Cell wall anchoring of the Streptococcus pyogenes M6 protein in various lactic acid bacteria.

The M6 protein from Streptococcus pyogenes is the best-characterized member of a family of cell envelope-associated proteins. Based on the observation that the C-terminal sorting signals of these proteins can drive cell wall anchoring of heterologous unanchored proteins, we have cloned and expressed the emm6 structural gene for the M6 protein in various lactic acid bacteria (LAB). The emm6 gene was successfully expressed from lactococcal promoters in several Lactococcus lactis strains, an animal-colonizing Lactobacillus fermentum strain, Lactobacillus sake, and Streptococcus salivarius subsp. thermophilus. The M6 protein was efficiently anchored to the cell wall in all strains tested. In lactobacilli, essentially all detectable M6 protein was cell wall associated. These results suggest the feasibility of using the C-terminal anchor moiety of M6 for protein surface display in LAB.     

Variation in M protein production among Streptococcus pyogenes strains according to emm genotype

M protein is an important virulence determinant in Streptococcus pyogenes, but the amounts of M protein in various strains of the species remain to be elucidated. To assess the amount of M protein in strains of each emm genotype, dot blot analysis was performed on 141 clinically isolated strains. Among the cell membrane-associated proteins, M protein was present in greater quantities in the emm1, 3, and 6 strains than in the other emm strains. In addition three strains, one each of the emm1, 3, and 6 types, showed prolific M protein production (M protein-high producers). These three emm genotypes are frequently isolated in clinical practice. Sequencing of the csrRS gene, one of the two-component signal transduction systems implicated in virulence, was performed on 25 strains bearing different amounts of M protein. CsrS mutations, in contrast to CsrR protein, were detected in 11 strains. The M protein-high producer strain of emm1 type carried two amino acid substitutions, whereas the other three emm1 strains carried only one substitution each. The M protein-high producer expressed its emm gene more strongly than the corresponding M protein-low producer did according to TaqMan RT-PCR. These observations suggest that the accumulation of amino acid substitutions in CsrS protein may contribute, at least in part, to the large amount of M protein production seen in several emm genotypes.     

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.     

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.     

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.      

Architecture of the vir regulons of group A streptococci parallels opacity factor phenotype and M protein class.

Group A streptococci have traditionally been categorized into two broad groups based on the presence or absence of serum opacity factor (OF). Recent studies show that these two groups vary in a number of properties in addition to the OF phenotype, including sequence variations in the constant region of the antiphagocytic M protein genes, the presence or absence of immunoglobulin G Fc receptor proteins, and the presence or absence of multiple M protein-like genes situated in a tandem array. The M protein genes (emm) in OF- streptococcal strains are known to be part of a regulon of virulence-related genes controlled by the trans-acting positive regulatory gene, virR, situated just upstream of emm. In OF+ strains, however, the region adjacent to virR is occupied by an M protein-related, type IIa immunoglobulin G Fc receptor gene (fcrA), and the relative position of emm has not been determined. To further define the vir regulon in OF+ streptococci, we used the polymerase chain reaction to show that fcrA49 is situated immediately upstream of emm49 in the OF+ type 49 strain CS101. This result shows for the first time the separate identity and genetic linkage of these two genes in the vir regulon of an OF+ group A streptococcal strain and confirms our previous hypothesis that emm49 exists as the central gene in a trio of emm-like genes. Additionally, using DNA hybridizations, we found considerable sequence divergence between OF- and OF+ group A streptococci in virR and in the noncoding sequences between virR and the emm or fcrA expression site. We found, however, a high degree of sequence conservation in this region within each of the two groups of strains.     

Identification of an intragenic integration site for foreign gene expression in recombinant Streptococcus gordonii strains

A new intragenic chromosomal integration site within the lacG gene of the lac operon has been identified in Streptococcus gordonii for use in the expression of foreign genes. Introduction of a portion of the Streptococcus pyogenes emm6 gene into the lacG locus resulted in the lactose-inducible surface expression of the S. pyogenes M6 protein. This result demonstrates the ability to modulate the in vitro or in vivo expression of a foreign gene in a S. gordonii recombinant using a biosynthetic metabolite.     

