Department of Medical Biochemistry, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland1
Author for correspondence: Jukka Hytönen. Tel: +358 2 333 7445. Fax: +358 2 333 7229. e-mail: jukka.hytonen{at}utu.fi
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ABSTRACT |
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Keywords: Streptococcus pyogenes, bacterial adhesion, transposon mutagenesis, glycoproteins
Abbreviations: ECL, enhanced chemiluminescence; HRP, horse radish peroxidase; THY, ToddHewitt medium containing 0·5% yeast extract
a Present address: Kennedy Institute of Rheumatology, Hammersmith, London W6 8LH, UK.
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INTRODUCTION |
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The adherence of bacteria to host cells is in many cases mediated by lectin-like adhesins on the bacterial surface binding to carbohydrate receptors (Beachey, 1981 ; Ofek & Sharon, 1990
), which are present on the host cell surface as part of the membrane glycoproteins and glycolipids. Escherichia coli is known to bind at least to eight different carbohydrate structures via its surface proteins (Karlsson, 1995
; Sharon, 1996
; Sharon & Lis, 1997
). Some of the carbohydrate structures have been used to inhibit the binding of Esc. coli to the host intestinal epithelia (Mouricout et al., 1990
). Likewise, streptococcal infections of the human pharyngeal epithelia, where local administration of the drug to the infection focus would be easy to carry out, would be a suitable target for anti-adhesive therapy. Unfortunately, very little is known about the streptococcal receptors on host cells.
A number of S. pyogenes surface proteins interacting with the human host have been identified. Some of these, like the fibronectin-binding proteins (Talay et al., 1991 , 1992
; Courtney et al., 1992
, 1994
; Hanski & Caparon, 1992
; Kreikemeyer et al., 1995
; Rakonjac et al., 1995
; Jaffe et al., 1996
; Rocha & Fischetti, 1999
), are adhesins, while others, for example M-like proteins (Cleary & Retnoningrum, 1994
) and C5a peptidase (Chen & Cleary, 1990
), are protective against the host immunodefence system. M-protein has a dual role, being the major antiphagocytic factor of S. pyogenes and also an adhesin that binds fibronectin, fibrinogen and albumin, among others (Fischetti et al., 1988
; Schmidt et al., 1993
; Gubbe et al., 1997
). The ability of S. pyogenes to infect and to persist in varied locations of the human host is in part controlled by the multiple-gene regulator of S. pyogenes, Mga, which controls the expression of the M-protein (emm), M-like proteins (fcrA, enn, sph) and C5a peptidase (scpA) genes (Podbielski et al., 1996a
). These genes are located in one regulon where the transcription of the genes downstream of the mga gene is monocistronically regulated. The genes encoding the opacity factor (sof), secreted inhibitor of the complement cascade (sic) and streptococcal cysteine protease (speB) are also regulated by Mga (Podbielski et al., 1996a
).
The purpose of the present study was to characterize glycoprotein-binding specificities of streptococci to understand the interaction between the bacteria and the potential carbohydrate receptor structures of the host cell surface. A glycoprotein-binding adhesion activity was shown to be present in the majority of the S. pyogenes strains studied. Using transposon mutagenesis, the novel glycoprotein-binding activity was shown to be regulated by Mga.
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METHODS |
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Streptococcal strains and Enterococcus faecalis were grown on ToddHewitt (Difco) plates or media supplemented with 0·5% yeast extract (THY; Biokar Diagnostics). Esc. coli was grown in Luria broth. All cultures were incubated at 37 °C in ambient air unless otherwise stated. All bacteria were stored at -70 °C in growth medium containing 15% (v/v) glycerol. The M2 71-676 and CS101 pSF152 mutants were grown in the presence of 60 µg spectinomycin ml-1. Recombinant Esc. coli JM109 was grown with 60 µg carbenicillin ml-1.
Materials.
Bovine and porcine thyroglobulin, bovine fetuin and asialofetuin, bovine submaxillar mucin, horse myoglobin, bovine erythrocyte carbonic anhydrase, BSA, trypsin, chicken-egg-white trypsin inhibitor, streptavidinHRP (horse radish peroxidase), diaminobenzidine, lysozyme, spectinomycin, streptomycin, tetracycline and carbenicillin were purchased from Sigma. Human thyroglobulin was from Biogenesis and human fibronectin from Chemicon. NHS-LC biotin was obtained from Pierce and the biotinylated derivatives were prepared according to the instructions of the manufacturer. Nitrocellulose membrane (0·45 µm) was from Schleicher & Schuell. Tween 20 and PEG (20000) were from Fluka. ECL Western blotting kit, Hybond-N+ membrane, Sequenase 2.0 sequencing kit, [-32P]ATP, [
-32P]dCTP and [
-35S]dATP were obtained from Amersham. Sephadex G-25 and Sephadex G-50 were purchased from Pharmacia Biotech. Tran35S-label was from ICN Biomedicals and Microbeta plates from Wallac. Hyperfilm ECL (Amersham) was used in ECL autoradiography. Sequencing gels were visualized on Fuji RX medical X-ray film. All molecular biology reagents were obtained from Promega.
