Skip to main content
  • Research article
  • Open access
  • Published:

Relationships between emm and multilocus sequence types within a global collection of Streptococcus pyogenes

Abstract

Background

The M type-specific surface protein antigens encoded by the 5' end of emm genes are targets of protective host immunity and attractive vaccine candidates against infection by Streptococcus pyogenes, a global human pathogen. A history of genetic change in emm was evaluated for a worldwide collection of > 500 S. pyogenes isolates that were defined for genetic background by multilocus sequence typing of housekeeping genes.

Results

Organisms were categorized by genotypes that roughly correspond to throat specialists, skin specialists, and generalists often recovered from infections at either tissue site. Recovery of distant clones sharing the same emm type was ~4-fold higher for skin specialists and generalists, as compared to throat specialists. Importantly, emm type was often a poor marker for clone. Recovery of clones that underwent recombinational replacement with a new emm type was most evident for the throat and skin specialists. The average ratio of nonsynonymous substitutions per nonsynonymous site (Ka) and synonymous substitutions per synonymous site (Ks) was 4.9, 1.5 and 1.3 for emm types of the throat specialist, skin specialist and generalist groups, respectively.

Conclusion

Data indicate that the relationships between emm type and genetic background differ among the three host tissue-related groups, and that the selection pressures acting on emm appear to be strongest for the throat specialists. Since positive selection is likely due in part to a protective host immune response, the findings may have important implications for vaccine design and vaccination strategies.

Background

A molecular arms race between pathogen and host often emerges when an immune response favors the selection of microorganisms displaying altered antigens on their surface. The mechanisms by which bacterial pathogens undergo immune escape include point mutation and replacement of antigen genes by homologous recombination following horizontal transfer of DNA between organisms of different strains. Genetic change provides the raw material upon which natural selection acts, and a host immune response is one of the strongest selection pressures a microbial pathogen will encounter.

The fibrillar M protein molecule present on the surface of Streptococcus pyogenes, a bacterial pathogen afflicting humans throughout the world, is often the target of a protective immune response mounted during infection [1–3]. M protein is an essential virulence factor and provides the basis for serotype [4]. A more recent typing scheme based on the nucleotide (nt) sequence at the 5' end of the emm gene, encoding the distal fibril tip, closely parallels serologic findings and has led to the identification of ~160 emm types [5]. Importantly, strong protective immunity to S. pyogenes infection is often M type-specific.

S. pyogenes strains can be divided into 3 major groups that roughly correspond to preferred tissue site for infection. Historically, it has been recognized that certain M types are strongly associated with cases of pharyngitis, whereas other M types are more often recovered from superficial skin infections (i.e., impetigo) [6–8]. Genotypic markers – known as emm patterns – are based on the phylogeny of the 3' end of emm genes, encoding the semi-conserved cell wall-spanning domain of M protein [9, 10]. Of biological relevance is the finding that emm pattern genotypes display strong associations with strains causing superficial infections at the throat or skin, whereby emm pattern A-C strains tend to cause pharyngitis (referred to as throat specialists), pattern D strains tend to cause impetigo (skin specialists), and pattern E strains as a group are often found in association with infections at both tissues (generalists) [11, 12]. Data from 11 population-based surveillance studies on streptococcal pharyngitis and/or impetigo, spanning all 6 major continents, provide strong support for these biological groupings (Table 1), even though tissue associations are not strict and occasionally deviate, particularly in communities having high rates of both pharyngitis and impetigo [11, 13–20]. Also, organisms found in association with throat carriage (versus infection) may not correlate as well with the emm pattern groupings [11, 17].

Table 1 Population-based surveillance of S. pyogenes.*

Extensive analysis of multilocus sequence typing (MLST) data for S. pyogenes strains assigned to the emm pattern-defined groups indicates that there is a history of ample flow of housekeeping genes between the 3 groups [21, 22]. Furthermore, there is a lack of housekeeping gene sequence clustering among isolates derived from patients known to have throat versus skin infection. The emm pattern-defined groups do not appear to represent deep ancestral lineages of S. pyogenes, based either on concatenated housekeeping gene trees or individual housekeeping gene tree topologies. Yet, for > 98% of emm types, all isolates examined that share an emm type are assigned to the same emm pattern group [23], suggesting that a given emm type is largely restricted to a single emm pattern group; this finding was recently validated in another study [20]. Experiments in which emm genes are swapped between strains of different emm pattern groups show that M protein function depends on interactions with other cell factors [24, 25]. Several emm pattern-linked traits encoded by physically distant loci have been identified and they may work in concert to play a critical role in adaptation of the organism to different ecological niches [26–29].

S. pyogenes is responsible for a large global burden of disease, and development of a preventative vaccine is a high priority [3, 30, 31]. The strong protective immunity elicited by M type-specific epitopes has led to efforts to develop an M type-based vaccine. In this report, genetic changes in emm type – due to mutation and/or recombination – are evaluated for strains defined for tissue site preference of infection.

Results

Characteristics of the strain sample set

Infection type is defined by a clear set of clinical criteria in each of the population-based surveillance studies listed in Table 1, upon which the strength of the association between infection type (pharyngitis, impetigo) and emm pattern genotype rests. However, in order to rigorously address the relationship between emm and genetic background on a global scale, a genetically diverse set of organisms spanning a wide time frame and geographic space is required instead. A genetically diverse set of S. pyogenes strains, isolated from > 25 countries throughout the world, was assembled for analysis (see Additional file 1).

Nucleotide (nt) sequence data was obtained for 7 housekeeping loci, providing ST assignments, and for the type-specific region of emm positioned at the 5' end of the locus, providing emm type and emm allele assignments. MLST and emm typing data was previously reported for 493 of the isolates under study [23, 32, 33]; an additional 89 isolates were included based on their large geographic distance relative to isolates sharing that emm type; emm alleles were determined for the majority of isolates (see Additional file 1).

