoney, 1985
). Clearly the role of FgBP and its IgG-binding properties in complications associated with this disease appear to warrant further investigation. Challenge trials in horses using S. equi fbp::
Km2 and similar derivatives should also provide a further insight into the role of FgBP and other potential S. equi virulence factors in the disease process (Anzai et al., 1999a
, b
; Chanter et al., 1999
; Flanagan et al., 1998
; Harrington et al., 2000
; Muhktar & Timoney, 1988
).
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ACKNOWLEDGEMENTS |
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Received 30 May 2001;
revised 1 August 2001;
accepted 9 August 2001.