INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Group A Streptococcus (GAS) causes human infections of the throat and soft tissue and systemic diseases (39). This broad spectrum of infected tissues indicates that GAS can adapt to changing environments during pathogen-host interactions. In addition, the wide range of infections caused by GAS suggests that virulence factor expression is a very complex regulated process. Indeed, transcriptional and posttranscriptional mechanisms that control expression of virulence genes have been described (1, 9, 17, 21, 31). For example, several streptococcal cell surface proteins expressed in exponential growth are regulated by Mga, a positive transcriptional activator protein (3, 23, 27, 32).

Microbial extracellular molecules interact with host proteins and often mediate adherence (26). Several cell surface proteins of gram-positive bacteria have structural similarities that include a variable amino terminus, a central region composed of repeating units, and a carboxy-terminal cell-associated region with an LPXTG cell wall anchor motif (8). GAS cell surface proteins have been identified as proven or potential virulence factors and include M protein (7), immunoglobulin (14) and fibronectin (13) binding proteins, serum opacity factor (4), C5a peptidase (44), and GRAB (34).

Recently, we identified a new GAS cell surface protein that contains a central region composed of variable numbers of Gly-X-X (GXX) collagen-like motifs (20). The gene (scl) encoding this streptococcal collagen-like (Scl) protein was present in all 50 GAS strains studied and was preferentially transcribed in the logarithmic phase of growth by a serotype M1 GAS strain. Although the exact role of Scl in human pathogenesis is not understood, an isogenic scl mutant had decreased adherence to human fibroblasts grown in culture and was attenuated for virulence in mice, as assessed by subcutaneous inoculation (20).

In this study, we characterized a second gene (scl2) encoding a collagen-like protein. The scl2 gene also was present in all 50 genetically diverse strains studied, together representing 21 distinct M protein serotypes. Expression of scl1 is controlled transcriptionally by Mga. In contrast, production of Scl2 is controlled at the level of translation by the number of CAAAA pentanucleotide repeats located immediately downstream from a GTG (Val) start codon. This form of regulation has not been described in GAS or other gram-positive pathogens.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Bacterial strains and growth. Fifty GAS strains isolated worldwide were used. The strain collection was described in a recent analysis of the molecular population genetics and virulence role of Scl1 (20). The 50 GAS strains represented 21 different M types, as verified by sequencing the emm gene fragment encoding the hypervariable amino terminus. MGAS6708 is identical to SF370, the serotype M1 strain used in a genome sequencing project (http://www.genome.ou.edu/strep.html). Isogenic M1 GAS strains JRS301 (wild type) and JRS403 (mga mutant) (28) were kindly provided by June R. Scott (Emory University).

Construction of scl2-phoZ fusions. Plasmid pDC123 (obtained from C. E. Rubens, University of Washington) and the scl2 genes from MGAS5005 (serotype M1) and MGAS6274 (serotype M28) were used. Shuttle vector pDC123 (2) contains the phoZ gene (16) transcribed from constitutively expressed tetM and cat tandem promoters. The phoZ gene present in pDC123 confers a blue-colony phenotype to E. coli and GAS grown on media supplemented with 5-bromo-4-chloro-3-indolylphosphate (XP or BCIP) (2). Intracellular alkaline phosphatase (AP) activity was minimal or negligible but increased substantially when AP was secreted, indicating that abundant AP activity was export dependent.

DNA methods. Standard molecular-biology techniques were used (36). Plasmid DNA was purified with the UltraClean kit (Mo Bio Laboratories, Inc., Solana Beach, Calif.). GAS chromosomal DNA was isolated as described previously (25). The presence of the scl2 gene in GAS strains was assessed by PCR. The entire scl2 open reading frame (ORF) was amplified with forward primer scl2-up (5'-CTTTCAATGGATGACGATACC; nucleotides 29 to 9 upstream of the sequence shown in Fig. 1) and reverse primer scl2-rev (5'-ACTTTCCATCAGTTAGGTAGC; nucleotide positions 1160 to 1140 in Fig. 1) using Taq polymerase (Life Technologies). DNA was denatured at 94°C for 1 min. Thirty amplification cycles were performed as follows: 1 min of denaturation at 94°C, 1 min of annealing at 55°C, and 1 min 45 s of extension at 72°C, followed by one cycle of 5 min at 72°C. The PCR products were analyzed by agarose gel electrophoresis and sequenced with internal primers and the Taq DyeDeoxy terminator cycle sequencing kit (Applied Biosystems, Inc., Foster City, Calif.) with an ABI 377 instrument. The DNA sequence data were analyzed with Sequencher, version 3.1.1 (Gene Codes Corporation, Inc., Ann Arbor, Mich.) and Lasergene (DNASTAR, Inc., Madison, Wis.) software.

