J Med Microbiol 57 (2008), 1238-1246; DOI: 10.1099/jmm.0.2008/001156-0
© 2008 Society for General Microbiology
ISSN 1473-5644
Superantigen genes in group A streptococcal isolates and their relationship with emm types
Robert Commons1,2,
Susan Rogers1,2,
Travis Gooding1,2,
Margie Danchin1,2,3,
Jonathan Carapetis1,2,3,
Roy Robins-Browne2,4 and
Nigel Curtis1,2,3
1 Department of Paediatrics, The University of Melbourne, Australia
2 Murdoch Children’s Research Institute, Royal Children’s Hospital Melbourne, Australia
3 Infectious Diseases Unit, Department of General Medicine, Royal Children’s Hospital Melbourne, Australia
4 Department of Microbiology & Immunology, The University of Melbourne, Australia
Correspondence
Nigel Curtis
nigel.curtis{at}rch.org.au
Received 3 March 2008
Accepted 13 June 2008
Superantigens are important virulence factors in the pathogenesis of invasive disease caused by group A streptococcus (GAS). There has been a recent re-emergence of this disease worldwide. A number of novel superantigens have been described recently. This study investigated 107 isolates of GAS for possession of each of the 11 currently known superantigen genes to determine the prevalence, co-occurrence and genetic restriction amongst different emm types of GAS. The results were compared with those in previously published studies. Superantigen genes were not randomly distributed amongst GAS isolates. Certain combinations of superantigen genes were more common and the majority of emm types showed restricted superantigen profiles. This is the first prevalence study of GAS isolates to include the complete range of known superantigen genes and their restriction amongst emm types. This study contributes to the understanding of the relationship between superantigen genes and emm types, and highlights the importance of comprehensive studies in different populations.
Abbreviations: GAS, group A streptococcus.
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INTRODUCTION
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Over the past 20 years, there has been a significant increase in the incidence of invasive disease caused by group A streptococcus (GAS) (Stevens et al., 1989; Johnson et al., 1992). Superantigens are believed to be important virulence factors of this pathogen. They are extracellular protein toxins that are pyrogenic, increase host susceptibility to endotoxic shock, suppress immunoglobulin production and have mitogenic activity for specific T-cell subsets (Curtis, 1996). This activity leads to extensive immune activation and a massive release of pro-inflammatory cytokines, such as tumour necrosis factor-
, interleukin-6, interleukin-2 and gamma interferon, which can cause shock and widespread organ damage. With the recent sequencing of the complete genomes of the emm1, emm3, emm6, emm18 and emm28 isolates of GAS, a number of novel superantigens have been discovered (Ferretti et al., 2001; Beres et al., 2002; Smoot et al., 2002a; Nakagawa et al., 2003; Banks et al., 2004; Green et al., 2005). A total of 11 superantigens have been identified in GAS to date: streptococcal pyrogenic exotoxin (SPE) A, SPEC, SPEG, SPEH, SPEI, SPEJ, SPEK, SPEL, SPEM, streptococcal mitogenic exotoxin (SME) Z and streptococcal superantigen (SSA).
Most GAS superantigen-encoding genes are associated with bacteriophages, except for speG, speJ and smeZ (Proft et al., 2000, 2003; Ferretti et al., 2001; Proft, 2003). The documented prevalence of bacteriophage-encoded superantigens varies widely geographically and temporally. GAS superantigens have also been associated with isolates of particular emm and M types. The emm-type-specific associations are hypothesized to reflect a selective influence of surface M protein on bacteriophage entry (Mylvaganam et al., 2000). The superantigen genes speG, speJ and smeZ are believed to be chromosomally encoded. This is supported by the reported 100 % prevalence of these genes in many studies (Proft et al., 2000, 2003; McCormick et al., 2001; Smoot et al., 2002a; Proft, 2003) and a genome sequencing study that showed no association of these genes with mobile elements in an M1 strain (Ferretti et al., 2001). However, smeZ and speG appear to have undergone horizontal transfer between species (Sachse et al., 2002; Igwe et al., 2003; Hashikawa et al., 2004). In addition, not all studies have found a 100 % prevalence rate for these genes.
This paper describes a study of GAS isolates collected during a short period within one metropolitan area. One hundred and seven isolates were emm-typed and tested for possession of each of the 11 superantigen genes. A previous study by us looked at the relationship between superantigens and virulence of GAS in the same collection of isolates (Rogers et al., 2007). This study focused on superantigen prevalence and the relationship of superantigens to emm type. It is one of the most comprehensive studies of superantigen gene prevalence to date.
