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.      

Insertional inactivation of virR in Streptococcus pyogenes M49 demonstrates that VirR functions as a positive regulator of ScpA, FcRA, OF, and M protein

Abstract Several reports have shown that Streptococcus pyogenes strains which produce opacity factor (OF+) have diverged significantly from OF⁻ serotypes. This study questions whether several surface proteins of an OF+ culture are regulated by the positive regulatory protein VirR, in a manner similar to OF~ strains. Interruption of the virR region of an OF⁺ S. pyogenes (strain CS101, M type 49) was performed using a temperature-sensitive plasmid containing a fragment of virR . Interruption of the virR region produced cultures with (indétectable amounts of M49 and ScpA proteins, and reduced the yield of FcRA protein. In addition, mutants had a significant reduction in detectable opacity factor. These results suggest that virR functions as a positive regulator of a variety of surface proteins in OF⁺ strains.     

Sequencing of genes within the vir regulon of Streptococcus pyogenes type M15 — an opacity factor-positive serotype with low opacity factor expression

Major virulence determinants of group A streptococci, such as M-protein, immunoglobulin Fc-receptors (FcRA, EmmL) and C5a peptidase, appear to be genetically co-regulated, their genes being located within a vir regulon. We studied the organization of these genes in a group A, type M15 strain of Streptococcus pyogenes, previously defined as OF^?, by hybridization analysis of chromosomal DNA and of an S. pyogenes gene library in Escherichia coli, and by gene sequencing. Within the vir regulon, in addition to the virR and scpA genes, three so-called emm-related genes were found: fcrA, emmL and enn. Whereas IgG Fc-binding proteins were encoded by fcrA and emmL, the product of enn was not identified. The presence of three emm-related genes in this region is reminescent of vir regulon organization in OF⁺ rather than OF^? strains as earlier defined by others. Furthermore, analysis of the deduced product of the emmL gene showed deletions and amino acid substitutions within the PGTS-rich domain and membrane anchor, which thus resembles corresponding products of OF⁺ rather than OF^? strains. In view of these findings, the opacity factor (OF) activity of the strain was tested using growth supernatant, with negative outcome. However, a concentrated SDS cell extract revealed definite OF activity. One of two other type M15 reference strains also showed definite OF activity in SDS extracts. We therefore propose that type M15 strains belong to the OF⁺ category but often show low levels of expression of OF.     

Genetic organisation of the M protein region in human isolates of group C and G streptococci: two types of multigene regulator-like (mgrC) regions

In addition to beta-haemolytic streptococci belonging to Lancefield group A (Streptococcus pyogenes, GAS), human isolates of group C (GCS) and group G (GGS) streptococci (S. dysgalactiae subsp. equisimilis) have been implicated as causative agents in outbreaks of purulent pharyngitis, of wound infections and recently also of streptococcal toxic shock-like syndrome. Very little is known about the organisation of the genomic region in which the emm gene of GCS and GGS is located. We have investigated the genome sequences flanking the emm gene in GCS by sequencing neighbouring fragments obtained by inverse PCR. Our sequence data for GCS strains 25287 and H46A revealed two types of arrangement in the emm region, which differ significantly from the known types of mga regulon in GAS. We named this segment of the genome mgrC (for multigene regulon-like segment in group C streptococci). In strains belonging to the first mgrC type (prototype strain 25287) the emm gene is flanked up-stream by mgc, a gene that is 61% identical to the mga gene of GAS. A phylogenetic analysis of the deduced protein sequences showed that Mgc is related to Mga proteins of various types of GAS but forms a distinct cluster. Downstream of emm, the mgrC sequence region is bordered by rel. This gene encodes a protein that functions in the synthesis and degradation of guanosine 3',5' bipyrophosphate (ppGpp) during the stringent regulatory response to amino acid deprivation. In the second mgrC type (prototype strain H46A), the genes mgc and emm are arranged as in type 1. But an additional ORF (orf) is inserted in opposite orientation between emm and rel. This orf shows sequence homology to cpdB, which is present in various microorganisms and encodes 2',3' cyclo-nucleotide 2'-phosphodiesterase. PCR analysis showed that these two mgrC arrangements also exist in GGS. Our sequence and PCR data further showed that both types of mgrC region in GCS and GGS are linked via rel to the streptokinase region characterised recently in strain H46A. A gene encoding C5a peptidase, which is present at the 3' end of the mga regulon in GAS, was not found in the mgrC region identified in the GCS and GGS strains investigated here.     

Identification of a divergent M protein gene and an M protein-related gene family in Streptococcus pyogenes serotype 49.

The antigenically variant M protein of Streptococcus pyogenes enhances virulence by promoting resistance to phagocytosis. The serum opacity factor (OF), produced by a subset of M serotypes, is also antigenically variant, and its antigenic variability exactly parallels that of M protein. OF-positive and OF-negative streptococci are also phenotypically distinguishable by a number of other criteria. In order to study the differences between OF-positive and OF-negative streptococci, we cloned and sequenced the type 49 M protein gene (emm49), the first to be cloned from an OF-positive strain. This gene showed evolutionary divergence from the OF-negative M protein genes studied previously. Furthermore, emm49 was part of a gene family, in contrast to the single-copy nature of previously characterized M protein genes.     