View larger version (52K): [in this window] [in a new window]   FIG. 1.   Nucleotide and amino acid sequences of the scl2 gene and inferred Scl2 protein in serotype M1 GAS (MGAS6708). The scl2 ORF consists of 951 bp (nucleotides 178 to 1129). The presumed scl promoter region has a predicted ribosome-binding site (RBS) and 10 and 35 regions. Dot at +1 inferred transcription start site. A potential transcription terminator (tt) consisting of two inverted repeats is located downstream of the ORF. The predicted GTG start codon (Val) and the TAA stop codon are in boldface. The inferred mature Scl2 polypeptide consists of 281 amino acids (nucleotides 284 to 1126). Four SSRs (CAAAA) located immediately after the GTG start codon would cause a frameshift in the downstream scl2 gene and result in premature termination of translation. SS, signal sequence; V, variable region; CL, collagen-like region consisting of 38 Gly-X-X triplet motifs (boxed); WM, cell wall membrane region containing the LPATG cell wall anchor motif (shaded). A TC point mutation (dot) in the TAA stop codon of the scl2 gene was present in all serotype M3 GAS strains. This polymorphism would extend the inferred Scl2 protein by 11 amino acid residues (italicized protein sequence between stars).

RNA methods. GAS strains were grown in THY medium and total RNA was isolated as described previously (19). Bacteria from 10-ml cultures were harvested and resuspended in Tris-EDTA buffer (10 mM Tris [pH 7.0], 1 mM EDTA). Cells were treated at 37°C for 5 min with mutanolysin (25 U) and lysozyme (1 mg/ml) in the presence of a 5 mM concentration of RNase inhibitor aurintricarboxylic acid. The cells were lysed by adding sodium dodecyl sulfate (SDS) (2% final concentration) and an equal volume of acid-phenol-chloroform at 65°C for 5 min. The samples were extracted with acid-phenol-chloroform, and RNA was precipitated with 2 volumes of ethanol in the presence of 0.2 M NaCl. DNA contamination was removed by digestion with DNase I, and the RNA was precipitated as described above.

Protein methods. The presence of the Scl1 and Scl2 proteins in culture supernatants and streptococcal cell wall fractions was studied. GAS strains were grown to exponential phase (optical density at 600 nm [OD₆₀₀] of ~0.5) in 150 ml of THY medium and pelleted by centrifugation, and total proteins in the culture supernatants were obtained by precipitation with trichloroacetic acid (TCA; 10% final concentration) on ice for 1 h. The TCA-precipitated protein samples were neutralized with saturated Tris before being loaded on an SDS-12% polyacrylamide gel electrophoresis (PAGE) gel. The cell wall-associated protein fractions were obtained from GAS cells resuspended in 2 ml of 20% sucrose with 10 mM Tris, pH 8.0, buffer containing 25 U of mutanolysin and 1 mg of lysozyme/ml. Cells were digested at 37°C for 1 h and pelleted by centrifugation, and the supernatants containing the cell wall fraction were used for subsequent analyses.