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METHODS
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Isolate collection and culture.
A total of 107 GAS isolates were studied, comprising 67 isolates randomly selected from the ‘Burden of Superficial GAS Diseases in Victoria Study’ at the Royal Children’s Hospital Melbourne, Australia, between August 2001 and December 2002, and 40 isolates collected during the same period as part of the ‘Invasive GAS Surveillance Study’ in metropolitan Melbourne. To prevent confounding from epidemiologically related isolates, those from related family members were excluded. Isolates were cultured on horse blood agar at 37 °C overnight with 5 % CO2. β-Haemolytic colonies were typed by latex agglutination to detect the presence of Lancefield group A antigen.
Purification and extraction of DNA.
DNA was extracted by mechanical agitation as preliminary experiments (data not shown) showed that PCR amplification using DNA produced by this method was significantly more sensitive than using DNA prepared by lysis (heating at 100 °C for 10 min; Beall et al., 1996). In brief, supernatants from overnight cultures at 37 °C in 10 ml brain heart infusion broth were resuspended in lysis buffer. DNA was extracted by agitation in a FastPrep FP120 cycler with glass beads. DNA was purified using conventional phenol/chloroform extraction and 2-propanol precipitation, and treated with RNase A. The pellet was resuspended in 50 µl dH2O. PCR detection of the speB gene was used to assess contamination in negative controls and also as a positive control for all isolates (Dmitrieva et al., 2002).
Verification of species of Streptococcus pyogenes.
Isolates in which speB was not detected were subjected to repeat agglutination testing and subsequent 16S rRNA gene sequencing with primers 63f (5'-CAGGCCTAACACATGCAAGTC-3') and 1387r (5'-GGGCGGTGTGTACAAGGC-3') (Marchesi et al., 1998).
emm gene typing.
Specific regions of the emm gene were amplified using the Qiagen HotStarTaq system and a Centers for Disease Control and Prevention protocol (www.cdc.gov/ncidod/biotech/strep/protocols.htm) with minor modifications. Cycling conditions were 15 min at 95 °C, 30 cycles of 45 s at 95 °C, 30 s at 40 °C and 120 s at 72 °C, and extension at 72 °C for 10 min. Products were purified using a QIAquick PCR purification kit (Qiagen) and sequenced with 2.5 pmol of the primer emmseq2 (5'-TATTCGCTTAGAAAATTAAAAACAGG-3'). emm types were identified using the CDC emm database.
PCR testing for superantigens.
Individual PCR amplifications were undertaken to detect the genes for each of the 11 superantigens and speB as a positive control. To minimize the chance of allelic variation affecting results, primers were designed or selected following analysis of all published sequences and targeting of a conserved site within alleles (Table 1
). Products of primers that have not been published previously were sequenced to verify that the correct gene was being targeted.
Each 50 µl reaction contained 20 pmol of each of the forward and reverse primers, PCR buffer (10 mM Tris/HCl, pH 8.3; 50 mM KCl), 1.5 mM MgCl2, 200 µM each dNTP, 2.5 U AmpliTaq Gold DNA polymerase and 10 µl genomic DNA (diluted 1 : 10 in dH2O). Cycling conditions were 10 min at 95 °C, 35 cycles of 30 s at 94 °C, 30 s at 50 °C and 60 s at 72 °C, and extension for 2 min at 72 °C. Staphylococcus aureus and Streptococcus pneumoniae genomic DNA were used as negative controls.
Eight isolates negative for speJ were retested with the primers used by Proft et al. (2001) (Table 1
). Samples in which the smeZ gene did not amplify under the above conditions were retested with 2.5 mM MgCl2 in the reaction mixture and an annealing temperature of 53 °C, using the conditions used by T. Proft (personal communication).
Statistical analysis.
Statistical pair-wise analysis was undertaken with 
2 and Fisher’s exact tests by comparing observed and expected associations.
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RESULTS AND DISCUSSION
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Prevalence of superantigen genes
The reported prevalence of superantigen genes in GAS isolates varies considerably. Laboratory technique is a possible source of false variation. Our pilot study showed diminished sensitivity for gene detection by PCR using DNA extraction by boiling compared with agitation. Allelic variation in primer-binding sites may also affect gene detection. However, there are also likely to be genuine geographical differences in superantigen prevalence, as well as differences over time.
