Infectious Disease Research Group, Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK1
Tel: +44 2476 528359. Fax: +44 2476 523701. e-mail: a.m.whatmore{at}warwick.ac.uk
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ABSTRACT |
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Abbreviations: SSR, short sequence repeat
The GenBank accession numbers for the sequences determined in this work are given in the text and Table 1.
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INTRODUCTION |
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A number of proteins harbouring cell wall sorting signals have now been characterized from S. pyogenes. These include C5a peptidase (Chen & Cleary, 1990 ), the T protein (Schneewind et al., 1990
), two distinct fibronectin-binding proteins SfbI/proteinF (Sela et al., 1993
; Talay et al., 1994
) and SfbII/OF (Kreikemeyer et al., 1995
; Rakonjac et al., 1995
) and a large number of proteins encoded by members of the emm gene family (reviewed by Navarre & Schneewind, 1999
). As might be expected, many of the surface proteins characterized to date have been postulated to play important roles in virulence either by modulating the host immune response and/or by involvement in adhesion to host surfaces (Jenkinson & Lamont, 1997
; Cunningham, 2000
). Many, notably members of the emm gene family, possess multiple binding activities for an array of host proteins that can include albumin, fibrinogen, IgG, IgA, kininogen, fibronectin, factor H, FHL-1, plasminogen and C4BP (Navarre & Schneewind, 1999
).
A further common feature of many surface proteins of Gram-positive bacteria is the presence of tandem repeat domains that can vary in size from just several to several hundred amino acids. Often strain-to-strain variations in the number of repeats are apparent, implying that gain or loss of repeats occurs as a result of intragenic recombination events or slipped-strand mispairing. Perhaps the best examples of such repetitive structures in S. pyogenes are members of the emm gene family, which can contain up to three distinct sets of tandem repeats (Kehoe, 1994 ). Analysis of successive isolates from patients demonstrates that variation in the anti-phagocytic M6 protein occurs as a result of alteration in the number of tandem repeats (Hollingshead et al., 1987
) and these changes can alter antigenic determinants, resulting in resistance to phagocytosis (Jones et al., 1988
). Variable repetitive structure is also apparent in a number of surface proteins of other streptococci, including the S. pneumoniae surface protein PspA (Yother & Briles, 1992
) and the alpha C protein encoding gene of S. agalactiae (Madoff et al., 1996
). A number of possible benefits, which may be of relevance in pathogenesis, of variation in repeat number can be envisaged. These include antigenic variation and subsequent immune escape, alteration in substrate binding properties and the simple physical displacement of parts of proteins further away from the immediate extracellular environment where they may be masked by other surface components. In several Gram-negative bacteria so-called short sequence repeats (SSRs), consisting of 220 nucleotide tandem repeats, can be involved in gene regulation either by altering the spacing between promoter domains or by affecting the integrity of ORFs (van Belkum et al., 1998
; Henderson et al., 1999
). However to date there is no strong evidence for the occurrence of similar repeats implicated in gene regulation in Gram-positive bacteria.
Sequencing of the complete genome of a serotype M1 invasive isolate of S. pyogenes (Roe et al., 1999 ) has facilitated the identification of previously unrecognized surface proteins. Such proteins are clearly of potential interest as many are likely to be involved in interactions with the host and may therefore play important roles in the pathogenic process. Elucidation of the nature of these proteins will facilitate a fuller understanding of the virulence of S. pyogenes and may identify novel therapeutic or vaccine targets. In this study, an ORF (sclB) identified during a screen of the genome sequence, potentially encoding a surface protein with a highly repetitive collagen-like structure, is described. In addition, the ORF is shown to be highly variable and to contain SSRs immediately downstream of the putative start codon that may regulate the expression of the corresponding SclB protein product.
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METHODS |
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PCR.