Glycoprotein-binding assay.
Bacteria grown overnight on THY plates were collected and washed twice by centrifugation in PBS and adjusted to a concentration that gave an OD600 of 0·5 with a Beckman DU 640 spectrophotometer and 1 cm path length cuvettes. Aliquots of 1 µl were pipetted onto gridded nitrocellulose membrane and nonspecific binding sites were saturated with 3% BSA, 0·1% Tween 20 in PBS at room temperature for 1 h. The biotinylated ligand was added to the membrane in 0·1% Tween 20 in PBS to give a final concentration of 0·31·0 µg ml-1 and incubated at room temperature for 30 min. The membrane was washed three times with PBS, and 0·1 µg ml-1 streptavidin-HRP was added to the membrane in 0·1% Tween 20 in PBS and incubated at room temperature for 30 min. The membrane was washed as above and the binding of the ligand was detected by ECL autoradiography. In inhibition experiments, the membrane containing a dilution series of the bacteria (corresponding to OD600 4·00·125) was preincubated with excess ligand at 1 mg ml-1 in 0·1% Tween 20 in PBS for 30 min, the biotinylated ligand was added directly to the preincubation solution and the incubation was continued for 30 min. The binding of the ligand was detected as above. The results shown in Tables 1, 2
and 3
are given as the reciprocal of the binding titre, defined as the last dilution showing clearly visible binding.
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Binding of radiolabelled streptococci to thyroglobulin on microtitre plates.
Bacteria were grown in 15 ml THY broth containing 0·5 mCi (18·5 MBq) Tran35S-label at 37 °C overnight. The bacteria were concentrated by centrifugation (3000 g for 10 min at 4 °C) to 1 ml PBS and the unbound label was removed by centrifuging the bacteria as above through 5 ml 6% BSA in PBS. Microtitre plate wells were coated with 100 µl of the ligand at 10 µg ml-1 at 37 °C for 2 h. The wells were rinsed with PBS and saturated with 2% BSA, 0·1% Tween 20 in PBS for 1 h at room temperature. The wells were overlaid with 100 µl bacteria in 0·1% BSA, 0·05% Tween 20 in PBS at a concentration that gave an OD600 of 0·3 (~4x108 c.f.u. ml-1), incubated for 1·5 h at 37 °C with gentle agitation and washed three times for 10 min with 200 µl PBS per well. In inhibition experiments, the bacteria were preincubated with excess ligand at 1 mg ml-1 in 0·1% BSA, 0·05% Tween 20 in PBS for 30 min and then added to the wells. Binding of the bacteria to wells coated with 2% BSA was used as control. Experiments were carried out in 12 parallel wells. The bound bacteria were detected using a liquid-scintillation counter.
Conventional DNA techniques.
Plasmid DNA preparation from Esc. coli, restriction enzyme digestions, ligations, transformations, agarose gel electrophoresis and Southern blotting were performed as described by Sambrook et al. (1989) . Chromosomal S. pyogenes DNA was prepared as described by Caparon & Scott (1991)
.
Transposon mutagenesis and analysis of the mutants.
Transposon mutagenesis was performed as described by Caparon & Scott (1991) . Briefly, a spontaneous streptomycin-resistant S. pyogenes strain (A8173sr) was grown overnight in 100 ml THY broth containing 1 mg streptomycin ml-1 at 37 °C. Donor strain Ent. faecalis CG110 (provided by D. Clewell, University of Michigan, Ann Arbor, USA) was grown similarly in 10 ml THY broth containing 10 µg tetracycline ml-1. Bacteria were harvested and A8173sr was resuspended in 1 ml THY. Ent. faecalis was resuspended in 10 ml THY. One millilitre of both suspensions were mixed together, plated on THY plates (no antibiotics) and grown overnight at 37 °C. Bacteria were collected from the overnight culture, plated on THY plates containing 1 mg streptomycin ml-1 and 5 µg tetracycline ml-1 and grown for 48 h at 37 °C. Colonies were blotted onto nitrocellulose membranes and were analysed by the glycoprotein-binding assay using diaminobenzidine as substrate. The number of transposons integrated into the genomes of the non-binding mutants was determined by Southern hybridization of HindIII-digested genomic DNAs using [
-32P]dCTP-labelled plasmid pAM120 (which carries the whole Tn916 transposon; provided by D. Clewell) as the probe.