Nucleotide sequence accession number. The scl2 sequence data reported here have been deposited in GenBank under accession no. AF317835.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Identification and analysis of the scl2 gene and inferred Scl2 protein in serotype M1 GAS. We recently described a GAS gene encoding a presumed cell-associated protein with a long region of Gly-X-X repeats (20). The protein was named Scl for streptococcal collagen-like, and the gene was designated scl. The protein sequence corresponding to the hydrophobic cell membrane domain of cell surface protein M6 (FFTAAALTVMATAGVAAVV) (7) was used as the search query. With the exception of a small part of the scl gene sequence with similarity to the emm6 gene sequence encoding the carboxy-terminal transmembrane domain, there was no homology between scl and other GAS genes encoding cell surface proteins. This result suggested that Scl represented a new class of GAS extracellular protein. Therefore, the M1 genome database was searched again with protein sequences corresponding to the signal peptide (amino-terminal 37 amino acids) and cell wall region (carboxy-terminal 82 amino acids) of Scl (20). One highly homologous region was identified for each query. The regions of homology were located 1 kb apart on the opposite side of the GAS chromosome relative to the location of the scl gene. Analysis of this region of the GAS chromosome identified a second gene encoding a collagen-like protein with a long region of Gly-X-X repeats. To avoid confusion, the original gene was renamed scl1 and the new gene was designated scl2.

Distribution and variation of the scl2 gene among GAS strains. The scl2 gene was amplified from strain MGAS6708 (identical to strain SF370 used for a streptococcal genome sequencing project) and was sequenced to verify the available genome data. The scl2 gene was present in all 50 GAS strains representing the breadth of species genetic diversity as assessed by multilocus enzyme electrophoresis (24). The size of the scl2 gene varied among strains representing different M serotypes. In addition, size variation in the scl2 gene was common among GAS strains with the same M serotype. This observation suggested that variation in the scl2 gene exceeded that found in the scl1 gene. For example, no scl1 sequence variation among serotype M3 GAS strains was identified (20), whereas the scl2 gene varied in size for all five M3 strains. Hence, the scl2 gene was commonly found in GAS and was polymorphic in size.

scl1 gene transcription and Scl production. We reported recently that the scl1 gene was transcribed in two genetically distinct serotype M1 GAS strains (MGAS6708 and MGAS5005). Moreover, the Scl1 protein was present in cell wall fractions prepared from these isolates (20). To determine if the scl1 gene was transcribed by strains of GAS representing more than one M protein serotype, we studied three M28 strains (MGAS6141, MGAS6143, and MGAS6274) and an M52 strain (MGAS6186) by Northern blot analysis (Fig. 2A). The M28 strains were used because they had three distinct scl1 alleles. Total RNA was isolated from bacteria grown to logarithmic phase (OD₆₀₀, ~0.5), a time when the scl1 gene was abundantly transcribed in M1 strains (20). A single transcript was made by all strains studied, and the length of each transcript corresponded to the predicted size obtained from the gene sequence.

View larger version (31K): [in this window] [in a new window]   FIG. 2.   scl1 gene expression in GAS. (A) Northern blot analysis of total RNA isolated from cultures of GAS serotype M1, M28, and M52 strains in the logarithmic (OD600, ~0.5) phase of growth. Total RNA (10 µg) was hybridized with biotinylated DNA probes amplified by PCR from the corresponding source strains. The blot was developed with streptavidin-AP conjugate, and the hybridizing bands were visualized by chemiluminescence. Single scl1 gene transcripts of the predicted sizes were made by all strains analyzed. RNA size markers were used to estimate the sizes of the scl1 transcripts. (B) Western blot analysis of the supernatant (S) and cell wall (W) protein samples prepared from exponential GAS cultures. Different scl1 gene alleles were translated into Scl1 protein variants. Immunoreactive bands were identified in the cell-free and cell-associated protein fractions in all strains tested. The Scl1 proteins migrated aberrantly slowly in SDS-PAGE gel. Molecular mass standards are shown.

Transcription of the scl2 gene by GAS strains. Sequence analysis (Fig. 1) identified a presumed promoter region upstream of the scl2 gene and a potential transcription terminator with two inverted repeats ~40 bp downstream of a TAA stop codon, suggesting that the scl2 gene encoded a monocistronic mRNA. Many streptococcal genes are temporally expressed during growth, including scl1. Therefore, transcription of the scl2 gene was studied with RNA samples extracted from GAS cells harvested in exponential (OD₆₀₀, ~0.5) and stationary (OD₆₀₀, ~0.9) phases. Two controls were included to test the stringency of hybridization with the scl1- and scl2-specific probes. First, MGAS321 was included on the basis of DNA sequence data predicting that the scl2 gene transcript would be more than 200 bp shorter than the mRNA for the scl1 gene. Therefore, we assumed that hybridizing bands would be easily resolved by electrophoresis. Second, an isogenic MGAS5005 scl mutant (20) was included as a negative control for the presence of the scl1-specific transcript.

