Department of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, Australia1
Menzies School of Health Research, Darwin, NT, Australia2
Department of Microbiology, GBF-National Centre for Biotechnology, Braunschweig, Germany3
Queensland Institute of Medical Research, Brisbane, Qld, Australia4
Cooperative Research Centre for Aboriginal and Tropical Health, Darwin, NT, Australia5
Author for correspondence: Mark J. Walker. Tel: +61 2 4221 3439. Fax: +61 2 4221 4135. e-mail: mwalker{at}uow.edu.au
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ABSTRACT |
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Keywords: SfbII, fibronectin-binding protein, virulence types, phylogeny
Abbreviations: GAS, group A streptococcus; HRP, horseradish peroxidase; SOF, serum opacity factor; VT, virulence type
The GenBank accession numbers for the sequences reported in this paper are AF367011 (sof VT3.2), AF367012 (sof VT3.1), AF367013 (sof VT2.2), AF367014 (sof VT21), AF367015 (sof VT37.1) and AF367016 (sof 13).
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INTRODUCTION |
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Adhesion of the streptococcal cell to host epithelial cells is a prerequisite for colonization and subsequent disease (Beachey, 1981 ). Group A streptococci possess a number of surface adhesins capable of interacting with the extracellular host protein fibronectin (Courtney et al., 1996
; Jaffe et al., 1996
; Kreikemeyer et al., 1995
; Talay et al., 1992
; Rocha & Fischetti, 1999
; Hanski & Caparon, 1992
), an interaction which has been shown to play a major role in the adherence of the streptococcal cell to the epithelial cells of the host (Talay et al., 1992
; Courtney et al., 1996
; Molinari et al., 1997
). The streptococcal serum opacity factor (SOF; SfbII) is a large extracellular and surface-bound protein of group A streptococci which is capable of binding fibronectin (Rakonjac et al., 1995
; Kreikemeyer et al., 1999
). SOF is unique among the group A streptococcus (GAS) fibronectin-binding proteins as it possesses enzyme activity which causes opalescence in mammalian serum (Rakonjac et al., 1995
). The two functional domains of SOF are located in separate regions of the protein. The enzyme domain of SOF is located in the variable N-terminus (Courtney et al., 1999
; Rakonjac et al., 1995
) and cleaves the ApoA1 portion of high-density lipoprotein (Saravani & Martin, 1990
). The fibronectin-binding domain is located in the conserved C-terminus of the SOF protein and binds fibronectin via highly homologous repeats (Kreikemeyer et al., 1995
; Rakonjac et al., 1995
). SOF has been shown to be a virulence determinant in group A streptococci which express a class II M protein; only those GAS isolates which express the class II M protein express the serum opacity activity (Courtney et al., 1999
).
Up to 60% of the GAS isolates circulating in the Northern Territory are non-typable by traditional serological methods (Relf et al., 1992 ); thus, a molecular method known as Vir-typing is used to genotypically characterize individual strains. Vir typing is a highly discriminatory typing method based on long-chain PCR of the highly variable virulence regulon of the genome (Gardiner et al., 1995
). Of all the clinical isolates from the Northern Territory examined, only 34% of the Vir types identified express SOF; however, over 96% of Aboriginal sera test positive for antibodies to SOF (Goodfellow et al., 2000
). This study characterizes the Aboriginal humoral immune response to SOF and assesses the variability of the sof gene in strains from different Vir types circulating in the Northern Territory.
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METHODS |
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Construction and purification of SOF fusion proteins.
Hexahistidyl (His6)-tagged SOF fusion proteins were constructed using the pQE30 vector system (Qiagen), thereby resulting in N-terminally His6-tagged fusion proteins that could be purified under denaturing conditions using Ni-NTA agarose according to the manufacturers instructions (Qiagen). Insert DNA was amplified from S. pyogenes strain 75490 chromosomal DNA using PCR primers containing a 5' in-frame BamHI restriction site (GGATCC) and a 3' EcoRI restriction site (GAATTC). The domains of SOF encompassed by each of the SOF fusion proteins are indicated in Fig. 1(a). Primers used for the construction of plasmids pSOF-4 (primer HT2) and pSOF-5 (primer HT1) have been previously described (Kreikemeyer et al., 1999
). PCR primers used for the construction of plasmids pSOF-1 and pSOF-6 were the 5' primers 5'-GGTTAATGCTGGATCCGAGACGAGTGC-3' (p-SOF1) and 5'-CCGATTGTCGGATCCGTCGAAGATA-3' (p-SOF6), and the 3' primers 5'-CCCTTAAATAGGAATTCTAATTGTTTC-3' (p-SOF1) and 5'-TCTTCGAATTCAGTAGCGTCATTTGAGC-3' (p-SOF6).
