Superantigenic activity of emm3 Streptococcus pyogenes is abrogated by a conserved, naturally occurring smeZ mutation.

Streptococcus pyogenes M/emm3 strains have been epidemiologically linked with enhanced infection severity and risk of streptococcal toxic shock syndrome (STSS), a syndrome triggered by superantigenic stimulation of T cells. Comparison of S. pyogenes strains causing STSS demonstrated that emm3 strains were surprisingly less mitogenic than other emm-types (emm1, emm12, emm18, emm28, emm87, emm89) both in vitro and in vivo, indicating poor superantigenic activity. We identified a 13 bp deletion in the superantigen smeZ gene of all emm3 strains tested. The deletion led to a premature stop codon in smeZ, and was not present in other major emm-types tested. Expression of a functional non-M3-smeZ gene successfully enhanced mitogenic activity in emm3 S. pyogenes and also restored mitogenic activity to emm1 and emm89 S. pyogenes strains where the smeZ gene had been disrupted. In contrast, the M3-smeZ gene with the 13 bp deletion could not enhance or restore mitogenicity in any of these S. pyogenes strains, confirming that M3-smeZ is non-functional regardless of strain background. The mutation in M3-smeZ reduced the potential for M3 S. pyogenes to induce cytokines in human tonsil, but not during invasive infection of superantigen-sensitive mice. Notwithstanding epidemiological associations with STSS and disease severity, emm3 strains have inherently poor superantigenicity that is explained by a conserved mutation in smeZ.

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

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 approximately 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.

Detecting key structural features within highly recombined genes.

Many microorganisms exhibit high levels of intragenic recombination following horizontal gene transfer events. Furthermore, many microbial genes are subject to strong diversifying selection as part of the pathogenic process. A multiple sequence alignment is an essential starting point for many of the tools that provide fundamental insights on gene structure and evolution, such as phylogenetics; however, an accurate alignment is not always possible to attain. In this study, a new analytic approach was developed in order to better quantify the genetic organization of highly diversified genes whose alleles do not align. This BLAST-based method, denoted BLAST Miner, employs an iterative process that places short segments of highly similar sequence into discrete datasets that are designated "modules." The relative positions of modules along the length of the genes, and their frequency of occurrence, are used to identify sequence duplications, insertions, and rearrangements. Partial alleles of sof from Streptococcus pyogenes, encoding a surface protein under host immune selection, were analyzed for module content. High-frequency Modules 6 and 13 were identified and examined in depth. Nucleotide sequences corresponding to both modules contain numerous duplications and inverted repeats, whereby many codons form palindromic pairs. Combined with evidence for a strong codon usage bias, data suggest that Module 6 and 13 sequences are under selection to preserve their nucleic acid secondary structure. The concentration of overlapping tandem and inverted repeats within a small region of DNA is highly suggestive of a mechanistic role for Module 6 and 13 sequences in promoting aberrant recombination. Analysis of pbp2X alleles from Streptococcus pneumoniae, encoding cell wall enzymes that confer antibiotic resistance, supports the broad applicability of this tool in deciphering the genetic organization of highly recombined genes. BLAST Miner shares with phylogenetics the important predictive quality that leads to the generation of testable hypotheses based on sequence data.

Identity and prevalence of multilocus sequence typing-defined clones of group A streptococci within a hospital setting.