In the present study, clearly defined, dense bands were detected for all superantigen genes, except for smeZ, which for some isolates was associated with faint bands. As expected, the putative chromosomally encoded superantigens speG and smeZ were the most prevalent, detected in 90 and 95 % of isolates, respectively. This contrasts with previous studies where these superantigens were found in 100 % of isolates using PCR-based detection (Proft et al., 2000, 2003).
Isolates in which these superantigens were not detected were found to be restricted to certain emm types. In accordance with previous studies (Murakami et al., 2002; Vlaminckx et al., 2003), this study found speG to be absent from emm4 isolates. This suggests that emm4 isolates may contain an allele with mutations in the primer-binding sites.
Polymorphisms in the primer-binding sites may also explain the variable and occasionally absent amplification in a number of isolates of the smeZ gene, which is known to be highly polymorphic (Proft et al., 2000). However, variation in the binding sites of the smeZ primers used in the present study has not been documented previously. The present study and two previous studies (Schmitz et al., 2003; Vlaminckx et al., 2003) found the absence of smeZ in emm3 isolates. A variation in binding sites in emm3 isolates would be consistent with evidence that smeZ alleles are linked to emm type (Proft et al., 2000). Interestingly, the smeZ gene and emm3 isolates have been linked independently to streptococcal toxic shock syndrome (Beres et al., 2002; Vlaminckx et al., 2003).
Despite also being putatively chromosomally encoded, speJ was found in only 51 % of isolates, consistent with some previous studies (Schmitz et al., 2003; Vlaminckx et al., 2003). However, this contrasts with the results of Proft and co-workers, who found a 100 % prevalence for speJ, suggesting this superantigen to be chromosomally encoded (Proft, 2003; Proft et al., 2003). This discrepancy was not attributable to the use of different primers, as results were unchanged in the present study when testing was repeated using the same primers as used by Proft and co-workers.
The reported prevalences of speA and speC are the most variable, perhaps attributable to the fact that they have been the most studied superantigens. The prevalence of speA in the present study was higher than that in previous Australian studies (DelVecchio et al., 2002; Norton et al., 2004). This could be due to three and two base mismatches, respectively, between the reverse primers used in these previous studies and the recently described alleles speA4 and speA5.
The prevalence of ssa in the present study was similar to that found in two other recent studies (Jing et al., 2006; Darenberg et al., 2007), but lower than the prevalence reported in earlier studies (Proft, 2003; Proft et al., 2003; Schmitz et al., 2003; Descheemaeker et al., 2000). This may be explained by the lower proportion of ssa-containing emm3 isolates. In contrast, the prevalence of the recently discovered superantigen genes speK, speL and speM was similar to that found in New Zealand (Proft et al., 2003). These three recently discovered superantigens have been associated with acute rheumatic fever (Smoot et al., 2002b; Proft et al., 2003).
The prevalence of superantigens in the present study is compared with previously published data in Table 2. A comparison of superantigen prevalence between invasive and non-invasive isolates from the present study has been published previously (Rogers et al., 2007).
Random or non-random association between superantigen genes?
To determine whether there was a non-random association between different toxin genes (due, for example, to gene co-location or emm restriction), pair-wise statistical analysis was undertaken. Some combinations of superantigen genes were detected at rates that differed to that predicted if they occurred independently (Table 3). For example, speH was detected in 18.6 % of isolates and speI in 16.8 %. The observed co-detection rate (16.8 %) was significantly higher than the expected rate (3.1 %) (P=0.0008). In contrast, the observed co-detection rate (1.9 %) for speA and speC was significantly lower than the expected rate (17.1 %) (P=0.0001).
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Table 3. Co-detection rates of superantigen genes amongst 107 GAS isolates
Bold depicts statistically significant associations.
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As found in previous studies, in the present study speL and speM were always co-detected (Igwe et al., 2003; Proft et al., 2003). This result was expected, as the genes are in adjacent positions on the same bacteriophage (
speL/M) (Smoot et al., 2002a). In contrast, the presence of speH was not always predictive of the presence of speI, despite their reported presence on the same bacteriophage (370.2) (Ferretti et al., 2001). This result confirmed the findings of previous studies (Proft et al., 2003; Ekelund et al., 2005a) and suggests that speI may be lost during integration into the genome or that there is another phage that carries only speH.
emm-type association with superantigen profiles
Twenty-six different superantigen profiles (unique combinations of superantigen genes) were present in the 107 isolates and these were distributed amongst 22 different emm types. When emm types and superantigen profiles were analysed together, the 107 isolates could be divided into 35 genetically distinguishable sets (Table 4).
