Differentiation between two biologically distinct classes of group A streptococci by limited substitutions of amino acids within the shared region of M protein-like molecules.

Group A streptococci can be categorized into two classes (I and II) based on immunodeterminants contained within a surface-exposed, conserved region (C repeat domain) of the major virulence factor, M protein. Previous studies have shown that several biological properties correlate strongly with streptococcal class, and thus, there is a strong impetus to precisely define the antigenic epitopes unique to class I and II M proteins. Using synthetic peptides, the binding sites of two class I-specific mAbs were mapped to distinct epitopes within the C repeat region of type 6 M protein (class I). A class II M protein-like gene (type 2) was cloned and sequenced, and the predicted amino acid sequence was compared for homology to class I and II molecules, whose sequences were previously reported. For a given C repeat block 35 amino acid residues in length, 20 residue positions were conserved among all sequences analyzed. Of the 15 variable amino acid positions, only four were class specific, and three of the four positions were localized in the area to which the class I-specific mAbs bound. The predicted secondary structures of class I and II C repeat blocks reveals that they are alpha-helical, except for a single area of disruption. In the class I molecules, the area of disruption corresponds to the class I-specific mAb binding sites. Importantly, the predicted conformational characteristics of this disruption differs for class I and II molecules. The data suggest that only limited changes in amino acid residues differentiate between class I and II molecules in the C repeat region. Therefore, selective (biological) pressures may have contributed to the evolution of these two classes of molecules.

Two-domain motif for IgG-binding activity by group A streptococcal emm gene products.

A biological role for the non-immune binding of human IgG by group A streptococci is evidenced by its strong association with a subpopulation of strains giving rise to tissue-specific infection. IgG-binding activity lies within many of the M and M-like surface proteins (encoded by emm genes), and several structurally distinct IgG-binding sites are known to exist. In this report, two adjacent IgG-binding domains, differing in their specificity for human IgG subclasses, are localized within the M-like protein, protein H. The putative coding regions for the two IgG-binding domains were mapped for 82 epidemiologically unrelated strains. Both coding regions are associated with phylogenetically distant emm genes, supporting a role for horizontal transfer and intergenomic recombination in the evolution of emm genes. In most instances, the two coding regions are tightly linked, suggesting that there exist strong selective pressures to maintain a two-domain binding motif. Both coding regions are found among all strains bearing emm gene markers associated with impetigo lesions as the principal tissue reservoir, but are absent from most strains that exhibit markers for a predominant nasopharyngeal reservoir. The data support the hypothesis that the pathogenic potential of an isolate is dictated, at least in part, by its unique array of multifunctional emm gene products.

Molecular markers for throat and skin isolates of group A streptococci.

In summary, the emm chromosomal patterns distinguish between the two principal tissue site reservoirs of group A streptococci--the nasopharyngeal mucosa and impetigo lesion. Strains derived from normally sterile tissue sites are probably transmitted to new hosts by respiratory droplets, at least in the Connecticut population. The speA gene provides an example of how genetic exchange between different strains of group A streptococci may be limited to a single tissue site or to a subset of emm chromosomal patterns.

Updated model of group A Streptococcus M proteins based on a comprehensive worldwide study.

Group A Streptococcus (GAS) M protein is an important virulence factor and potential vaccine antigen, and constitutes the basis for strain typing (emm-typing). Although >200 emm-types are characterized, structural data were obtained from only a limited number of emm-types. We aim to evaluate the sequence diversity of near-full-length M proteins from worldwide sources and analyse their structure, sequence conservation and classification. GAS isolates recovered from throughout the world during the last two decades underwent emm-typing and complete emm gene sequencing. Predicted amino acid sequence analyses, secondary structure predictions and vaccine epitope mapping were performed using MUSCLE and Geneious software. A total of 1086 isolates from 31 countries were analysed, representing 175 emm-types. emm-type is predictive of the whole protein structure, independent of geographical origin or clinical association. Findings of an emm-type paired with multiple, highly divergent central regions were not observed. M protein sequence length, the presence or absence of sequence repeats and predicted secondary structure were assessed in the context of the latest vaccine developments. Based on these global data, the M6 protein model is updated to a three representative M protein (M5, M80 and M77) model, to aid in epidemiological analysis, vaccine development and M protein-related pathogenesis studies.

Localization of immunoglobulin A-binding sites within M or M-like proteins of group A streptococci.