The complete data set contains 582 isolates represented by 259 sequence types (STs) and 156 emm types (Table 2). ST is used as a marker to distinguish among isolates with different genetic backgrounds. Approximately 97% of the known emm types of S. pyogenes [34] are included in the sample set. Nearly all of the isolates (577 of 582) can be assigned to one of the 3 emm pattern groups [23], a genotype that corresponds well to throat specialists (pattern A-C; N = 156), skin specialists (pattern D; N = 181) and generalists (pattern E; N = 240).

Table 2 Summary of S. pyogenes sample (sub)sets analyzed in this study.

Each emm pattern-defined group is highly diverse, as evidenced by Simpson's diversity index (D) values of 0.950, 0.985 and 0.990 for emm pattern A-C, D and E isolates, respectively. For the D value calculation, S. pyogenes clones are defined by their combination of emm type and ST. A D value equal to one signifies that the genotyping method distinguishes between all isolates, whereas a D value equal to zero means that all isolates are the same clone.

In summary, the strain sample set is characterized as being both comprehensive, including representatives of most known genotypes, and highly diverse, containing relatively few isolates that represent identical clones.

Diversifying selection in emm alleles

Because the M type-specific region is a target of a protective immune response by the human host [1, 2], nt substitutions at nonsynonymous sites and insertions or deletions (indels) have the potential to modify the antigenic structure of the surface protein and render an immune response ineffective.

Alignment of nt sequences assigned to the same emm type were generated by Clustal W. Fifty-one of the 582 isolates corresponded to emm types that are unique to a single isolate and were not aligned (see Additional file 1). Two or more isolates were sampled for 105 emm types (see Additional file 2). The 105 separate alignments include emm sequences from 520 isolates (11 of the 531 sequences were not assessed), represented by 189 distinct emm alleles. For each of the 105 Clustal W alignments of emm type, whereby each alignment contains 150 nt sites and at least 2 emm sequences, Ka and Ks values were calculated. Nonsynonymous substitutions (measured by Ka) result in an amino acid change, whereas synonymous substitutions (measured by Ks) are silent. Since 58 of the 105 emm type alignments were devoid of nt polymorphisms, the average mean of the Ka and Ks values for all 105 alignments was determined. The ratio of the average mean Ka value to the average mean Ks value was 1.96 (Table 3), indicative of positive diversifying selection acting on the type-specific region of emm genes.

Table 3 Synonymous and nonsynonymous nucleotide substitutions within the emm type region (150 nt), based on Clustal W alignments corresponding to each emm type

The impact of diversifying selection was next examined for emm types in accordance with emm pattern group. The average mean of the Ka and Ks values was calculated and yielded a Ka to Ks ratio of 4.92 for the pattern A-C subset of emm types, but only 1.53 and 1.26 for the patterns D and E groups, respectively (Table 3). Pair wise comparisons between raw Ka and Ks values were significantly different for the pattern A-C emm types (t < 0.01, paired t-test, 2 tailed), but not for the pattern D or E emm types (see Additional file 2). Also, the raw Ka values were significantly different for pattern A-C emm types versus either pattern D or E emm types (t < 0.05, unpaired t-test, 2-tailed); no significant differences were found for the Ks values.

The average mean Ka to Ks ratio for pattern A-C emm types exceeded the values observed for pattern D and E emm types by 3- to 4-fold. The data provide evidence that diversifying selection is strongest for pattern A-C emm types. Because the emm types associated with S. pyogenes are largely restricted to this bacterial species [35, 36], the observed genetic changes most likely originated as mutations within the S. pyogenes population, rather than having arisen by lateral transfer from another species.

Small indels within the emm type-specific region can lead to alterations in the phenotypic surface expression of M protein. Only 2 isolates belonging to the complete data set – both of which are pattern A-C and not included among the 520 isolates comprising the emm gene sequence alignments – have indels within the emm type region that lead to frame shift mutations and premature termination of the translated M protein products (see Additional file 2). Conceivably, loss of M protein via a frame shift may be a strategy for immune escape that is largely restricted to pattern A-C strains however, the number of events is too small to draw conclusions.

In-frame indels are observed in 11 (10%) of the 105 emm type alignments. Four and 7 pattern D and E emm type alignments, respectively, each contain 1 allele having an indel (see Additional file 2). Indels most likely arise via slipped strand mispairing during DNA replication or homologous recombination resulting in an unequal crossover. Thus, epitope loss or gain mediated via small indels may constitute a strategy used by a small proportion of pattern D and E strains to alter antigenic structure and evade a specific immune response.

Recombination involving emm type: STs associated with multiple emm types

An important strategy for escape from a protective host immune response directed towards M protein is the recombinational replacement of an emm type following a horizontal gene transfer (HGT) event, whereby the donor and recipient strains differ in emm type. In general terms, interstrain gene exchange is favored by close physical proximity between the donor and recipient cells, which may occur during a co-infection taking place at either the throat or skin [6, 37].

Of the 259 STs identified in the set of 582 isolates, 14 STs were recovered in association with > 1 emm type; they are referred to as emm-variable STs. Although the 14 emm-variable STs account for only a small percentage of the total STs (5.4%), > 20% of the emm types (N = 35) were found in association with these few STs (Table 4). The emm pattern D subset had the greatest number of emm-variable STs (N = 9), which collectively, were recovered in association with 23 different emm types. Only 6.6% of the emm types assigned to pattern E were present among emm-variable STs. The number of recombinational replacements of emm type per ST was at least 7- to 9-fold higher for the patterns A-C and D subsets (0.119 and 0.154, respectively) as compared to pattern E strains (0.016) (Table 4). The data are consistent with the idea that substitution with a completely new emm type may be an important adaptive strategy for the specialist strains, and less critical for the generalists.