In those emm types for which there were sufficient numbers of isolates represented in the study for further analysis, namely emm1, emm4, emm12, emm28 and emm75, superantigen profiles were relatively restricted. Conserved superantigen profiles in specific emm types have been reported previously (Schmitz et al., 2003; Vlaminckx et al., 2003; Darenberg et al., 2007; Luca-Harari et al., 2008).
Two previous studies (Descheemaeker et al., 2000; Vlaminckx et al., 2003) have suggested that the majority of emm1 isolates contain speA, speG, speJ and smeZ but do not possess speC, ssa and speH (profile D). This is consistent with the present study in which 27 of the 29 emm1 isolates possessed profile D. The presence of speC in emm1 isolates has also been reported as variable (Ekelund et al., 2005a). In the present study, emm1 isolates did not possess speI (as previously reported; Ekelund et al., 2005a) or speK, speL or speM (not previously been reported). The speJ gene was present in 97 % of emm1 isolates in the present study, which contrasts with the variable prevalence reported in two previous studies (Schmitz et al., 2003; Darenberg et al., 2007). emm1 isolates have been associated with streptococcal toxic shock syndrome in numerous studies (Musser et al., 1991; Vlaminckx et al., 2003).
Other emm types that were present in significant numbers also showed conserved superantigen profiles (Table 5), including a number of novel associations in emm types previously thought not to be associated with smeJ and smeZ. All but one of the ten emm4 isolates possessed profile B. Of the 17 emm12 isolates, 14 possessed superantigen profile H. Although the remaining three isolates each had a different profile, it was noteworthy that 16 of the 17 isolates possessed speG, speH, speI and smeZ. All but two of the 14 emm28 isolates possessed superantigen profile C. The profile of the remaining two isolates differed only by the addition of speK.
In comparison, emm75 appeared to have a greater diversity in that only four of the eight emm75 isolates shared a superantigen profile. However, the superantigens in this profile (speG, speL, speM and smeZ) were present in three of the remaining four emm75 isolates. Superantigens commonly associated with emm75 isolates have not been reported previously.
Why are superantigen profiles restricted amongst different emm types?
Conserved superantigen profiles for different emm types, especially the presence or absence of bacteriophage-encoded superantigens, support the contention that surface M proteins selectively influence the entry of bacteriophages, including those encoding superantigens (Mylvaganam et al., 2000). For example, speA or speJ were found together with speH or speI in the same isolate less frequently than expected. This suggests that the characteristics of the M protein in isolates containing these genes allow entry of mobile elements containing speA and speJ and prevent entry of bacteriophages containing speH and speI.
Conserved superantigen profiles could be an evolutionary event. For example, bacteriophages encoding superantigens may have become associated with specific emm types at the time of emm-type differentiation. However, a number of factors argue against this. Strains before the mid-1980s have tested negative for speK (Beres et al., 2002; Ikebe et al., 2002). This superantigen gene was found in all emm3 isolates in the present study, consistent with the finding that most contemporary emm3 isolates carry speK (Beres et al., 2002; Ikebe et al., 2002) and confirming the dissemination of the speK-carrying phage in emm3 strains in Melbourne. Furthermore, one study showed a progressive change in the proportion of emm1 isolates carrying speA over a 4-year period (Ekelund et al., 2005b), suggesting that variation in prevalence amongst isolates may occur over a relatively short time.
Variation in emm28 superantigen profiles from different geographical locations suggests that the presence of a superantigen in a given emm type may be influenced by more than just M protein. This variation, in association with the dissemination of speK in the early 1980s, suggests that the properties of the M protein could change with time. This could be due to the acquisition of other bacteriophages encoding surface proteins, minor nucleotide variations in the M protein itself or changes in the bacteriophage to increase its infective capacity or host range.
Previous studies suggest that there is geographical variation in the association between superantigen gene possession and emm type (Table 2), although temporal changes may provide an alternative explanation (Ikebe et al., 2002). This is illustrated by the fact that, in contrast to the results of the present study, speL and speM were found in emm4, emm22, emm28 and 73 % of emm89 isolates, and speK in emm89 isolates, from New Zealand (Proft et al., 2003).
This study provides detailed data about the prevalence of and relationships between superantigens, and their association with specific emm types. The importance and relevance of these relationships needs further investigation by clarifying their role in the outcome of infection with GAS.
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ACKNOWLEDGEMENTS
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We gratefully acknowledge laboratory assistance provided by Gowri Selvaraj and Andrea Bigham. This work was funded by grants from the National Heart Foundation of Australia and the National Health and Medical Research Council, Australia.
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