Many strains of group A streptococci are capable of binding human immunoglobulin A (IgA) by a nonimmune mechanism. M or M-like proteins constitute a family of structurally diverse molecules which form surface fibrillae, and some of the M or M-like protein forms are responsible for the IgA-binding activity. In this report, the binding site for IgA is localized within two structurally distinct M or M-like proteins, ML2.2 and Arp4. Apart from those structural domains which are common to all M and M-like proteins, ML2.2 and Arp4 lack significant levels of amino acid sequence homology, with the exception of a short segment (ALXGENXDLR) located at residues 21 to 30 of the mature ML2.2 protein. Recombinant fusion polypeptides containing portions of the ML2.2 and Arp4 proteins were expressed in Escherichia coli and tested for binding of human myeloma IgA. A 58-residue polypeptide containing residues 14 to 71 of ML2.2 bound human IgA. The IgA-binding site of Arp4 could be localized to a 53-residue polypeptide containing residues 43 to 95, which encompasses the ALXGENXDLR consensus sequence of Arp4 positioned at residues 50 to 59. Site-specific mutagenesis at three codons within the ALXGENXDLR coding sequence of both the ML2.2 and Arp4 recombinant polypeptides leads to a loss in IgA-binding activity. Thus, the ALXGENXDLR consensus sequence is essential for the nonimmune binding of IgA by both ML2.2 and Arp4. However, the failure to bind IgA by polypeptides which partially overlap the 58- and 53-residue IgA-binding polypeptides of ML2.2 and Arp4, yet contain the ALXGENXDLR consensus sequence, strongly suggests that flanking regions are also critical for IgA binding. In summary, the results indicate that common functional domains bearing significant sequence homology are distributed within regions of M or M-like molecules that are otherwise highly divergent.

Nucleotide sequences of two adjacent M or M-like protein genes of group A streptococci: different RNA transcript levels and identification of a unique immunoglobulin A-binding protein.

M protein is a key virulence factor present on the surface of group A streptococci. M protein is defined by its antiphagocytic function, whereas M-like proteins, while structurally related to M proteins, lack an established antiphagocytic function. Group A streptococci can be divided into two main groups (class I and II) on the basis of the presence or absence of certain antigenic epitopes within the M and M-like molecules, and importantly, the two classes correlate with the disease-causing potential of group A streptococci. In an effort to better understand this family of molecules, a 2.8-kb region containing the two M protein-like genes from a class II isolate (serotype 2) was cloned and sequenced. The two genes lie adjacent to one another on the chromosome, separated by 211 bp, and have many structural features in common. The emmL2.1-derived product (ML2.1 protein) is immunoreactive with type-specific antiserum, a property associated with M proteins. The cloned product of the downstream gene, emmL2.2 (ML2.2 protein), is an immunoglobulin A (IgA)-binding protein, binding human myeloma IgA. Interestingly, the RNA transcript levels of emmL2.1 exceed that of emmL2.2 by at least 32-fold. Northern (RNA) hybridization and primer extension studies suggest that the RNA transcripts of emmL2.1 and emmL2.2 are monocistronic. The ML2.1 and ML2.2 proteins exhibit 53% amino acid sequence identity and differ primarily in their amino termini and peptidoglycan-spanning domains and in a Glu-Gln-rich region present only in the ML2.1 protein. However, the previously described M-like, IgA-binding protein from a serotype 4 isolate (Arp4) displays a higher level of amino acid sequence homology with the ML2.1 molecule than with the IgA-binding ML2.2 protein. Amino acid sequence alignments between all M and M-like proteins characterized to date suggest the existence of two fundamental M or M-like gene subclasses within class II organisms, represented by emmL2.1 and emmL2.2. In addition, IgA-binding activity can be found within both types of molecules.

Allelic polymorphism of emm loci provides evidence for horizontal gene spread in group A streptococci.

Group A streptococci have a virulence regulon containing a single emm locus or two or three distinct and adjacent loci of structurally related emm family genes. The products of the emm gene cluster consist of fibrillar surface proteins, at least some of which are known to contain determinants of type specificity located in their NH2-terminal regions, lying distal to the cell surface. The emm genes can be categorized into four major subfamilies (SFs), based on structural differences within their 3' regions encoding the peptidoglycan-spanning domain. In this study, we investigate the polymorphism within the 5' region of SF-4 and SF-3 emm genes (which occupy the first and last emm positions of the gene cluster, respectively) in 22 strains representing different serotypes. Our findings indicate that unlike the centrally positioned SF-1 or SF-2 genes, SF-3 and SF-4 genes each display only limited polymorphism in their 5' regions, suggesting that their gene products may not be major contributors to type specificity. Two forms of the SF-3 gene (SF3a, SF3b) and two forms of the SF-4 gene (SF4a, SF4b) are found to exist in all four possible combinations (SF3aSF4a, SF3aSF4b, SF3bSF4a, SF3bSF4b), strongly suggesting that horizontal gene spread has contributed to the evolution of emm genes and to the generation of emm gene diversity in group A streptococci.