Cloning of the Tn916-chromosomal DNA junction fragment.
Chromosomal DNA from Streptococcus pyogenes mutated with Tn916 was cleaved with HindIII and the fragments were separated by agarose gel electrophoresis. A 14 kb fragment containing the 5' end of Tn916 was extracted from the gel, cleaved with HincII and ligated to pBS13+ (Promega) The ligation mixture was transformed into Esc. coli JM109, the bacteria were grown overnight and the colonies transferred to a nylon membrane. The membrane was probed with [-32P]ATP labelled oligonucleotide Tn1 (5'-GAGTGGTTTTGACCTTGATA-3') which binds to the 5' end of Tn916 at position 5372. Plasmid DNA from hybridizing colonies was prepared and the DNA sequenced.
DNA sequencing.
DNA was sequenced with a Sequenase 2.0 kit according to the instructions of the manufacturer. Primer Tn1 was used in sequencing reactions. The products were analysed by gel electrophoresis and visualized by autoradiography. The sequence data were analysed with DNAstar software.
Generation of an mga-complemented strain.
Plasmid pJRS2050 (Andersson et al., 1996 ; carries mga and associated regulatory sequences in pLZ12-Spec; provided by J. Scott, Emory University Health Sciences Center, Atlanta, USA) was electroporated into A8173-1 to generate A8173-1(pJRS2050). The electroporation method described by Simon & Ferretti (1991)
was used with one modification: THY medium was used instead of ToddHewitt supplemented with horse serum. As a control, the plasmid pLZ12-Spec (an Esc. coliS. pyogenes shuttle vector) was introduced into A8173-1 to generate A8173-1(pLZ12-Spec).
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RESULTS |
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Six S. pyogenes strains representing different infection foci and different geographical origins, six streptococcal strains from the Lancefield groups C and G (wound and throat infections) and six strains from group D were analysed for binding to glycoprotein ligands. Thyroglobulin, fetuin and asialofetuin bound to several S. pyogenes, group C and group G strains, and submaxillary mucin to S. pyogenes and group C strains (Fig. 1). No binding of the control non-glycosylated proteins horse myoglobin and carbonic anhydrase to the bacteria was detected.
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Extractability of the binding activity
To characterize the properties of the glycoprotein-binding molecule, bacteria were subjected to various treatments, and the binding of thyroglobulin to a dilution series of the bacteria was analysed (Table 2). Trypsin digestion of the bacteria using different enzyme concentrations either reduced or abolished the binding activity, which suggests that the interaction is mediated by a protein on the bacterial surface. Incubation of the bacteria at 80 °C or in the presence of 2 M NaCl, 10 mM DTT or 1% Triton X-100 had only a minor effect on the thyroglobulin-binding activity, which indicated that the protein is heat stable and tightly attached to the surface of the bacteria.
Adhesion of bacteria to immobilized ligands
Adhesion of bacteria to thyroglobulin immobilized on microtitre plate wells was investigated to find out whether the glycoprotein-binding activity could support the adhesion of bacteria to glycan-containing surfaces. Metabolically labelled bacteria adhered to the wells coated with thyroglobulin, and the adhesion was inhibited with excess ligand (Fig. 2a). Essentially, no binding was detected to the control BSA-coated surface. These results indicate that S. pyogenes can adhere to surfaces coated with thyroglobulin.
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Further evidence for the dependence of the S. pyogenes A8173 glycoprotein-binding activity on Mga was obtained by analysing independent, previously characterized mga mutants for their binding to thyroglobulin. As with the A8173 strain, thyroglobulin bound to the M2 71-676 and CS101 wild-type strains. Thyroglobulin showed no binding to the mga gene insertion mutants of these strains (Table 3). In contrast, binding of thyroglobulin to the emm and fcr mutants of M2 71-676, and to a mrp mutant of CS101 was as strong as to the parental strains, which indicated that neither the M-protein nor the IgG-binding protein, which are regulated by the mga gene, is responsible for the binding activity.
Prevalence of the binding activity
Prevalence of the binding activity among S. pyogenes was investigated by studying the binding of thyroglobulin to 46 S. pyogenes strains. The activity was present in at least 32 of the 46 strains studied (Fig. 5). There appeared to be no major difference in the presence of the activity between bacteria from various infection foci (throat, skin and blood), opacity factor positive and negative strains or strains of different T serotypes (not shown), or from different geographical origins (Finland, USA, Spain).