Primer pairs used for initial amplification of RST00068 and RST00596 products were 210up (5'-CCAAGCCTAATCGCTTAGTCT-3') with 210dn (5'-TACTTTCCATCAGTTAGGTAGCA-3') and 174up (5'-TTTGCTTATCAGTAAGGTCTCTC-3') with 174dn (5'-CGTTCAGGAGTTACCAAAAGAAT-3') respectively. Primer pair sclup (5'-ATGTTGACATCAAAGCACCA-3') and scldn (5'-GTTGTTTTCTTTGCGTTTTGT-3') was used for amplification of sclA. PCR was performed under standard conditions with 32 cycles of 95 °C for 1 min, x °C for 1 min and 72 °C for 1 min where x °C represents an annealing temperature appropriate for the particular primer set.
Sequencing.
Full-length RST00068 ORFs were sequenced from PCR products amplified using primer 210up, described above, in conjunction with primer Rst69 (5'-TTTAACTCATCTGATGTTTCCAT-3') located approximately 200 bp downstream of the RST00068 sequence in the genome sequence. Sequencing was performed using a range of internal primers and dye-labelled dideoxy terminator cycle sequencing kits (Beckman Coulter) with the CEQ2000 automated DNA analysis system.
DNA sequence analysis.
Preliminary analysis of sequences was peformed using the DNASTAR package and multiple sequence alignments were performed using the CLUSTAL package (http://www.ebi.ac.uk).
Predicted structure of SclB.
The structure of the putative protein encoded by sclB was predicted using the PROT EAN program within the DNA Star package. This package uses the ChouFasman (Chou & Fasman, 1978 ) and GarnierRobson (Garnier et al., 1978
) algorithms to predict alpha, beta and turn regions, the GarnierRobson algorithm for predicting coil regions and the KyteDoolittle algorithm (Kyte & Doolittle, 1982
) for calculating hydrophilicity. Protein secondary structure was confirmed using the PSIPRED program at http://insulin.brunel.ac.uk/psipred (Jones, 1999
). The presence and predicted cleavage site of signal peptide was predicted using the SignalP program at http://www.cbs.dtu.dk/services/SignalP (Nielsen et al., 1997
).
RT-PCR.
Total cell RNA was prepared from S. pyogenes cultures grown to mid-exponential phase in ToddHewitt broth at 37 °C. A 10 ml culture was harvested by centrifugation and the pellet was resuspended in 100 µl nuclease-free water. RNA was isolated using the Hybaid Ribolyser Blue Kit and Ribolyser according to the manufacturers instructions and was resuspended in 50 µl nuclease-free water. The RNA was then treated with 5 U RQ1 RNase-free DNase (Promega) for 30 min at 37 °C. Following inactivation of the DNase at 75 °C for 5 min, RNA was stored at -70 °C in the presence of 40 U RNasin (Promega) until required. RT-PCR was performed using the Access RT-PCR system (Promega) according to the manufacturers instructions. First-strand cDNA synthesis was performed at 48 °C for 45 min while second-strand cDNA synthesis and PCR amplification were performed using 36 amplification cycles at an annealing temperature of 53 °C. Positive control primers gtrup and gtrdn, designed to amplify an internal 598 bp fragment of the housekeeping gene glnQ (glutamine-transport ATP-binding protein), were used. Primer pairs 655Exp (5'-GGATCCGATGGTGAAGATGCCCAA-3'), corresponding to the extreme N-terminus of the mature protein, with 210dn (described above) and SigP (5'-TTWGGAGGTGYAAGYGCRGTT-3'), corresponding to the signal peptide, with Rst69 (described above) were used to detect an sclB-specific transcript.
emm sequence typing.
This was performed as described by Beall et al. (1996) using primers 1 and 2, which were previously described by Whatmore & Kehoe (1994)
. The identity of emm sequences was confirmed by interrogation of the Centers for Disease Control emm sequence database at http://www.cdc.gov/ncidod/biotech/strepinfo.html.