Table 4 Recombinational replacement of emm with a new emm type.

Recombination involving emm type: emm types on distant STs

The same emm type present on STs differing at ≥ 5 housekeeping alleles can arise from HGT of emm to a distant ST or alternatively, by genetic changes in most housekeeping loci in the absence of a corresponding shift in emm type assignment. Recovery of emm types on STs of intermediate genetic distances, or the lack thereof, can help to distinguish between horizontal movement of an emm type to a distant ST versus genetic diversification at most housekeeping loci.

The 105 emm types having ≥ 2 isolates represented, and together comprising a sample set of 531 isolates, were examined for the distribution of emm type among different STs (Figure 1A). Twenty-four emm types were exclusively associated with a single ST. Another 17 emm types were associated with multiple STs that were assigned to the same clonal complex (CC), defined as sharing ≥ 5 of the 7 housekeeping alleles. Thus, for these 41 emm types, there is no evidence for HGT of emm type to a distant genetic background.

Figure 1
figure 1

Differences in the number of housekeeping alleles between isolates sharing an emm type. The y-axis shows the numbers of emm types represented by are each category, as defined by the x-axis. (A), The minimum number of differences in housekeeping alleles between isolates sharing an emm type are: zero (singleton STs), one or two (1 CC with multiple STs), three or four (multiple CCs with STs of intermediate distance), and five (multiple CCs whereby all STs are distant; represents HGT). (B), Distribution of the maximum number of differences in housekeeping alleles between isolates sharing an emm type. Clonal complex (CC) is defined by STs sharing at least 5 of 7 housekeeping alleles

In contrast to the above findings, 52 emm types were found in association with distant STs and ≥ 2 CCs (Figure 1A), whereby ≥ 5 housekeeping allele differences are evident for all possible pair wise comparisons of the STs assigned to different CCs. Distant STs are defined as those differing at ≥ 5 housekeeping alleles. Since intermediate MLST genotypes could not be identified among organisms sharing these 52 emm types, it is reasonably argued that they likely arose via horizontal transfer of an emm type to a distant ST in a single genetic step, rather than by diversification of an ST at ≥ 5 housekeeping loci involving ≥ 5 independent genetic steps.

Among the 52 emm types associated with distant STs, 63 HGT events to distant STs could be distinguished (Table 5). About half (51%) of the HGT events involved identical emm alleles and therefore, may have occurred within the relatively recent evolutionary past.

Table 5 emm types associated with distant STs.

Only 12 emm types are associated with STs that were assigned to 2 CCs that differ in as few as 3 or 4 housekeeping alleles (i.e., STs of an intermediate genetic distance) (Figure 1A). Therefore, the majority of emm types were either restricted to a single CC (39%) or associated only with distant STs (50%), with relatively few emm types falling into the intermediate category (11%). This finding is further underscored by the distribution of the maximum number of differences in the 7 housekeeping alleles between STs sharing an emm type (Figure 1B). Thirty-six (34%) of the emm types are associated with STs having 0 or 1 housekeeping gene difference, whereas 47 (45%) emm types are associated with STs having 6 or 7 housekeeping gene differences. Only 21% of the emm types lie in between these two extremes, with 2, 3, 4 or 5 housekeeping gene differences.

The horizontal transfer of an emm type to a distant ST was compared for the 3 emm pattern-defined groups of isolates (Table 5). Only 3 of the 18 pattern A-C emm types (17%) were recovered in association with distant STs. In contrast, 48 and 61% of the pattern D and E emm types, respectively, displayed evidence for HGT. Eight emm types – 4 each from the pattern D and E groups – were found in association with ≥ 3 distant STs of distinct CCs, indicative of multiple HGT events involving these emm types. The average mean number of HGT events per emm type is calculated as 0.17, 0.60 and 0.75 for the pattern A-C, D and E groups, respectively.

Using a 2 × 2 test for independence (Fisher's exact, 2-tailed) with Table 5 data, the difference between the emm pattern A-C and D strain groups in terms of the number of emm types having undergone HGT, versus restriction to a single ST or CC, is significant (p = 0.015). The difference is highly significant between the emm pattern A-C and E strain groups (p < 0.005), but not significant when pattern D and E strains are compared.

The strikingly higher number of HGT events uncovered for pattern D and E emm types is not likely a consequence of sampling bias (Table 5). If there was sampling bias, it is expected that more extensive sampling of a given emm type would result in an increase in the number of HGT events detected simply by chance. Yet, the mean average number of isolates sampled per emm type was highest for pattern A-C strains, the group which displayed the fewest HGT events. The average number of countries sampled per emm type was slightly higher for the pattern A-C group as compared to the other subsets (Table 5) and therefore, geographic distance does not appear to explain the higher levels of HGT observed for the pattern D and E groups. For emm types with no HGT detected, irrespective of emm pattern group, an average of 2.25 (s.d., 1.24) countries were sampled per emm type, whereas for emm types displaying ≥ 1 HGT event, a similar average of 2.38 (s.d., 0.796) countries were sampled per emm type (data not shown).

The findings on HGT of emm indicate that recovery of the same emm type on distant STs is most probable for isolates belonging to the skin specialist (pattern D) and generalist (pattern E) groups. Importantly, the data also show that emm type can be a poor marker for clone (i.e., ST) or clonal complex (CC). The higher prevalence of HGT of emm type among the pattern D and E groups may be a consequence of higher intrinsic recombination rates, different selection pressures and/or a combination of both genetic change and selection effects.