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DISCUSSION |
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Cell adhesion mechanisms characterized for other streptococci include the interactions between Streptococcus pneumoniae and the oligosaccharide structures Galß1-4GlcNAcß1-3Galß1-4Glc (Andersson et al., 1983 ), GalNAcß1-4Galß1- (Krivan et al., 1988
), GalNAcß1-3Gal
1-4Galß1-4Glc-Cer (Cundell & Tuomanen, 1994
) and NeuAc
2-3/6Galß1- (Barthelson et al., 1998
) of human respiratory epithelial cells. The oligosaccharides were used as inhibitors of binding of pneumococci to epithelial cells. The binding of the meningitis-associated Streptococcus suis to Gal
1-4Gal and to sialic acid-containing oligosaccharides are other well characterized interactions of streptococci with oligosaccharide receptors (Liukkonen et al., 1992
; Haataja et al., 1993
, 1994
). Streptococcus sanguis expresses the SSP-5 adhesin, which mediates binding to sialic acid-containing salivary glycoproteins (Demuth et al., 1988
, 1990
). Anti-adhesion experiments have been carried out in animal models using lacto-N-neo-tetraose and its derivatives to attenuate the course of pneumococcal pneumonia and to prevent colonization of the nasopharynx (Idänpään-Heikkilä et al., 1997
; Barthelson et al., 1998
).
The results of the present study suggest the presence of a novel glycoprotein-binding activity in S. pyogenes. The presence of the activity is demonstrated both by the binding of soluble glycoproteins to immobilized bacteria, and by the binding of bacteria to immobilized glycoproteins. The activity is different from the S. pyogenes fibronectin-binding activity, since the binding of thyroglobulin to A8173 was not inhibited by fibronectin (J. Hytönen & J. Finne, unpublished results). The glycoprotein-binding activity is present in the majority of S. pyogenes strains studied. A similar activity is also found in group C and G streptococci.
There are no major protein sequence similarities between the glycoproteins studied. The cross-inhibitions of the glycoproteins and the absence of binding to non-glycosylated proteins therefore suggest that the receptor structure may be a carbohydrate. On the other hand, the observation that the binding of thyroglobulin is only partially inhibited by fetuin, asialofetuin and mucin may suggest the presence of one or more additional binding activities. Bovine thyroglobulin contains only N-linked glycans (Rawitch et al., 1993 ) and bovine submaxillary mucin mainly O-linked glycans (Strous & Dekker, 1992
). Fetuin is glycosylated with both N- and O-linked sialic acid-containing oligosaccharides (Spiro & Bhoyroo, 1974
; Yet et al., 1988
). Preliminary experiments using a selection of mono-, di- and trisaccharides as inhibitors of binding did not reveal the binding specificity of the adhesin (P. Isomäki & J. Finne, unpublished results). The receptor structure could therefore be a more complex or extended structure included in the N- and/or O-glycans of these glycoproteins, or a structure not apparent in gross peptide sequence comparison analysis.
As indicated by the resistance of the binding protein to solubilizations by salt, detergent, reducing agent and heat treatment, it appears to be tightly anchored to the bacterial surface. Only trypsin digestion removed the activity from the bacteria. These physical characteristics parallel those described for some other streptococcal surface proteins like streptococcal surface dehydrogenase, which was shown to be resistant to 2% SDS and 2 M NaCl treatments but sensitive to trypsin digestion (Pancholi & Fischetti, 1992 ). Unfortunately, analysis of the trypsin extract of streptococci revealed that the activity was rapidly inactivated and the molecule was therefore not amenable to further purification.
In transposon-mutagenesis experiments, the adhesion-inactivating insertion was found to be in the mga gene. The finding that the S. pyogenes binding-activity expression is controlled by the mga gene was confirmed by studying the adhesion of the glycoproteins to independent mga mutant strains, and by complementation with a wild-type mga gene. That the complementation was partial may be related to the fact that the mga gene used originated from another strain of S. pyogenes. On the other hand, there was binding of the glycoproteins to mutant strains deficient in M-protein and IgG-binding protein, which indicates that these proteins do not mediate the binding activity. The fact that the mga gene is a regulator of important S. pyogenes virulence factors, together with the high prevalence of the glycoprotein-binding activity among S. pyogenes and group C and G strains, suggests that it may have a role in the pathogenicity of streptococci. Further studies including the cloning of the gene are needed in order to characterize the molecular properties of this strepadhesin activity, its receptor specificity and its role in streptococcal virulence.
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ACKNOWLEDGEMENTS |
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Received 6 September 1999;
accepted 22 September 1999.