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RESULTS |
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In the case of RST00068 the reverse primer was designed immediately downstream of the putative stop codon that could be clearly defined on the basis of the wall-associated sequence seen in many Gram-positive bacteria. However, the start of the gene was more difficult to predict the WIT database predicted a TTG start codon, but there were no convincing RBS or promoter elements associated with this predicted start codon (see Fig. 1). Because of this uncertainty, the upstream primer (210up) was designed within a conserved 3' region of a gene upstream of RST00068 which had significant similarity to GTP-binding proteins from other Gram-positive bacteria, such as Bacillus and Mycobacterium. Initially, 17 randomly selected strains were tested by PCR and a PCR product was amplified from 15 of these interestingly these products were extremely variable in size. In both negative reactions the integrity of the DNA was confirmed by the amplification of the emm gene (Whatmore et al., 1994
) from the same preparations. The presence of the sequence was then examined in more extensive studies using strains associated with acute rheumatic fever (6), acute post-streptococcal glomerulonephritis (16), throat infection and carriage (19) and impetigo (9). Forty-five of these 50 isolates (90%) gave a PCR product and these reactions confirmed the extensive size diversity of this product, with bands ranging from some 600 bp to approximately 2 kb. There was no clear relationship between the presence of this gene and the ecological association of the isolates and again emm could be amplified from all isolates that were PCR negative for the RST00068 ORF.
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Sequence of RST00068
To further characterize the nature of the genetic diversity apparent in ORF RST00068, sequencing of the complete ORF and flanking regions was undertaken from 11 isolates, while an incomplete 5' sequence was obtained from a further seven isolates. The isolates chosen were from diverse diseases and various geographical localities and represented diverse emm types as determined by sequencing of the 5' emm gene (Whatmore et al., 1994 ).
Identification of predicted start codon and control elements. As mentioned above, the rarely used TTG start codon predicted by WIT was not convincing. Fig. 1(a) shows a comparison of the sequences from three isolates, 650, 655 and 733, with the RST00068 sequence. The TTG is marked but no convincing control elements could be identified. On comparing sequences it is also clear that the TTG is located in the middle of a highly conserved region encoding a putative signal peptide as described below. Comparison of sequences at the 5' of the ORF enabled the identification of much more likely elements. A much more promising GTG start codon was identified farther upstream GTG is used as an initiation codon at about 5% the frequency of ATG. A purine-rich tract was located immediately upstream of this start codon where a RBS would be expected. Located some 55 bp upstream of this were putative -35 and -10 elements which exactly matched, in both sequence and spacing, the TTTACT/A and TATAAT consensus sequences determined from other S. pyogenes genes (unpublished data). Fig. 1(b)
shows an alignment downstream of the predicted stop codon. An 11 bp inverted repeat followed by a run of T residues resembling a classical transcription terminator was located downstream of the putative coding sequence.
Identification of pentameric repeats within the ORF. A further interesting feature of this region was the presence of a tract of pentameric CAAAA repeats immediately after the putative start codon. Although only four of these repeats were seen in the SF370 genome sequence, the number of these repeats was found to vary from a minimum of 4 copies up to 15 copies (Table 1). It is clear that variation in repeat number resulted in the ORF being either in or out of frame with respect to the predicted start codon, as illustrated in Fig. 2
. ORFs containing 5, 8, or 14 repeats were in-frame while other numbers of repeats resulted in the generation of stop codons almost immediately downstream of the CAAAA tract. The exceptions were two isolates, each containing nine repeats (733 and 740), that also appear to be in-frame as a result of minor sequence variation at the end of the CAAAA tract. Thus, these repeats may represent a mechanism to modulate expression of this ORF at the translational level.
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Detection of expression of the RST00068 ORF
To determine whether a transcript corresponding to RST00068 was present in S. pyogenes, RT-PCR was performed using specific primers. Representative results using RNA isolated from strain 655 are shown in Fig. 5. An RNA transcript of approximately the correct size was detected using primer pair 655Exp/210dn located within the predicted RST00068 transcript and predicted from the sequence analysis to give a product of 815 bp. In contrast, when using the primer set SigP/Rst69, where Rst69 is located downstream of the predicted transcription terminator, no RNA transcript was detected in the DNase-treated preparation. In the RNA fraction that was not subjected to DNase treatment, a band of approximately 1 kb was seen, equating to the 1011 bp product which would be predicted assuming the region downstream of RST00068 in strain 655 was of identical length to the equivalent sequence in strain SF370. Primers to the housekeeping gene glnQ were used as a positive control and detected a transcript specific for this gene. Similar results to those obtained using the RNA from isolate 655 were also obtained using RNA from three other isolates, P180, P19 and 733, confirming the presence of a transcript corresponding to ORF RST00068 in S. pyogenes in culture in vitro.