Estimate of relative levels of recombination based on housekeeping genes

Recovery of STs associated with multiple emm types was highest for throat specialists (Table 4), whereas recovery of emm types associated with multiple STs was highest for generalists (Table 5). Estimates of the relative levels of recombination among housekeeping genes for the emm pattern-defined groups might help to discern between the contribution of genetic change and the effects of selection. The Ka to Ks ratios for each of the 7 housekeeping genes is less than one (range, 0.033 to 0.493; data not shown), and lower than the values obtained for emm type sequences (Table 3).

eBURST is a clustering algorithm that has been widely used to assess the mechanisms of genetic change within a bacterial population, based on MLST of housekeeping genes [38]. An estimate of the number of recombination versus mutations events can be made based on the nature of the nt differences between the mismatched allele of a single locus variant (SLV) pair. In a previous report on S. pyogenes, ≥ 28 of the 48 SLVs were attributed to recombination in accordance with a count-based method that was conservative for scoring recombination [23]. In this larger strain sample set of 582 isolates, the same approach used in the prior study was applied; 56 SLVs were detected by eBURST, wherein ≥ 33 genetic changes were estimated to have arisen following recombination (Table 6).

Table 6 Estimate for the minimum number of recombinational events involving housekeeping genes.

The minimum number of recombination events per housekeeping locus per ST was calculated as 0.007, 0.025 and 0.017 for emm pattern groups A-C, D and E, respectively (Table 6). These findings are in agreement with the general trend from previous estimates of recombination based on the congruency of housekeeping gene tree topologies [22], whereby emm pattern A-C strains showed the highest level of congruence and thereby, the lowest relative level of recombination, and emm pattern D strains displayed the lowest level of congruence and highest recombination [22]. Thus, based on the conservative estimates derived from SLV pairs, the relative rate of intrinsic recombination appears to be lower for the emm pattern-defined throat specialist group.

Recombination involving emm type: Recovery of donors and recipients

emm-variable STs represent new clones and their progenitors (i.e., the recipient), whereas distant STs sharing an emm type represent new clones and their donors. The recovery of all 3 genotypes involved in the HGT event – donor, recipient, and new clone – from the extant S. pyogenes population was evaluated, based on the findings presented in Tables 3 and 4.

For the 8 pattern A-C emm types associated with 3 emm-variable STs, it is expected that 5 of the 8 emm types will have originated from donor strains; however, only 1 donor emm type (emm14) was recovered on a distant ST (20%). In contrast, of the 4 pattern E emm types associated with 2 emm-variable STs, potential donor strains were recovered for both HGT events (100%). Among pattern D strains, 23 emm types were recovered in association with 9 emm-variable STs (Table 4) and therefore, 14 donor emm types are to be expected; 9 emm types (64% of the total possible) were found on distant STs representing putative donors (see Additional file 1).

The number of recipient STs recovered from the natural population can also be evaluated for distant ST pairs that share the same emm type. Only 3 distant ST pairs sharing the same pattern A-C emm type were found among the collection of 582 strains, and a potential recipient ST was recovered for one of them (33%). A similar proportion (38%) of recipient STs were recovered for the 24 HGT events involving pattern D strains. In contrast, very few (2 of 33; 6%) recipient STs matching the pair of the pattern E emm donor and new clone were found.

The findings show that the proportion of STs associated with multiple emm types was highest for throat specialists, whereas the proportion of emm types associated with distant STs was highest for generalists. Although some of the numbers are quite small, the data reveal a trend whereby relatively fewer genotypes corresponding to pattern A-C donor and pattern E recipient strains were recovered by sampling than were expected.

Discussion

S. pyogenes strains that are grouped according emm pattern genotype share a predilection for causing infection at particular tissue sites (Table 1). Analysis of emm pattern-defined strains for relationships between emm type and housekeeping genes reveals that the throat infection specialist group is distinct from the skin infection specialists and generalists in several key characteristics. This trend provides support for the idea that strains which tend to cause pharyngitis display different evolutionary dynamics.

Despite the possibility of underestimating positive selection using Ka to Ks ratios, the average ratios of Ka to Ks > 1 observed for emm type sequences of each emm pattern group provide evidence for positive diversifying selection. A likely source of the selection pressure is host immunity, whereby amino acid changes within the M type-specific region lead to alterations in antigenic structure that allow mutants to escape the protective immune response in at least some hosts. An important mechanism underlying protective immunity against S. pyogenes infection involves M type-specific antibodies that mediate opsonization and thereby, overcome the antiphagocytic property of M protein [1].

There is direct experimental evidence in support of amino acid changes in the M type-specific region that allow for immune escape [39–41]; this mechanism may even explain the recent emergence of an important M3 type clone. However, other findings that measure opsonophagocytosis with hyperimmune rabbit sera raised to the polypeptide product of one emm allele show high levels of bactericidal activity for strains of numerous emm alleles of the same emm type, arguing against the likelihood of immune escape mutants emerging in a vaccinated population [42]. Combined with the population genetics findings of this report, there is support for the notion that in a naturally infected human host population, the immune selection pressures on emm type may be somewhat lower in intensity and/or the immune response more variable in specificity, as compared to what can potentially be achieved in a vaccinated population.