Differential recognition of surface proteins in Streptococcus pyogenes by two sortase gene homologs

The interaction of Streptococcus pyogenes (group A streptococcus [GAS]) with its human host requires several surface proteins. In this study, we isolated mutations in a gene required for the surface localization of protein F by transposon mutagenesis of the M6 strain JRS4. This gene (srtA) encodes a protein homologous to Staphylococcus aureus sortase, which covalently links proteins containing an LPXTG motif to the cell wall. The GAS srtA mutant was defective in anchoring the LPXTG-containing proteins M6, protein F, ScpA, and GRAB to the cell surface. This phenotype was complemented when a wild-type srtA gene was provided in trans. The surface localization of T6, however, was unaffected by the srtA mutation. The M1 genome sequence contains a second open reading frame with a motif characteristic of sortase proteins. Inactivation of this gene (designated srtB) in strain JRS4 affected the surface localization of T6 but not M6, protein F, ScpA, or GRAB. This phenotype was complemented by srtB in trans. An srtA probe hybridized with DNA from all GAS strains tested (M types 1, 3, 4, 5, 6, 18, 22, and 50 and nontypeable strain 64/14) and from streptococcal groups C and G, while srtB hybridized with DNA from only a few GAS strains. We conclude that srtA and srtB encode sortase enzymes required for anchoring different subsets of proteins to the cell wall. It seems likely that the multiple sortase homologs in the genomes of other gram-positive bacteria have a similar substrate-specific role.     

Genome sequence of a nephritogenic and highly transformable M49 strain of Streptococcus pyogenes

The 1,815,783-bp genome of a serotype M49 strain of Streptococcus pyogenes (group A streptococcus [GAS]), strain NZ131, has been determined. This GAS strain (FCT type 3; emm pattern E), originally isolated from a case of acute post-streptococcal glomerulonephritis, is unusually competent for electrotransformation and has been used extensively as a model organism for both basic genetic and pathogenesis investigations. As with the previously sequenced S. pyogenes genomes, three unique prophages are a major source of genetic diversity. Two clustered regularly interspaced short palindromic repeat (CRISPR) regions were present in the genome, providing genetic information on previous prophage encounters. A unique cluster of genes was found in the pathogenicity island-like emm region that included a novel Nudix hydrolase, and, further, this cluster appears to be specific for serotype M49 and M82 strains. Nudix hydrolases eliminate potentially hazardous materials or prevent the unbalanced accumulation of normal metabolites; in bacteria, these enzymes may play a role in host cell invasion. Since M49 S. pyogenes strains have been known to be associated with skin infections, the Nudix hydrolase and its associated genes may have a role in facilitating survival in an environment that is more variable and unpredictable than the uniform warmth and moisture of the throat. The genome of NZ131 continues to shed light upon the evolutionary history of this human pathogen. Apparent horizontal transfer of genetic material has led to the existence of highly variable virulence-associated regions that are marked by multiple rearrangements and genetic diversification while other regions, even those associated with virulence, vary little between genomes. The genome regions that encode surface gene products that will interact with host targets or aid in immune avoidance are the ones that display the most sequence diversity. Thus, while natural selection favors stability in much of the genome, it favors diversity in these regions.     

Role of M3 protein in the adherence and internalization of an invasive Streptococcus pyogenes strain by epithelial cells

Streptococcus pyogenes utilizes multiple mechanisms for adherence to and internalization by epithelial cells. One of the molecules suggested of being involved in adherence and internalization is the M protein. Although strains of the M3 serotype form the second largest group isolated from patients with severe invasive diseases and fatal infections, not much information is known regarding the interactions of M3 protein with mammalian cells. In this study we have constructed an emm3 mutant of an invasive M3 serotype (SP268), and demonstrated that the M3 protein is involved in both adherence to and internalization by HEp-2 cells. Fibronectin promoted both adherence and internalization of SP268 in an M3-independent pathway. Utilizing speB and speB/emm3 double mutants, it was found that M3 protein is not essential for the maturation of SpeB, as was reported for the M1 protein. Increased internalization efficiency observed in both the speB and emm3/speB mutants suggested that inhibition of S. pyogenes internalization by SpeB is not related to the presence of an intact M3 protein. Thus, other proteins in SP268, which serve as targets for SpeB activity, have a prominent role in the internalization process.