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Western blotting.
SDS-PAGE was performed according to the method of Laemmli (1970) . SDS-PAGE gels were Western blotted (Burnette, 1981
) onto a PVDF membrane (Micron Separations) by using a wet Western transfer apparatus (Bio-Rad) overnight at 20 V.
To detect the fibronectin-binding ability of proteins, PVDF membranes were blocked in PBS containing 3% (w/v) BSA, pH 7·4, for 90 min at room temperature, washed three times for 10 min in PBS containing 0·05% (v/v) Tween 20, and reacted with biotinylated fibronectin (100 µg ml-1 in PBS containing 3%, w/v, BSA for 90 min at room temperature). Human fibronectin (Sigma) was biotinylated by mixing 1 mg fibronectin in 100 mM Na2CO3, pH 8·5, with 0·25 mg Biotin-X-NHS (40 mg ml-1 in DMSO; Sigma) and incubating with gentle rocking for 2 h at room temperature. The sample was then dialysed against PBS containing 0·2% azide for 48 h with four buffer changes. Binding of biotinylated fibronectin was detected by incubating with a streptavidinHRPbiotin conjugate. Five micrograms HRPbiotin conjugate (1 mg HRPbiotin conjugate ml-1, 0·01% thimerosol, 1 mg BSA ml-1) was mixed with 5 µg streptavidin (1 mg streptavidin ml-1, 0·01% thimerosal, 1 mg BSA ml-1; Roche) prior to incubation with the membrane. The blot was then developed with 4-chloro-1-naphthol (Bio-Rad).
To detect immunoglobulins specific for recombinant SOF protein, membranes were blocked in PBS containing 5% (w/v) skimmed milk, pH 7·4, for 60 min at room temperature, washed three times for 10 min in PBS and reacted with rabbit or human sera (1:1000 PBS containing 1%, w/v, skimmed milk for 60 min). The blots were then washed in PBS, reacted with HRP-conjugated goat anti-rabbit IgG (Bio-Rad) or goat anti-human IgG or IgA (Bio-Rad, USA) (1:1000 in PBS with 1%, w/v, skimmed milk for 60 min) and washed with PBS prior to development with 4-chloro-1-naphthol.
Generation of antisera.
New Zealand White rabbits were injected with an equal mix of Freunds adjuvant (complete for primary immunization and incomplete for subsequent immunizations) and purified recombinant protein. One hundred micrograms protein in a total volume of 500 µl was administered intramuscularly at two sites, every 3 weeks for a total of four injections. Rabbits were exsanguinated one week post-immunization.
ELISA.
Microtitre wells were coated for 90 min at 37 °C with purified recombinant fusion proteins (5 µg ml-1) in carbonate buffer (15 mM sodium carbonate, 35 mM sodium bicarbonate, pH 9·6). Wells were then blocked with 5% (w/v) skimmed milk in Tris-buffered saline (TBS) for 90 min at 37 °C, and reacted with sera diluted 1:10000 in TBS containing 5% (w/v) skimmed milk, or with Aboriginal saliva diluted 1:30 in TBS containing 5% (w/v) skimmed milk (100 µl per well) for 90 min at 37 °C. Plates were washed five times with TBS containing 0·05% Tween 20, prior to the addition of 100 µl of a 1:2500 dilution of anti-human IgG HRP-conjugate for sera (Sigma), or a 1:2500 dilution of anti-human IgA HRP-conjugate (Calbiochem) for saliva and incubated for a further 90 min at 37 °C. Plates were washed five times with TBS containing 0·05% Tween 20 prior to development with o-phenylenediamine dihydrochloride (Sigma) at room temperature, in the dark for 30 min. Reactions were stopped with 30 µl 3 M HCl, and the plates were analysed by measuring A490 using a Spectramax 250 plate reader (Molecular Devices). Antibody responses to recombinant SOF domains were compared using the two-tailed Tukey test and ANOVA (Zar, 1984 ).
DNA sequence analysis.