The average ratio of Ka to Ks is ~3- to 4-fold higher for the emm pattern A-C group of emm types, suggesting that the throat specialists may be subject to stronger positive selection pressures. Several experimental findings may help to explain the lower Ka to Ks ratios observed for the skin specialists and generalists. A serological typing scheme based on the serum opacity reaction was developed as an alternative to M serotyping in order to circumvent difficulties encountered in determining the M type for many emm pattern E strains [10, 43, 44]; perhaps emm pattern E strains are more difficult to M type because of weaker immunogenicity and/or higher cross-reactivity. Hyperimmune rabbit antiserum raised to M type-specific antigens show that more pattern A-C emm types consistently elicit antiserum having a strong bactericidal effect, as compared to pattern E emm types [45]. The M type-specific regions of most S. pyogenes strains bind the complement regulator C4b-binding protein (C4BP). C4BP binding is achieved in the absence of a shared amino acid sequence motif and even though substitutions can introduce antigenic change without altering C4BP binding activity [46], it is conceivable that there are some functional constraints on sequence variation. Of probable relevance is the finding that most isolates lacking C4BP binding activity have emm types characteristic of pattern A-C strains (egs., M types 1, 3, 5, 6, 12, 19, 24, 26, 30, 39) [23, 46], suggestive of higher levels of purifying (negative) selection on pattern D and E emm types in order to maintain C4BP binding activity.

For emm variable STs, whereby ST is defined by 7 housekeeping alleles, emm type can be regarded as an 8th locus, wherein the related organisms are SLVs that arose via recombinational replacement of emm. Throat specialists appear to have the lowest level of recombination involving housekeeping genes, yet they display a relatively high number of recombinational replacements of emm type per ST. The observed imbalance in the relative levels of recombination affecting housekeeping genes versus emm genes supports the likely conclusion that pattern A-C emm types are subject to stronger positive selection. Thus, for throat specialists, immune escape mutants – arising by either mutation or recombination – appear to have a strong selective advantage, as compared to skin specialists and generalists.

The disproportionately low recovery rate for pattern A-C donor and pattern E recipient genotypes might be explained by several factors, which include competition between the new clone and the donor or recipient genotype as mediated through host or herd immunity [47]. For eg., if a new pattern A-C clone has a higher transmission rate than the donor genotype with which it shares an emm type, it may outcompete the donor by reducing the number of susceptible (i.e., nonimmune) hosts available to it. Likewise, if non-emm genes are critical for protective immunity against pattern E strains, the new clone may outcompete its progenitor if it has a higher transmission rate. Conceivably, immune-mediated competition may reduce the transmission success of the less fit organism to the point that it becomes rare and much more difficult to recover through sampling.

A previous study showed that many of the associations between emm type and ST differed for isolates collected from a remote Australian community, when compared to strains recovered from the United States and Europe [32]. The present study includes representatives of most known emm types, and demonstrates that emm type is a reasonably good marker for ST or CC among the throat specialist strains, but a rather poor marker for clone among skin specialists and generalists. This finding underscores the importance of multilocus typing methods for defining S. pyogenes strains [33].

The strong positive selection pressures acting on emm types of throat specialists are consistent with their role as targets of strongly protective immunity. Thus, an M type-based vaccine directed against pattern A-C strains is expected to have high efficacy, provided that the avenues for immune escape are blocked. This might be achieved by including numerous emm types in the vaccine formulation to help prevent the spread of new clones arising by recombinational replacement of emm type, and by eliciting a polyspecific immune response that protects against the spectrum of emm allelic variants that arise by mutation. Since only a small fraction of STs are associated with multiple emm types, targeting non-emm gene products of those STs, whereby the non-emm products elicit (partial) protection, could provide a sound complementary strategy.

The relatively weaker positive selection observed for emm types of pattern D and E strains is consistent with the possibility that they may be less ideal candidates for an M type-based vaccine. The pattern E generalists comprise about half of all known emm types [23], and account for ~40 to 50% of isolates collected in population-based surveys in many parts of the world (Table 1). An efficacious vaccine targeting this highly prevalent group of S. pyogenes organisms may require additional antigens in its formulation.

Conclusion

This study provides a comprehensive population analysis of strains representing nearly all emm types; for the majority of emm types, multiple isolates recovered from distant geographic locations were studied. The relationships between emm type and genetic background differ among the 3 groups of emm pattern-defined genotypes which roughly correspond to host tissue site preferences for infection. Furthermore, the selection pressures acting on emm appear to be strongest for the throat specialists. Since a protective host immune response is probably a key factor driving positive selection, the findings provide important new insights that may aid in vaccine design and vaccination strategies.

Methods

Bacterial strains

Nearly all (493 of 495) isolates of S. pyogenes that were previously described in [23] are also included in this study. In an effort to expand the number of isolates sharing an emm type with isolates of the previous data set, and also having been recovered from a distant geographic location, 89 bacterial isolates representing 65 different emm types were added to the analysis, comprising the complete sample set of 582 isolates (see Additional file 1).

emm based genotypes

emm type and emm allele were established by nucleotide (nt) sequence determination of PCR amplicons [5]. emm type is a character state whereby a unique emm type is defined as sharing < 92% sequence identity over the first 90 bases encoding the deduced processed M protein of the type reference strain, allowing for small indels [34]; emm type strongly correlates with M protein serotype, as previously established by immunoreactivity with typing sera [5, 48]. emm allele assignments are based on the first 150 nt corresponding to the 5' end region encoding the mature M protein molecule [34]; each allele has a unique sequence, and is equivalent to "emm subtype." emm pattern was ascertained by PCR-based mapping, or was inferred based on emm type, as described [23].

MLST

Internal fragments of 7 housekeeping genes (gki, gtr, murI, mutS, recP, xpt, yqiL) were amplified by PCR and the nt sequence determined using primers and conditions described previously [33]. For each locus, distinct allele numbers were assigned to each unique sequence, generating a seven integer allelic profile for each isolate; isolates with identical allelic profiles were assigned to the same ST. A complete database of alleles, allele sequences and STs is maintained on the internet [49]. A total of 38 new STs are reported.