Selective distribution of a high-affinity plasminogen-binding site among group A streptococci associated with impetigo.

Group A streptococci can be classified according to their tendency to cause either impetigo, pharyngitis, or both types of infection. Genotypic markers for tissue site preference lie within emm genes, which encode fibrillar surface proteins that play a key role in virulence. emm gene products (M and M-like proteins) display an extensive array of binding activities for tissue and plasma proteins of the human host. In a previous study, a high-affinity binding site for human plasmin(ogen) was mapped to the emm53 gene product. In this report, a structurally similar plasminogen-binding domain is found to be widely and selectively distributed among group A streptococci harboring the emm gene marker for the skin as the preferred tissue site for infection. The findings are highly suggestive of a central role for bacterial modulation of host plasmin(ogen) during localized infection at the epidermis.

Humanized in vivo model for streptococcal impetigo.

An in vivo model for group A streptococcal (GAS) impetigo was developed, whereby human neonatal foreskin engrafted onto SCID mice was superficially damaged and bacteria were topically applied. Severe infection, indicated by a purulent exudate, could be induced with as few as 1,000 CFU of a virulent strain. Early findings (48 h) showed a loss of stratum corneum and adherence of short chains of gram-positive cocci to the external surface of granular keratinocytes. This was followed by an increasing infiltration of polymorphonuclear leukocytes (neutrophils) of mouse origin, until a thick layer of pus covered an intact epidermis, with massive clumps of cocci accumulated at the outer rim of the pus layer. By 7 days postinoculation, the epidermis was heavily eroded; in some instances, the dermis contained pockets (ulcers) filled with cocci, similar to that observed for ecthyma. Importantly, virulent GAS underwent reproduction, resulting in a net increase in CFU of 20- to 14,000-fold. The majority of emm pattern D strains had a higher gross pathology score than emm pattern A, B, or C (A-C) strains, consistent with epidemiological findings that pattern D strains have a strong tendency to cause impetigo, whereas pattern A-C strains are more likely to cause pharyngitis.

Structural heterogeneity of the emm gene cluster in group A streptococci.

One or more distinct copies of emm genes lie within a gene cluster that is located downstream from a transcriptional regulatory gene (mry). Mry is a positive regulator for the genes in this cluster and for the downstream gene, scpA. The objective of this study is to examine the structure of this cluster and the distribution of specific alleles within the cluster among group A streptococcal isolates of 32 different serotypes. The peptidoglycan (PG)-spanning domain, which exists in four divergent forms, was used to identify specific alleles of the genes within the emm cluster. Gene content of the cluster was determined by Southern hybridization with allele-specific oligonucleotides. Five different chromosomal patterns for this cluster were observed. Sequence heterogeneity in the adjacent mry locus was demonstrated by the ability of some of the isolates to hybridize with a whole mry gene probe, but not with mry-based oligonucleotide probes. A PCR-based chromosomal mapping technique was used to examine further the gene order within the emm gene clusters. Structural heterogeneity of the emm gene cluster was found within class I isolates in this study, while class II isolates were relatively homogeneous at this chromosomal locus and distinct from class I.

Directional gene movement from human-pathogenic to commensal-like streptococci.

Group A streptococci (GAS) are highly pathogenic for humans, and their closest genetic relatives, group C and G streptococci (GCS and GGS, respectively), are generally regarded as commensals, although they can be found in association with human disease. As part of an effort to better understand the evolution of virulence, the phylogenetic relationships between GAS, GCS, and GGS were examined. The nucleotide sequence was determined for an internal portion of seven housekeeping (neutral) loci among >200 isolates of GAS and 34 isolates of GCS or GGS obtained from human subjects. Genotypic analysis failed to show support for the separation of GCS and GGS into two distinct populations. Unlike GAS, there was poor concordance between emm type and genetic relatedness among GCS and GGS. All housekeeping genes within GAS displayed relatively low levels of sequence diversity. In contrast, individual GCS and GGS strains had mosaic genomes, containing alleles at some loci that were similar or identical to GAS alleles, whereas the alleles at other loci were about 10 to 30% diverged. The data provide evidence for a history of recent interspecies transfer of neutral genes that exhibits a strong net directionality from GAS donors to GCS and GGS recipients. A model for the evolution of GAS and of GCS and GGS is described.

Multilocus sequence typing of Streptococcus pyogenes and the relationships between emm type and clone.