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DISCUSSION |
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This study also identified a novel repetitive element apparently located within the sclB coding sequence. Repetitive DNA elements, often called SSRs have been increasingly recognized in prokaryotes in recent years. As a result of slipped-strand mispairing altering the number of such repeats they can be involved in gene regulation both when they are positioned upstream of the gene, thus influencing transcription, or within the gene, influencing translation (Henderson et al., 1999 ). There are now several examples of regulation of genes occurring at the translational level as a result of variation in the number of SSRs at the 5' of genes, similar to those seen in sclB, which alter the reading-frame. For example, the adhesins encoded by the opa genes of Neisseria gonorrhoeae and Neisseria meninigitidis achieve an onoff phase variation at the translational level by alterations in the reading frame of a semi-variable hydrophobic portion of the leader sequence consisting of a run of CTCTT repeats which can vary as a result of slipped-strand mispairing (Stern et al., 1986
). In a similar manner translational control as a result of slipped-strand mispairing within a run of cytosine residues in the ORF has been shown to regulate expression of the Bordetella pertussis bvgS gene, a major regulator of virulence genes in this organism (Stibitz et al., 1989
). Similar events have been implicated in the regulation of genes in a variety of other Gram-negative organisms, including Vibrio cholerae, Haemophilus influenzae, Helicobacter pylori, Mycoplasma hyorhinis, Haemophilus somnus, Pasteurella haemolytica and Mycoplasma fermentans (Henderson et al., 1999
; van Belkum et al., 1999
). Recent analysis of whole genome sequences has led to suggestions that translational slipped-strand mispairing may be a more important and widespread mechanism of regulation than has previously been realized. A substantial number of genes in both H. pylori and N. meninigitidis contain such SSRs and are potentially regulated by this mechanism (Saunders et al., 1998
, 2000
). There is currently no direct evidence that sclB is regulated at the translational level by slipped-strand mispairing of the CAAAA repeats. However, circumstantial evidence in the form of the location of these repeats at the 5' of the signal peptide encoding sequence and immediately downstream of a putative start codon suggests that this is a probable scenario. Although no attempt was made to demonstrate an alteration in repeat number in culture, different numbers of CAAAA repeats were found to be associated with sequences sharing otherwise identical sclB sequences (669 and P195), providing additional support for this scenario. To date, translational regulation by slipped-strand mispairing has not been demonstrated in Gram-positive bacteria although slipped-strand mispairing of internal sequence repeats has been implicated in the antigenic variation of some surface proteins, notably the highly repetitive emm genes which encode one of the major virulence proteins of S. pyogenes (Harbaugh et al., 1993
; Whatmore et al., 1994
).
The striking and extensive collagen-like structure of the putative SclB protein appears unprecedented amongst the prokaryotic proteins characterized to date. The extensive run of GXYn repeats resembles the remarkably regular structure of collagen, where virtually every third residue is glycine. It is thus tempting to speculate that SclB may exist in the same triple-helix conformation as collagen, composed of three protein units wound around each other to form a stiff cable. The three-residue glycine periodicity is crucial in such a structure as there are three residues per turn of the helix and the only amino acid small enough to fit in an interior position is glycine. Previously only very limited runs of collagen-like sequence have been reported in bacteria, such as in a hydrolase of Klebsiella pneumoniae, which contains up to six tripeptide repeats (Charalambous et al., 1988 ), and in the Streptococcus sanguis platelet-aggregation-associated protein (Erickson & Herzberg, 1993
). Interestingly, a phage-encoded hyaluronidase found in S. pyogenes can contain a run of ten GXY repeats although this region is often missing, implying that it is not important for hyaluronidase activity (Hynes et al., 1995
). During the course of this work Lindmark & Guss (1999)
reported a novel fibronectin-binding protein, SFS, from Streptococcus equi, which also shows high scores against collagen on similarity searches. The basis of this identity was the high content of glycine, serine and proline present in both proteins and a ten-residue motif (QGERGEAGPP) seen in both collagen and the fibronectin-binding domain of SFS. Although SclB and SFS share some common characteristics in terms of amino acid composition there are clear differences between the proteins. While the collagen-like GXY repeats of SclB are virtually perfect and very extensive, SFS has only very limited runs of perfect three-residue periodicity. In addition, although SFS does possess a signal peptide it does not contain any of the sequence motifs known to mediate attachment to the cell wall.