Genomic DNA was extracted using Instagene (Bio-Rad) according to the manufacturers instructions. To amplify specific regions of DNA from S. pyogenes strains for DNA sequence comparison, Taq polymerase (Qiagen) was used to generate PCR products, which were purified from 0·8% (w/v) agarose gels using a Minelute gel extraction kit (Qiagen). Purified PCR products were then subjected to DNA sequence analysis. DNA sequence reactions were carried out using a dye terminator ready reaction mix (Perkin Elmer) and electrophoresed on a model 377 DNA sequencer (Perkin Elmer). Primers were designed based on the DNA sequence similarity observed between three published sequences obtained from GenBank [accession nos X83303 (Kreikemeyer et al., 1995 ), AF082074 and AF057697 (Courtney et al., 1999
)]. The primers used for PCR and for sequencing of the sof genes were as follows: 5'-ATAATAAAGTTCGGAACAATT-3' (SOF U); 5'-ATGACAAATTGTAAGTATAAAC-3' (SOF 1); 5'-GATGGACGTGGAACAGTATA-3' (SOF F1); 5'-ACTTTAACAGGCGAGCCAAC-3' (SOF F5); 5'-TCATAACTAAAGGTTGACTCG-3' (SOF 2); 5'-CATATCGTCCTGTTCTCTCAA-3' (SOF 3); 5'-CGAGTCAACCTTTAGTTATGA-3' (SOF F6); 5'-ACATCAACGGAATAGAAGGT-3' (SOF R5); 5'-ACCTTCTATTCCGTTGATGT-3' (SOF F8); 5'-TCTTTGGAAATAGTCCAAGTGAG-3' (SOF R3); 5'-CTCACTTGGACTATTTCCAAAGA-3' (SOF F3); 5'-GACATCACCGAAGATACCCAA-3' (SOF F4); 5'-GGTTGGGTATCTTCAGTGATA-3' (SOF R4); 5'-TTAGTTTCCTTCGGTGTCGCG-3' (SOF 6); 5'-CCTTCAACCGTAAGGGATACC-3' (SOF d). Various other non-conserved primers were also used for sequence analysis.
All DNA sequence data were analysed using ABI Prism DNA sequencing analysis software (Applied Biosystems), assembled using Autoassembler DNA sequence assembly software and translated using Macvector (version 4.14).
Phylogenetic analyses.
Nucleotide sequences were translated to amino acid sequences and the latter were aligned with CLUSTAL W. Nucleotide sequences were then aligned, using the amino acid alignment as a guide, with DNAstacks (version 1.1; Eernisse, 1992 ). The phylogeny of the SOF virulence types (VTs) and SOF sequences was determined under a range of neighbour-joining and maximum-parsimony methods. Phylogenetic analyses were performed using PAUP*. Bootstrapping was performed as an indication of the support for various nodes in the resolved phylogeny using the consensus of 100 resampled datasets.
Nucleotide sequence accession numbers.
The sequences generated in this study were deposited in GenBank under accession nos AF367011 (sof VT3.2), AF367012 (sof VT3.1), AF367013 (sof VT2.2), AF367014 (sof VT21), AF367015 (sof VT37.1) and AF367016 (sof 13).
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RESULTS |
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The predicted molecular masses of the SOF protein domains, based on the amino acid sequence of SOF, were 45 kDa (SOF-1), 91 kDa (SOF-4), 113 kDa (SOF-5) and 22 kDa (SOF-6). The migratory distance of the different SOF protein domains on SDS-PAGE suggested that the proteins were of a higher apparent molecular mass than predicted from the amino acid sequence. This has been reported previously by Rakonjac et al. (1995) and may be due to the high content of acidic amino acid residues, within the SOF protein domains, which would reduce the SDS-binding capacity (Tung & Knight, 1972
).
Horse serum agarose overlay studies indicated that the purified recombinant SOF protein displayed enzyme activity and readily opacified horse serum. However, not all bands of recombinant SOF-4 (SOF N-terminus) and recombinant SOF-5 expressed opacity factor activity. Two bands of SOF-5 protein, one of which corresponded to the full-length SOF-5 protein of 115 kDa, displayed opacity factor activity. Three bands of recombinant SOF-4, one of which corresponded to the full length SOF-4 of 105 kDa and two which corresponded to degraded SOF-4 protein of 99 kDa and 76 kDa, displayed opacity factor activity. One of the bands of SOF-4 which showed the strongest opacity activity was the 76 kDa degraded form of recombinant SOF-4. Enzyme activity has been previously localized to a region in the N-terminus of SOF encompassed by amino acid residues 148 and 813 (Courtney et al., 1999 ; Rakonjac et al., 1995
); this region has a predicted molecular mass of 76 kDa. The degraded form of SOF-4 which displayed the strongest opacity factor activity may contain only the active enzyme region of SOF and therefore display a comparatively greater activity. Thus, it is likely that only those recombinant SOF-4 and SOF-5 proteins that contained this region in its entirety displayed the opacity factor enzyme activity (Fig. 1c
).