Multilocus sequence typing (MLST) is a tool that can be used to study the molecular epidemiology and population genetic structure of microorganisms. A MLST scheme was developed for Streptococcus pyogenes and the nucleotide sequences of internal fragments of seven selected housekeeping loci were obtained for 212 isolates. A total of 100 unique combinations of housekeeping alleles (allelic profiles) were identified. The MLST scheme was highly concordant with several other typing methods. The emm type, corresponding to a locus that is subject to host immune selection, was determined for each isolate; of the >150 distinct emm types identified to date, 78 are represented in this report. For a given emm type, the majority of isolates shared five or more of the seven housekeeping alleles. Stable associations between emm type and MLST were documented by comparing isolates obtained decades apart and/or from different continents. For the 33 emm types for which more than one isolate was examined, only five emm types were present on widely divergent backgrounds, differing at four or more of the housekeeping loci. The findings indicate that the majority of emm types examined define clones or clonal complexes. In addition, an MLST database is made accessible to investigators who seek to characterize other isolates of this species via the internet (http://www.mlst.net).

Genetic correlates of throat and skin isolates of group A streptococci.

The existence of discrete populations of throat and skin strains of group A streptococci has long been recognized; however, a molecular basis for this distinction is not known. The emm gene structure was analyzed for 105 isolates obtained from patients with well-defined group A streptococcal diseases: uncomplicated pharyngitis, impetigo, and acute rheumatic fever. Four emm gene sub-family forms, defined by nucleotide sequence differences in regions encoding the peptidoglycan-spanning domain of M and M-like surface proteins, were found to exist in five different chromosomal patterns among naturally occurring isolates. Strong correlations were made between disease and the number and arrangement of emm subfamily genes. These findings provide a genetic basis for the historical references to "throat," "skin," and "rheumatogenic" types.

Molecular evolution of a multigene family in group A streptococci.

The emm genes are members of a gene family in group A streptococci (GAS) that encode for antiphagocytic cell-surface proteins and/or immunoglobulin-binding proteins. Previously sequenced genes in this family have been named "emm," "fcrA," "enn," "arp," "protH," and "mrp"; herein they will be referred to as the "emm gene family." The genes in the emm family are located in a cluster occupying 3-6 kb between the genes mry and scpA on the chromosome of Streptococcus pyogenes. Most GAS strains contain one to three tandemly arranged copies of emm-family genes in the cluster, but the alleles within the cluster vary among different strains. Phylogenetic analysis of the conserved sequences at the 3' end of these genes differentiates all known members of this family into four evolutionarily distinct emm subfamilies. As a starting point to analyze how the different subfamilies are related evolutionarily, the structure of the emm chromosomal region was mapped in a number of diverse GAS strains by using subfamily-specific primers in the polymerase chain reaction. Nine distinct chromosomal patterns of the genes in the emm gene cluster were found. These nine chromosomal patterns support a model for the evolution of the emm gene family in which gene duplication followed by sequence divergence resulted in the generation of four major-gene subfamilies in this locus.

A genetic-based evaluation of the principal tissue reservoir for group A streptococci isolated from normally sterile sites.

The primary sites of infection and principal reservoirs for transmission of group A streptococci are the nasopharyngeal mucosa and the impetigo lesion. However, pharyngitis and impetigo are rarely observed prior to invasive disease, and, thus, the origin of invasive strains is largely unknown. As part of an active surveillance program, group A streptococci were obtained from normally sterile tissue sites of Connecticut residents during a 6-month period. Organisms were analyzed for genetic markers that distinguish between strains that use the nasopharynx versus an impetiginous lesion as their primary site for infection. The nasopharyngeal marker was observed for most sterile-site isolates, suggesting that the upper respiratory tract is the principal reservoir from which organisms causing invasive disease are disseminated. Genotypic analyses of sterile-site isolates support the view that additional factors, aside from a recent emergence of a few virulent clones, are important contributors to invasive group A streptococcal disease.

Numerous eruptive lesions of panniculitis associated with group A streptococcus bacteremia in an immunocompetent child.

A previously healthy 13-month-old boy developed group A beta-hemolytic streptococcus bacteremia coinciding with numerous eruptive subcutaneous lesions primarily on his extremities. Skin biopsy revealed infectious panniculitis; gram-positive cocci were present within both fat lobules and septa. Molecular genetic analysis of an isolate from the patient's blood revealed an emm type 4 organism displaying the emm chromosomal pattern E that is characteristic of opacity factor-producing strains; the organism also harbored the gene encoding for streptococcal pyrogenic exotoxin C (speC). To our knowledge, this clinical presentation has not yet been described in the spectrum of infections directly caused by group A beta-hemolytic streptococci.