View larger version (37K): [in this window] [in a new window]   FIG. 3.   Transcription of the scl2 gene in GAS. (A) Northern blot analysis of the total RNA isolated from cultures of GAS serotype M1, M4, and M28 strains in the logarithmic (L; OD600, ~0.5) or stationary (S; OD600, ~0.9) phase of growth. Ten micrograms of total RNA was hybridized with biotinylated DNA probes prepared from the corresponding source strains by PCR. Hybridization was identified with streptavidin-AP conjugate with a chemiluminescence detection method. The scl2 gene was transcribed by all GAS strains in the exponential phase. RNA size markers were used to estimate the sizes of the scl2 transcripts.

Transcription of scl1 is regulated by mga. Several GAS cell surface proteins expressed in exponential growth are regulated by Mga, a positive transcriptional activator protein (1, 3, 30). The scl1 gene promoter region has a potential Mga binding site (20). To directly test the hypothesis that transcription of scl1 is regulated by Mga, we used an isogenic M1 mutant strain in which mga had been insertionally inactivated (28). Northern blot analysis showed that an scl1 transcript was made by wild-type strain JRS301 but not by the isogenic mutant strain, JRS403 (Fig. 4). No difference between early- and mid-logarithmic-phase cultures was identified. In contrast, an scl2 transcript was made by the wild-type and mutant organisms, a result indicating that this gene is not regulated by Mga.

View larger version (38K): [in this window] [in a new window]   FIG. 4.   Control of scl1 gene expression by Mga. The effect of inactivation of the mga gene on transcription of scl1 and scl2 was studied. Shown is a Northern blot analysis of the total RNA isolated during the early (OD600, ~0.3) and middle (OD600, ~0.4) logarithmic phases of growth from wild-type strain JRS301 and the isogenic mga mutant, JRS403. scl1 and control emm1 genes were transcribed in an mga-dependent fashion. In contrast, transcription of scl2 and the control gene recA did not require a functional mga gene.

Scl2 protein production is controlled by the number of CAAAA nucleotide repeats. The sequence of the scl2 gene in serotype M1 GAS strains indicated that the full-length Scl2 protein would not be produced due to premature translation termination. We also observed that the number of CAAAA pentanucleotide repeats located next to the presumed GTG start codon varied among the 25 GAS strains sequenced for scl2 (Table 1). In principle, variation in the number of these repeats would either permit full-length translation of the scl2 transcript or cause premature termination. Control of gene expression at the level of translation by variation in the number of SSRs has been reported for many gram-negative bacterial species (40), but this mechanism of gene regulation has not been described for GAS or gram-positive organisms.

View larger version (30K): [in this window] [in a new window]   FIG. 5.   Scl2 protein production depends on the number of CAAAA pentanucleotide repeats. (A) Schematic of the scl2 gene GTG start codon and CAAAA repeat region in four serotype M28 strains. The scl2 genes in MGAS6141 and MGAS6180 have 16 and 10 CAAAA nucleotide repeats, respectively, resulting in early translation termination. In contrast, scl2 genes in MGAS6143 and MGAS6274 have 11 and 3 CAAAA repeats, respectively, and should produce the extracellular Scl2 protein. Star, stop codon. (B) Immunoblot analysis showing the presence of Scl2 in the supernatant (S) and cell wall (W) protein samples obtained from the logarithmic GAS cultures. As anticipated, the Scl2 proteins were identified in cell-free and cell-associated fractions prepared from MGAS6143 and MGAS6274. Molecular mass standards are shown.