Ligand blot analysis, using biotinylated fibronectin, indicated that the recombinant SOF protein and recombinant SOF domains displayed the ability to bind fibronectin. In this study it was found that the full-length recombinant SOF (SOF-5) and a peptide encompassing only the fibronectin-binding repeats (SOF-6) were able to bind fibronectin (Fig. 1d). Again, not all of the recombinant SOF-5 protein present exhibited the ability to bind fibronectin, but full length SOF-5 (113 kDa) and two degraded forms of SOF-5 (106 kDa and 99 kDa) did bind fibronectin. Fibronectin-binding has previously been localized to a repeat region in the C-terminus of the SOF protein (Kreikemeyer et al., 1995
; Rakonjac et al., 1995
). It is likely that only recombinant SOF-5 breakdown proteins containing this region bind fibronectin. Thus, the recombinant SOF protein domains and recombinant full-length SOF protein purified under denaturing conditions retain the functions of native streptococcal SOF.
ELISA and Western blot analysis of the Aboriginal humoral immune response to SOF
Sera and saliva samples from human subjects were tested by ELISA using purified full-length SOF protein and recombinant SOF domains as antigens (Fig. 2). Sera from Aboriginal adults and children had significantly higher SOF-specific serum IgG responses against SOF-1, SOF-4 and SOF-5 than did sera from non-Aboriginal subjects (P<0·001), reflecting the endemic exposure to GAS in Aboriginal communities (Fig. 2a
). No significant difference between the three populations was found for salivary IgA against the full-length recombinant SOF (SOF-5) and the recombinant SOF domains (SOF-1, SOF-4 and SOF-6) (Fig. 2b
). The anti-SOF salivary IgA response was elevated in the Aboriginal children compared with the other groups, but not significantly so.
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Western blot analysis of purified full-length SOF (SOF-5) and the SOF domains (SOF-1, SOF-4 and SOF-6) (Fig. 3) was performed using pooled Aboriginal adult sera. The immune response against the fibronectin-binding domain of SOF was observed to be reduced using the Aboriginal adult sera sample (Fig. 3b
).
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Identity and similarity of the SOF from SOF VT2.2, SOF VT3.1, SOF VT3.2, SOF VT21 and SOF VT37.1 with SOF 2, SOF 13, SOF 22, SOF 28, SOF 49, SOF 63 and SOF 75
The deduced amino acid sequences of SOF from a number of the most common Vir types circulating in the Northern Territory of Australia were compared with other SOF amino acid sequences listed in the GenBank database. The optimal alignment of SOF sequences was determined using the CLUSTAL W alignment program (alignment not shown). SOF sequences derived in this study were designated based upon Vir type (VT) results: NS 733, SOF VT2.2; NS 297, SOF VT3.1; NS 192, SOF VT3.2; BL28, SOF VT21; NS 473, SOF VT37.1. The sequence SOF 13, also derived in this study, has been designated based upon M type, as this is the sequence of the SOF protein from an M13 reference strain and not from an isolate from within the Aboriginal community. Sequences obtained through GenBank, SOF 2 (Courtney et al., 1999 ), SOF 22 (Rakonjac et al., 1995
), SOF 28 (Courtney et al., 1999
), SOF 49 (Courtney et al., 1999
), SOF 63 (Katerov et al., 2000
) and SOF 75 (Kreikemeyer et al., 1995
), have been designated based upon M type, as Vir type data were not available for all of these sequences.
In concurrence with previous studies, it was found that a large degree of variability exists among the SOF protein of different strains, with similarity between the deduced amino acid sequences of SOF ranging from 52·6 to 99·6% over the full length of the protein. From the results obtained it is apparent that the N-terminus of SOF, that is the entire molecule except the fibronectin-binding domain and membrane-spanning regions (Rakonjac et al., 1995 ; Kreikemeyer et al., 1999
), is the region where most of the variability in the SOF protein exists. While the N-terminal domains of SOF VT2.2 and SOF VT25 show 96% similarity and the N-terminal domains of SOF 22 and SOF 75 share 99·5% sequence similarity, comparison of the N-terminus of the other strains examined in this study reveals similarity ranging from 42·9 to 72·3% (Fig. 4a
). In contrast, the C-terminus of the SOF protein, containing the fibronectin-binding domain and membrane-spanning regions, is highly conserved. Sequence similarity between the different Vir types ranges from 84·6 to 100% within the fibronectin-binding repeats and C-terminus (Fig. 4b
).