The function of SclB remains unknown. Attempts to express the protein in Escherichia coli have so far proved unsuccessful, perhaps reflecting the unusual structure of SclB. It is possible that SclB is involved in either adhesion to the cell matrix or binding of host proteins from the extracellular milieu. A further intriguing possibility, given the structural similarity to such a ubiquitous host protein as collagen, is the involvement of this molecule in the autoimmune sequelae, particularly acute rheumatic fever, associated with S. pyogenes. Although the sequences of SclB are rather variable, the M protein precedent demonstrates that S. pyogenes proteins displaying little or no primary sequence similarity can have identical activities (Johnsson et al., 1998 ). In light of the similarity to SFS described above it is possible that SclB represents a fibronectin-binding protein, particularly as collagens of types I to V have been shown to bind fibronectin (Engvall et al., 1978
). S. pyogenes is already known to harbour at least two fibronectin-binding proteins, SfbI/protein F (Sela et al., 1993
; Talay et al., 1994
) and SfbII/OF (Kreikemeyer et al., 1995
; Rakonjac et al., 1995
) although neither are found in all strains (Goodfellow et al., 2000
). In addition, some proteins encoded by certain members of the emm gene family are believed to possess fibronectin-binding activity (Frick et al., 1995
; Reichardt et al., 1995
). However, it is becoming apparent that binding of fibronectin by bacterial surface proteins is a common property and multiple fibronectin-binding proteins have now been reported from a number of other Gram-positive bacteria (Joh et al., 1999
).
The confirmation that at least two surface proteins with collagen-like structure are present in at least some isolates of S. pyogenes identifies a new family of variable proteins within this organism. Although limited sequence data are available as yet for SclA, it is likely that SclA and SclB share the same highly repetitive structure with a variable-length tract of GXYn repeats following an N terminus displaying extensive sequence diversity. However, SclA appears to lack the pentameric nucleotide repeats seen within the signal-peptide region of SclB. This family appears to share a number of common features with members of the emm gene family. Both families have variable N termini that, at least in part, reflect a positive selection for the accumulation of nonsynonymous point mutations (Whatmore et al., 1994 ). Both, in common with many Gram-positive surface proteins, also encode extensive tandem amino acid repeats within the structural gene. It seems likely that genetic variation in members of the scl family can be generated by the gain or loss of repeats as a result of intragenic recombination events and/or slipped-strand mispairing as seen in members of the emm gene family (Whatmore et al., 1994
). The presence of two genes sharing considerable sequence similarity further raises the possibility that variation could also be generated as a result of intergenic exchange of DNA between sclA and sclB, as has been noted between enn and emm gene representatives in the emm gene family (Whatmore & Kehoe, 1994
; Podbielski et al., 1994
). It is also possible that one scl gene may serve as a silent cassette acting as a potential reservoir of diversity for antigenic variation.
The results presented in this paper represent an initial characterization of a novel S. pyogenes gene, sclB, potentially encoding a surface protein and belonging to a family of previously uncharacterized proteins. Future work will be aimed at expressing and elucidating the activities of SclB, determining whether post-infection human sera contain antibodies to SclB, confirmation of the role of the pentameric repeats in modulation of expression, and primer extension studies to confirm the location of control elements predicted by sequence comparisons.
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ACKNOWLEDGEMENTS |
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Received 25 August 2000;
revised 10 October 2000;
accepted 16 October 2000.