Computations

Simpson's diversity index (D) was calculated as described [50]. The 150 nt emm type-specific sequence derived from > 2 isolates assigned to the same emm type were aligned using the Clustal W algorithm, implemented in Megalign (DNASTAR, Lasergene, Inc.). The number of nonsynonymous substitutions per nonsynonymous site (Ka) and the number of synonymous substitutions per synonymous site (Ks) were calculated for each Clustal W alignment, using DnaSP version 4.10 [51]. The eBURST clustering algorithm [38] for analyzing relationships between STs was applied using software available at http://eburst.mlst.net. Single locus variants (SLVs) were identified with a user-defined setting of 6 of 7 shared housekeeping alleles. The method for estimating recombination events was previously described [23].

References

  1. Lancefield RC: Current knowledge of the type specific M antigens of group A streptococci. J Immunol 1962, 89:307-313.

    CAS  PubMed  Google Scholar 

  2. Beachey EH, Seyer JM, Dale JB, Simpson WA, Kang AH: Type-specific protective immunity evoked by synthetic peptide of Streptococcus pyogenes M protein . Nature 1981, 292:457-459. 10.1038/292457a0

    Article  CAS  PubMed  Google Scholar 

  3. Bisno AL, Rubin FA, Cleary PP, Dale JB: Prospects for a group a streptococcal vaccine: rationale, feasibility, and obstacles – report of a National Institute of Allergy and Infectious Diseases workshop. Clinical Infectious Diseases 2005,41(8):1150-1156. 10.1086/444505

    Article  CAS  PubMed  Google Scholar 

  4. Cunningham MW: Pathogenesis of group A streptococcal infections [review]. Clinical Microbiology Reviews 2000,13(3):470. 10.1128/CMR.13.3.470-511.2000

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Beall B, Facklam R, Thompson T: Sequencing emm -specific PCR products for routine and accurate typing of group A streptococci. J Clin Microbiol 1996, 34:953-958.

    PubMed Central  CAS  PubMed  Google Scholar 

  6. Bisno AL, Stevens D: Streptococcus pyogenes (Including streptococcal toxic shock syndrome and necrotizing fasciitis). In Principles and Practice of Infectious Diseases. Volume 2. 5th edition. Edited by: Mandell GL, Douglas RG, Dolin R. Philadelphia: Churchill Livingstone; 2000:2101-2117.

    Google Scholar 

  7. Carapetis J, Currie B, Kaplan E: Epidemiology and prevention of group A streptococcal infections: Acute respiratory tract infections, skin infections, and their sequelae at the close of the twentieth century. Clin Infect Dis 1999, 28:205-210. 10.1086/515114

    Article  CAS  PubMed  Google Scholar 

  8. Wannamaker LW: Differences between streptococcal infections of the throat and of the skin. N Engl J Med 1970, 282:23-31.

    Article  CAS  PubMed  Google Scholar 

  9. Hollingshead SK, Readdy T, Arnold J, Bessen DE: Molecular evolution of a multi-gene family in group A streptococci. Mol Biol Evol 1994, 11:208-219.

    CAS  PubMed  Google Scholar 

  10. Hollingshead SK, Readdy TL, Yung DL, Bessen DE: Structural heterogeneity of the emm gene cluster in group A streptococci. Mol Microbiol 1993, 8:707-717. 10.1111/j.1365-2958.1993.tb01614.x

    Article  CAS  PubMed  Google Scholar 

  11. Bessen DE, Carapetis JR, Beall B, Katz R, Hibble M, Currie BJ, Collingridge T, Izzo MW, Scaramuzzino DA, Sriprakash KS: Contrasting molecular epidemiology of group A streptococci causing tropical and non-tropical infections of the skin and throat. J Infect Dis 2000, 182:1109-1116. 10.1086/315842

    Article  CAS  PubMed  Google Scholar 

  12. Bessen DE, Sotir CM, Readdy TL, Hollingshead SK: Genetic correlates of throat and skin isolates of group A streptococci. J Infect Dis 1996, 173:896-900.

    Article  CAS  PubMed  Google Scholar 

  13. Dicuonzo G, Gherardi G, Lorino G, Angeletti S, DeCesaris M, Fiscarelli E, Bessen DE, Beall B: Group A streptococcal genotypes from pediatric throat isolates in Rome, Italy. J Clin Microbiol 2001, 39:1687-1690. 10.1128/JCM.39.5.1687-1690.2001

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  14. Alberti S, Garcia-Rey C, Dominguez MA, Aguilar L, Cercenado E, Gobernado M, Garcia-Perea A: Survey of emm gene sequences from pharyngeal Streptococcus pyogenes isolates collected in Spain and their relationship with erythromycin susceptibility. J Clin Microbiol 2003,41(6):2385-2390. 10.1128/JCM.41.6.2385-2390.2003

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Brandt CM, Spellerberg B, Honscha M, Truong ND, Hoevener B, Lutticken R: Typing of Streptococcus pyogenes strains isolated from throat infections in the region of Aachen, Germany. Infection 2001,29(3):163-165. 10.1007/s15010-001-1106-x

    Article  CAS  PubMed  Google Scholar 

  16. Espinosa LE, Li ZY, Barreto DG, Jaimes EC, Rodriguez RS, Sakota V, Facklam RR, Beall B: M protein gene type distribution among group A streptococcal clinical isolates recovered in Mexico City, Mexico, from 1991 to and Durango, Mexico, from 1998 to 1999: Overlap with type distribution within the United States. Journal of Clinical Microbiology 2000,41(1):373-378. 10.1128/JCM.41.1.373-378.2003

    Article  Google Scholar 

  17. Sakota V, Fry AM, Lietman TM, Facklam RR, Li ZY, Beall B: Genetically diverse group A streptococci from children in Far-Western Nepal share high genetic relatedness with isolates from other countries. Journal of Clinical Microbiology 2006,44(6):2160-2166. 10.1128/JCM.02456-05