Isolated DNA repeat region from fcrA76, the Fc-binding protein gene from an M-type 76 strain of group A streptococci, encodes a protein with Fc-binding activity

The DNA repeat region of fcrA76, the gene encoding a group A streptococcal Fc-binding protein, was subcloned in-frame into an Escherichia coli plasmid expression vector. The expressed protein product displayed the same Fc-binding properties as the full-length Fc-binding protein expressed from fcrA76. The affinity-purified, full-length Fc-binding protein was found to compete with staphylococcal protein A or streptococcal protein G for binding to beads coated with human IgG. These results are consistent with earlier studies suggesting that the binding sites on human IgG for protein A, protein G and the type II Fc-binding protein from group A streptococci are located at the interface of the CH2 and CH3 domains of the Fc region.     

Three different types of organization of the vir regulon in group A streptococci

Summary The DNA of group A streptococci (GAS) encodes several important virulence factors such as the antiphagocytic M protein, the Ig-Fc-binding M-related proteins (FcrA-like and EnnX-like) and the complement factor-inactivating C5a peptidase. The corresponding genes emm, fcrA, ennX, and scpA, respectively, were assumed to be located close together in the GAS genome. Additionally, emm and scpA have been found to be under the positive, coordinate control of the virR locus, which led to the designation vir regulon for the corresponding genomic segment. In order to map the vir regulons of many GAS serotypes and to analyse any correlation between the organization of vir regulons and circumscribed heterogeneities within the emm, virR, and scpA genes, an approach using several distinct sets of polymerase chain reaction (PCR) experiments was chosen. By examination of the genomic DNA of 42 GAS isolates from 36 different M serotypes three patterns of vir regulon topography were found. The first, designated large vir regulon (LVR), consists of virR -fcrA(-like) emm - ennX(-like) - scpA. The second, designated small vir regulon (SVR), contains virR - emm- scpA, and the last, designated unusual vir regulon (UVR), resembles SVR but contains additional heterogeneous sequences between emm and scpA. The patterns correlate with heterogeneities at the 3 ends of the virR and scpA genes, with the M classification system and the occurrence of specific non-coding intervening sequences within the vir regulons. The potential impact of these patterns on models to account for generation of vir regulons is discussed.     

Horizontal gene transfer in the evolution of group A streptococcal emm-like genes: gene mosaics and variation in Vir regulons

Most M type 5 group A streptococcal strains were found to contain a single emm-like gene between virR and scpA (the Vir reguion), but two distinct emm-like genes were identified in the Vir regulon of the MS strain NCTC8193. The complete sequences of both of these genes were determined. One, called emm5.8193, was shown to be a minor variant of the previously described emm5 gene from strain Manfredo. The second, designated enn5.8193, expresses an IgG-binding protein when cloned in Escherichia coli. A comparison of enn5.8193 with emm-like gene sequences from other strains indicated that it has a mosaic structure, consisting of distinct segments originating from emm-like genes in different OF⁺ and OF⁻strains. These data provide the first clear evidence that the horizontal transfer of emm-like sequences between distinct strains contributes to the evolution of group A streptococcal emm-like genes and Vir regulons.     

Nucleotide substitutions and small-scale insertion produce size and antigenic variation in group A streptococcal M1 protein

The presence of M protein on the surface of group A streptococci (GAS) confers the ability of the cell to resist phagocytosis in the absence of type-specific antibodies. It undergoes antigenic variation with more than 80 different serotypes having been defined. We have sequenced the M protein gene (emm1.1) from strain CS190 and present evidence that individual nucleotide substitutions are responsible for sequence variation in the N-terminal non-repeat region of emm1.1 and these substitutions have altered antibody recognition of opsonic epitopes. The N-terminal non-repeat domains of two other closely related strains, 71-155 and 76-088, were found to have sequence identical to emm1.1 with the addition of a 21 bp insert. This study provides the first evidence that nucleotide substitutions and small insertions are responsible for size and antigenic variation in the N terminal non-repeat domain of the M protein of GAS.     

Limited repertoire of the C-terminal region of the M protein in Streptococcus pyogenes

We have amplified genomic sequences (emm) that may encode M protein from strains of Streptococcus pyogenes using the polymerase chain reaction (PCR). Genomic DNA from 22 isolates representing 14 M serotypes was selected for the study. Primers which corresponded to the observed N-terminal signal sequence and the variable C-terminal sequences of emm6, emm49 and ennX were used. PCR products using emm6 and emm49 oligonucleotides were classified into two mutually exclusive groups which correspond to the presence or absence of serum opacity factor. These findings support the concept of limited heterogeneity in the C-terminal sequences of the M protein.     

The F1 genes of the F1F0 ATP synthase from the acidophilic bacterium Thiobacillus ferrooxidans complement Escherichia coli F1unc mutants

Abstract Because of the allelic variations within the M protein gene ( emm gene) of group A streptococci, reliable typing of this important human pathogen can be accomplished by the use of emm gene-specific oligonucleotide probes. Two technical modifications (a reverse dot blot and a reverse line blot hybridization assay) of a novel approach for the type-specific identification of emm genes have been developed. Both procedures involved amplification of an emm gene by polymerase chain reaction. The non-radioactively labeled amplicon was subsequently hybridized to a membrane carrying an array of immobilized emm gene-specific oligonucleotide probes, thus allowing the simultaneous analysis of the gene polymorphism in a single hybridization reaction. The feasibility of these rapid and easy to perform methods was shown for the unequivocal identification of reference strains and clinical isolates belonging to 16 different M serotypes.