View larger version (32K): [in this window] [in a new window]   FIG. 6.   Analysis of translation control of Scl2 production by the number of CAAAA pentanucleotide repeats with PhoZ reporter constructs. (A) Schematic representation of the fusions between the region of the scl2 gene encoding the Scl2 signal sequence and phoZ (drawing not to scale). (Bottom) Plasmid pDC123 contains the phoZ gene under the control of the tetM and cat tandem promoters. The signal sequence (SS) and multiple cloning site (MCS) are located at the amino terminus of the functional PhoZ protein. pDC123 was digested with Eco47III and SphI restriction enzymes. (Middle) Schematic of the GAS scl2 gene, including the promoter (Pscl2) and regions encoding the signal sequence (SS), variable region (V), collagen-like region (CL), and cell wall membrane region (WM). A DNA fragment of scl2 containing the promoter region and encoding the signal sequence of Scl2 was amplified by PCR, digested with SmaI and SphI, and cloned into pDC123. In the resulting construct, the secretion signal sequence of PhoZ is replaced by the secretion signal sequence of Scl2; hence, production of the full-length Scl2-PhoZ chimeric protein is dependent on the presence of an in-frame number of CAAAA nucleotide repeats located immediately downstream of the GTG start codon. (Top) Amino acid sequences at the amino termini of the Scl2 signal sequences in two GAS isolates and associated nucleotide sequences. Different numbers of CAAAA nucleotide repeats located downstream of the GTG start codon are shown. Four CAAAA repeats cause a frameshift in the downstream DNA resulting in premature termination of translation in MGAS5005. In contrast, three CAAAA repeats present in the scl2 gene from MGAS6274 encode a functional signal sequence, resulting in Scl2 production. (B) Blue-white-colony phenotype depends on the number of CAAAA pentanucleotide repeats. AP activity was detected in both E. coli and GAS only when the phoZ reporter was fused to the scl2 signal sequence with the correct (in-frame) number of CAAAA repeats, scl26274-phoZ.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The data presented in this paper and another very recent contribution (20) indicate that GAS strains have two genes that encode collagen-like proteins (Table 2). The Scl1 and Scl2 proteins have several features in common with other GAS cell surface proteins (8), including a secretion signal sequence, variable domain at the amino terminus of the mature protein, repetitive central part, and conserved cell wall membrane domain with an LPXTG cell wall anchor motif. All 50 GAS strains tested, which together represent the breadth of species diversity in GAS, have both the scl1 and scl2 genes. This finding differs from what was found for emm and emm-like genes encoding streptococcal cell surface proteins M and M-like, respectively. All GAS strains have the emm gene encoding type-specific M protein, but other emm family members (enn, fcrA, and sph) are present only in some strains (5). The emm and emm family genes are located contiguously and their transcription is coordinately controlled by Mga. The two scl genes are expressed in the exponential phase of growth; however, the scl1 gene is regulated by Mga, whereas scl2 is not. The scl1 and mga genes are located ~30 kb apart in the chromosome of an available serotype M1 GAS strain. At least one other gene controlled by Mga (sof, encoding serum opacity factor) is located outside of the region of the chromosome that contains mga and genes immediately downstream of mga controlled by it (23). Our study provides no insight into the molecular mechanism controlling temporal regulation of scl2 gene expression. However, Mga-independent but growth phase-dependent expression has been reported for the slo (streptolysin O) and plr genes (22).

After this paper was submitted, Rasmussen et al. reported that the scl1 gene was under Mga control in GAS strain AP1 (serotype M1) by using a transposon-inactivated mga mutant (33).

Although we found that the scl2 gene was transcribed by all six GAS strains tested in this study, not all of these organisms produced full-length Scl2 protein. Our data indicate that failure to produce Scl2 by some or all strains was due to premature translation termination caused by variable numbers of CAAAA pentanucleotide repeats located immediately downstream from the GTG (Val) start codon. Analysis of the scl2 genes in four M28 serotype GAS strains with 3 to 16 CAAAA repeats predicted that only two of these four strains should produce the full-length Scl2 protein. Immunoblot and phoZ reporter fusion analyses fully supported this prediction. Only the correct number of CAAAA repeats in a signal sequence resulted in Scl2 protein production by GAS or in a blue-colony phenotype by phoZ fusion. In-frame expansion of the number of CAAAA repeats would result in elongation of the signal sequence, a process that could detrimentally affect Scl2 secretion. However, structural predictions made for the longest variant of the Scl2 signal sequence (made by the allele with 17 CAAAA repeats) indicated that this is not expected to be the case. Expansion of the CAAAA repeats would result in production of additional QNKTK pentapeptides and extend the charged domain of the secretion signal sequence. Of note, an scl2 gene with 11 CAAAA repeats is present in MGAS6143 and this strain secreted the Scl2 protein, a result indicating that elongation of the charged domains of the secretion signal sequence is not an impediment to extracellular production of Scl2.