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DISCUSSION |
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ELISA and Western blot analyses in this study suggested that the significant majority of antibodies against SOF are specific for epitopes in the N-terminus of the SOF protein rather than the C-terminus. This includes not only human IgG produced as a result of natural infection with GAS, but also rabbit IgG produced as a result of immunization with full-length recombinant SOF. Both forms of antibody strongly reacted with fusion proteins encompassing N-terminal domains of the SOF protein but reacted only weakly, if at all, with the SOF-6 fusion protein encompassing the C-terminal fibronectin-binding repeats of SOF. DNA sequence analysis indicates that the fibronectin-binding repeats of group A streptococcal strains from the Northern Territory of Australia are highly conserved in comparison to the group A streptococcal strain from which the recombinant fibronectin-binding repeats are derived (M75). Hence, the observed lack of reactivity against the fibronectin-binding repeats was, in all probability, not due to sequence variation between strains.
While it appears that the immune response is preferentially directed towards the N-terminus of the SOF protein, this study indicates that the fibronectin-binding repeats of SOF are antigenic. Immunization of rabbits with SOF-6 fusion protein encompassing the fibronectin-binding domain of SOF engendered a strong and specific immune response. Antibodies generated against recombinant SOF fibronectin-binding repeats failed to react strongly with any N-terminal portion of SOF, but strongly reacted with recombinant SOF protein encompassing the fibronectin-binding repeats in Western blot analysis. The fibronectin-binding repeats of SOF have been shown to be necessary for fibronectin binding in S. pyogenes strains that express SOF (Kreikemeyer et al., 1995 ). It may be this functional importance of the fibronectin-binding repeats that results in the immune response to SOF directed against the N-terminus of the SOF protein, which would potentially be beneficial to the survival of SOF-positive S. pyogenes strains. Similarly, the fibronectin-binding repeats of another GAS fibronectin binding protein, SfbI, have been shown to be immunogenic. The fibronectin-binding repeats of SfbI stimulate strong secretory IgA resulting in protective immunity in a mouse model (Schulze et al., 2001
).
It is well established that antibodies against SOF inhibit the enzyme opacity activity of SOF in a serologically specific manner (Gooder, 1961 ; Top & Wannamaker, 1968
). However, in this study it is apparent that anti-SOF antibodies are capable of recognizing proteins from heterologous SOF serotypes. Beall et al. (2000)
suggested that the type-specific anti-opacity factor epitopes of the SOF protein reside within 450 residues of the C-terminal of the enzyme domain (
950 residues long), with initial sequence comparison indicating a possible 107 residue stretch within this region representing the sole region determining anti-opacity factor type specificity. Thus, a large portion of the enzyme region of SOF contains non-type-specific epitopes, which may explain the recognition of heterologous SOF protein by Aboriginal and non-Aboriginal antibodies.
GAS strains causing invasive and non-invasive disease from the Top End of the Northern Territory are genotypically different to isolates deposited in Western World strain collections (Relf et al., 1994 ). Yet, the phylogenetic analysis of sof genes indicates that there is no distinct geographical segregation between the sof genes from Australian isolates and sof genes of other international isolates examined and that isolates frequently share common ancestry, based on parsimony analysis. Previous analysis of the first 270 residues of the SOF protein (Beall et al., 2000
) indicated that sof genes can be conserved between strains of different genetic backgrounds (different emm type, Vir type), a phenomenon also observed in this study. This may contribute to the lack of geographical segregation observed when examining the relatedness of sof genes in S. pyogenes. This phylogenetic analysis confirms that the reduced immune recognition of the fibronectin-binding domain was not due to the geographical segregation of Australian SOF isolates and the German M75 SOF from which the purified recombinant SOF domains were derived. The phylogenetic analysis in this study indicates a close relationship between SOF of Australian and SOF of international isolates (neighbour-joining and parsimony analysis). Thus, patterns of immune response observed among the Aboriginal people may also occur in populations from regions where GAS infection is not endemic, an observation that will impact on future vaccine studies.
SOF is required for virulence of group A streptococci expressing class II M protein (Courtney et al., 1999 ). The fibronectin-binding repeats of this streptococcal protein are highly conserved, required for fibronectin-binding by SOF (Kreikemeyer et al., 1995
) and are capable of engendering an immune response not observed as a result of natural group A streptococcal infection. Given these observations, and recent work suggesting the fibronectin-binding repeats of S. pyogenes SfbI (protein F) are protective (Schulze et al., 2001
), the fibronectin-binding repeats of SOF may prove to be a useful target for a vaccine against GAS.
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ACKNOWLEDGEMENTS |
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Received 22 June 2001;
revised 22 August 2001;
accepted 24 August 2001.