    Article  PubMed Central  PubMed  Google Scholar 

  18. Shulman S: Group A streptococcal pharyngitis serotype surveillance in North America, 2000–2002. Clin Infect Dis 2004, 39:325-332. 10.1086/421949

    Article  PubMed  Google Scholar 

  19. Smeesters PR, Vergison A, Campos D, de Aguiar E, Deyi VY, Van Melderen L: Differences between Belgian and Brazilian group A streptococcus epidemiologic landscape. PLoS ONE 2006, 1:e10. 10.1371/journal.pone.0000010

    Article  PubMed Central  PubMed  Google Scholar 

  20. McDonald MI, Towers RJ, Andrews R, Benger N, Fagan P, Currie BJ, Carapetis JR: The dynamic nature of group A streptococcal epidemiology in tropical communities with high rates of rheumatic heart disease. Epidemiol Infect 2007, 1-11.

    Google Scholar 

  21. Feil EJ, Holmes EC, Bessen DE, Chan MS, Day NPJ, Enright MC, Goldstein R, Hood DW, Kalia A, Moore CE, Zhou JJ, Spratt BG: Recombination within natural populations of pathogenic bacteria: Short-term empirical estimates and long-term phylogenetic consequences. Proceedings of the National Academy of Sciences of the United States of America 2001,98(7):4276.

    Article  CAS  Google Scholar 

  22. Kalia A, Spratt BG, Enright MC, Bessen DE: Influence of recombination and niche separation on the population genetic structure of the pathogen Streptococcus pyogenes . Infect Immun 2002, 70:1971-1983. 10.1128/IAI.70.4.1971-1983.2002

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  23. McGregor KF, Spratt BG, Kalia A, Bennett A, Bilek N, Beall B, Bessen DE: Multi-locus sequence typing of Streptococcus pyogenes representing most known emm -types and distinctions among sub-population genetic structures. J Bacteriol 2004, 186:4285-4294. 10.1128/JB.186.13.4285-4294.2004

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  24. Kotarsky H, Thern A, Lindahl G, Sjobring U: Strain-specific restriction of the antiphagocytic property of group A streptococcal M proteins. Infect Immun 2000, 68:107-112.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  25. Husmann LK, Scott JR, Lindahl G, Stenberg L: Expression of the Arp protein, a member of the M protein family, is not sufficient to inhibit phagocytosis of Streptococcus pyogenes . Infect Immun 1995, 63:345-348.

    PubMed Central  CAS  PubMed  Google Scholar 

  26. Bessen DE, Izzo MW, Fiorentino TR, Caringal RM, Hollingshead SK, Beall B: Genetic linkage of exotoxin alleles and emm gene markers for tissue tropism in group A streptococci. J Infect Dis 1999, 179:627-636. 10.1086/314631

    Article  CAS  PubMed  Google Scholar 

  27. Kalia A, Bessen DE: Natural selection and evolution of streptococcal virulence genes involved in tissue-specific adaptations. J Bacteriol 2004,186(1):110-121. 10.1128/JB.186.1.110-121.2004

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. Bessen DE, Manoharan A, Luo F, Wertz JE, Robinson DA: Evolution of transcription regulatory genes is linked to niche specialization in the bacterial pathogen Streptococcus pyogenes . Journal of Bacteriology 2005,187(12):4163-4172. 10.1128/JB.187.12.4163-4172.2005

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  29. Kratovac Z, Manoharan A, Luo F, Lizano S, Bessen DE: Population genetics and linkage analysis of loci within the FCT region of Streptococcus pyogenes . J Bacteriol 2007, 189:1299-1310. 10.1128/JB.01301-06

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  30. Carapetis JR, Steer AC, Mulholland EK, Weber M: The global burden of group A streptococcal diseases [Review]. The Lancet Infectious Diseases 2005,5(11):685-694. 10.1016/S1473-3099(05)70267-X

    Article  PubMed  Google Scholar 

  31. Carapetis JR: Rheumatic heart disease in developing countries. N Engl J Med 2007,357(5):439-441. 10.1056/NEJMp078039

    Article  CAS  PubMed  Google Scholar 

  32. McGregor K, Bilek N, Bennett A, Kalia A, Beall B, Carapetis J, Currie B, Sriprakash K, Spratt B, Bessen D: Group A streptococci from a remote community have novel multilocus genotypes but share emm-types and housekeeping alleles. J Infect Dis 2004, 189:717-723. 10.1086/381452

    Article  PubMed  Google Scholar 

  33. Enright MC, Spratt BG, Kalia A, Cross JH, Bessen DE: Multilocus sequence typing of Streptococcus pyogenes and the relationship between emm -type and clone. Infect Immun 2001, 69:2416-2427. 10.1128/IAI.69.4.2416-2427.2001

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  34. Beall B: Atlanta. 2007. [http://www.cdc.gov/ncidod/biotech/strep/strepindex.htm]

    Google Scholar 

  35. Kalia A, Enright MC, Spratt BG, Bessen DE: Directional gene movement from human-pathogenic to commensal-like streptococci. Infect Immun 2001, 69:4858-4869. 10.1128/IAI.69.8.4858-4869.2001

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Davies MR, McMillan DJ, Beiko RG, Barroso V, Geffers R, Sriprakash KS, Chhatwal GS: Virulence profiling of Streptococcus dysgalactiae subspecies equisimilis isolated from infected humans reveals two distinct genetic lineages which do not segregate with their phenotypes or propensity to cause diseases. Clin Infect Dis 2007, in press.

    Google Scholar 

  37. Carapetis J, Gardiner D, Currie B, Mathews JD: Multiple strains of Streptococcus pyogenes in skin sores of Aboriginal Australians. J Clin Microbiol 1995, 33:1471-1472.