Between July and October 2003, 121 clinical isolates of group A streptococci (GAS) were collected from a London hospital and characterized by multilocus sequence typing (MLST) to determine the identity and prevalence of clones circulating within this setting. A total of 39 sequence types (ST), of which 20 were represented by a single isolate, were identified. The eight most prevalent clones among the 121 GAS were ST117/emm81 (16%), ST39/emm4 (9%), ST62/emm87 (7%), ST28/emm1 (6%), ST36/emm12 (6%), ST46/emm22 (5%), ST334/emm82 (5%), and ST101/emm89 (4%). Compared to those in the MLST database (http://spyogenes.mlst.net), 12 (31%) of the 39 STs had not been previously identified, although 7 of these differed from recognized STs at only a single locus, suggesting they were closely related to previously recognized strains. Resistance to erythromycin and tetracycline was seen in 7 and 20% of isolates, respectively, with four isolates resistant to both agents. GAS strains with higher (>80) emm types accounted for 45% of GAS isolates collected during this study. Continuing GAS surveillance, using easily comparable methods, is important for detecting changes in the character of disease-causing isolates.

Multilocus sequence typing of Streptococcus pyogenes representing most known emm types and distinctions among subpopulation genetic structures.

A long-term goal is to characterize the full range of genetic diversity within Streptococcus pyogenes as it exists in the world today. Since the emm locus is subject to strong diversifying selection, emm type was used as a guide for identifying a genetically diverse set of strains. This report contains a description of multilocus sequence typing based on seven housekeeping loci for 495 isolates representing 158 emm types, yielding 238 unique combinations of sequence type and emm type. A genotypic marker for tissue site preference (emm pattern) revealed that only 17% of the emm types displayed the marker representing strong preference for infection at the throat and that 39% of emm types had the marker for skin tropism, whereas 41% of emm types harbored the marker for no obvious tissue site preference. As a group, the emm types bearing the emm pattern marker indicative of no obvious tissue site preference were far less likely to have two distinct emm types associated with the same sequence type than either of the two subpopulations having markers for strong tissue tropisms (P < 0.002). In addition, all genetic diversification events clearly ascribed to a recombinational mechanism involved strains of only two of the emm pattern-defined subpopulations, those representing skin specialists and generalists. The findings suggest that the population genetic structure differs for the tissue-defined subpopulations of S. pyogenes. The observed differences may partly reflect differential host immune selection pressures.

Group A streptococci from a remote community have novel multilocus genotypes but share emm types and housekeeping alleles with isolates from worldwide sources.

Group A streptococci (GAS) cause several human diseases that differentially affect distinct host populations. Genotypes were defined by multilocus sequence typing and emm typing for 137 organisms collected from individuals in a remote aboriginal island community in tropical Australia and compared with >200 isolates obtained from sources elsewhere in the world. The majority of aboriginal-derived isolates shared emm types and housekeeping alleles with GAS isolates recovered from outside Australia, but these emm types and alleles were in novel combinations. There were many examples in which isolates from aboriginal and non-Australian subjects shared the same emm type, but for approximately 50% of emm types, the multilocus genotypes of isolates of the same emm type but from different regions were very different. A single emm type may typically define a single clone within the United States and on the remote island that is the focus of this study, but in many cases, these clones will be different, and this finding has implications for attempts to make global associations between emm types and certain disease manifestations.

Group A streptococcal infections in Sweden: a comparative study of invasive and noninvasive infections and analysis of dominant T28 emm28 isolates.

Surveillance of group A streptococcus (GAS) infections in Sweden during 1996-1997 indicated that T28 isolates were dominant, whereas T1M1 infections were uncommon. Circulating T28 isolates were nearly all emm28, MLST52, and these clones had also been prevalent 10 years earlier. Isolates from invasive and noninvasive infections were of similar types and prevalences. The average national incidence of invasive episodes was 2.9/100,000 population but varied between 0 and 8.3/100,000 population in different counties. It increased markedly with age, reaching 22.9 episodes/100,000 among people aged > or =90 years. The incidence of puerperal sepsis was higher than expected (22.4/100,000 of those at risk), with 1 death. Overall mortality was 16% and was associated with preexisting chronic disease (P=.002). Streptococcal toxic shock syndrome (STSS) developed in approximately 15% of patients with invasive episodes, with a mortality rate of 45%. The use of nonsteroidal anti-inflammatory drugs was not found to be associated with the development of STSS.