Control of protein expression by variation in the number of SSRs such as CAAAA has not been reported for GAS or other gram-positive bacteria. There are several examples of genes whose expression is controlled by SSRs in gram-negative bacteria. For example, variation in the number of CAAT tetranucleotide repeats in lic1, lic2, and lc3 regulates lipopolysaccharide production by Haemophilus influenzae (42, 43). Cell surface variation in Neisseria gonorrhoeae is caused by early translation termination of the lsi-2 (lipopolysaccharide synthesis) and opa (opacity protein) genes and is due to variation in the length of a polyguanidine tract and the number of CTCTT repeats, respectively (6, 38). Phase and antigenic variation in these microorganisms affects important biological traits such as the ability of bacteria to colonize the host mucosal surface, to evade the host immune response, and to cause disease.

The frequency with which the number of CAAAA repeats varies in clonal descendants of a GAS strain is unknown. Consequently, it is not known if extracellular production of Scl2 undergoes classical, high-frequency phase variation. However, the gene sequence data provide strong indirect evidence that phase variation occurs (Table 1). Four of the six serotype M12 strains (MGAS6139, MGAS6144, MGAS6198, and MGAS6259) have scl2 genes that differ only by the number of CAAAA repeats. As a result, strains MGAS6139 (8 CAAAA repeats) and MGAS6144 (11 CAAAA repeats) are expected to express the same mature Scl2 protein variant whereas translation of Scl2 by strains MGAS6198 and MGAS6259 is expected to terminate prematurely (both strains have 6 CAAAA repeats). These four strains have the same multilocus enzyme electrophoretic type and multilocus gene sequence type (unpublished data) and hence have a recent common ancestor. Interestingly, these strains were isolated from patients in Texas during an outbreak of invasive disease that occurred in the winter of 1997 to 1998. Similarly, the scl2 genes in serotype M28 strains MGAS6274 and MGAS6180 also differed only by the number of CAAAA pentanucleotide repeats. As expected, MGAS6274 produces extracellular Scl whereas MGAS6180 does not because of premature translation termination (Fig. 5). These two strains also have a recent common ancestor, as assessed by multilocus enzyme electrophoresis and comparative sequencing of several other genes. Although these data are limited, taken together they suggest that Scl2 extracellular production can be modulated by cells that have recently descended from a common ancestor. We speculate that phase variation of Scl2 extracellular production in the course of pathogen-host interaction provides a survival advantage or enhances durability. In this regard, we note that variation in capsule production by Neisseria meningitidis is due to 1-bp deletions and insertions occurring in contiguous cytosine residues present in siaD (polysialyltransferase gene). Insertion or deletion of one cytosine residue results in a frameshift that produces premature translation termination or full-length translation of this enzyme, which participates in capsule synthesis (12). Spontaneous reversion of the capsule-deficient variant occurred in vitro at the high frequency of 10³. Similarly, size variation in several variable-number-of-tandem-repeat loci has been reported among strains of H. influenzae isolated from patients in an outbreak of lung infections (41). Variation in the number of SSRs is presumed to occur by slipped-strand mispairing during replication (18), and there is evidence that amplification of the number of SSRs by slipped-strand mispairing increases the likelihood of subsequent slippage events. Hence, infrequent expansion of the CAAAA present in scl2 might accelerate the frequency of insertions and deletions. Therefore, it is possible that an scl2 allele with 2 to 4 CAAAA repeats would be more stable than an scl2 allele with 8 to 10 CAAAA repeats. Additional experimental and epidemiological studies are needed to fully understand molecular events controlling production of Scl2 by GAS.