    PubMed Central  CAS  PubMed  Google Scholar 

  38. Feil EJ, Li BC, Aanensen DM, Hanage WP, Spratt BG: eBURST: Inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data. J Bacteriol 2004, 186:1518-1530. 10.1128/JB.186.5.1518-1530.2004

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  39. Jones KF, Hollingshead SK, Scott JR, Fischetti VA: Spontaneous M6 protein size mutants of group A streptococci display variation in antigenic and opsonogenic epitopes. ProcNatlAcadSciUSA 1988, 85:8271-8275.

    Article  CAS  Google Scholar 

  40. Beres SB, Richter EW, Nagiec MJ, Sumby P, Porcella SF, DeLeo FR, Musser JM: Molecular genetic anatomy of inter- and intraserotype variation in the human bacterial pathogen group A Streptococcus. Proc Natl Acad Sci USA 2006,103(18):7059-7064. 10.1073/pnas.0510279103

    Article  PubMed Central  PubMed  Google Scholar 

  41. Eriksson BKG, Villasenor-Sierra A, Norgren M, Stevens DL: Opsonization of T1M1 group A Streptococcus: Dynamics of antibody production and strain specificity. Clinical Infectious Diseases 2001,32(2):E24-E30. 10.1086/318448

    Article  CAS  PubMed  Google Scholar 

  42. Dale JB, Penfound T, Chiang EY, Long V, Shulman ST, Beall B: Multivalent group A streptococcal vaccine elicits bactericidal antibodies against variant M subtypes. Clinical & Diagnostic Laboratory Immunology 2005,12(7):833-836. 10.1128/CDLI.12.7.833-836.2005

    CAS  Google Scholar 

  43. Haanes EJ, Heath DG, Cleary PP: Architecture of the vir regulons of group A streptococci parallel opacity factor phenotype and M protein class. JBacteriol 1992, 171:4967-4976.

    Google Scholar 

  44. Johnson D, Kaplan E, Sramek J, Bicova R, Havlicek J, Havlickova H, Motlova J, Kriz P: Laboratory diagnosis of group A streptococcal infections. Geneva: World Health Organization; 1996.

    Google Scholar 

  45. Hu MC, Walls MA, Stroop SD, Reddish MA, Beall B, Dale JB: Immunogenicity of a 26-valent group A streptococcal vaccine. Infect Immun 2002,70(4):2171-2177. 10.1128/IAI.70.4.2171-2177.2002

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  46. Persson J, Beall B, Linse S, Lindahl G: Extreme sequence divergence but conserved ligand-binding specificity in Streptococcus pyogenes M protein. PLoS Pathog 2006,2(5):e47. 10.1371/journal.ppat.0020047

    Article  PubMed Central  PubMed  Google Scholar 

  47. Gupta S, Anderson R: Population structure of pathogens: the role of immune selection. Parasitol Today 1999, 15:497-501. 10.1016/S0169-4758(99)01559-8

    Article  CAS  PubMed  Google Scholar 

  48. Li ZY, Sakota V, Jackson D, Franklin AR, Beall B: Array of M protein gene subtypes in 1064 recent invasive group A streptococcus isolates recovered from the active bacterial core surveillance. J Infect Dis 2003,188(10):1587-1592. 10.1086/379050

    Article  CAS  PubMed  Google Scholar 

  49. Aanensen D: Multi locus sequence typing. 2007. [http://www.mlst.net/]

    Google Scholar 

  50. Grundmann H, Hori S, Tanner G: Determining confidence intervals when measuring genetic diversity and the discriminatory abilities of typing methods for microorganisms. Journal of Clinical Microbiology 2001,39(11):4190-4192. 10.1128/JCM.39.11.4190-4192.2001

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  51. Rozas J, Sanchez-DelBarrio JC, Messeguer X, Rozas R: DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 2003,19(18):2496-2497. 10.1093/bioinformatics/btg359

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank Bernie Beall and the many investigators worldwide who provided bacterial strains to the C.D.C., the investigators (W. Tewodros, K. Sriprakash, B. Currie, W. Kabat, S. Shulman) who provided strains to D.E.B., Brian Spratt and Jim Dale for helpful discussions, and Alicia Bennett for technical assistance. This work was supported by funding from the National Institutes of Health (GM060793, AI053826 and AI061454, to D.E.B.), the American Heart Association (Grant-in-Aid, to D.E.B.), and a Wellcome Trust Research Fellowship in Biodiversity (Grant Number 53589, to A.M.W.).

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Debra E Bessen, Karen F McGregor or Adrian M Whatmore.

Additional information

Authors' contributions

KM and AW generated and analyzed sequence data. DB conceived the study, participated in its design and coordination, generated and analyzed some sequence data, performed computational analysis, and wrote draft of the manuscript. All authors read and approved the final manuscript.

Electronic supplementary material

12866_2007_492_MOESM1_ESM.xls

Additional file 1: Characteristics of all 582 S. pyogenes isolates under study. The data provided represent numerous key characteristics of the strains under study. (XLS 158 KB)

12866_2007_492_MOESM2_ESM.xls

Additional file 2: Analysis of nt substitutions among alleles assigned to the same emm type. The data provided represent the raw data used to calculate values in Table 3. (XLS 30 KB)

Authors’ original submitted files for images

Below are the links to the authors’ original submitted files for images.

Authors’ original file for figure 1

Rights and permissions

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reprints and permissions

About this article

Cite this article

Bessen, D.E., McGregor, K.F. & Whatmore, A.M. Relationships between emm and multilocus sequence types within a global collection of Streptococcus pyogenes. BMC Microbiol 8, 59 (2008). https://doi.org/10.1186/1471-2180-8-59

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/1471-2180-8-59

Keywords