Close correlation of streptococcal DNase B (sdaB) alleles with emm genotypes in Streptococcus pyogenes

DNase B is a major nuclease and a possible virulence factor in Streptococcus pyogenes. The allelic diversity of streptococcal DNase B (sdaB) gene was investigated in 83 strains with 14 emm genotypes. Of the 15 alleles identified, 11 alleles carried only synonymous nucleotide substitutions. On the other hand, 4 alleles had a non-synonymous substitution other than synonymous substitutions, resulting in the substitution of a single amino acid. The distribution of each allele was generally emm genotype-specific. Only sdaB7 was found in both emm2 and emm4. The promoter region was highly conserved and DNase B protein was similarly expressed in all alleles.     

A single emm gene-specific oligonucleotide probe does not recognise all members of the Streptococcus pyogenes M type 1

Abstract Serological typing of the streptococcal M protein has recently been challenged by a number of unique molecular methodologies based on oligonucleotide recognition of allelic variations within the M protein ( emm ) gene. In these methods, stringent hybridization of an oligonucleotide probe to a polymerase chain reaction amplified emm gene is used as confirmation of specific M type identity. A sample of 17 isolates from 7 previously defined distinct genotypes were tested using a single M1 oligonucleotide probe. Isolates from only three of the genotypes hybridized with the probe. The results demonstrate that a single emm -specific oligonucleotide probe can not identify all members of M type 1, as defined by conventional serotyping using polyclonal antisera.     

Two DNA-binding domains of Mga are required for virulence gene activation in the group A streptococcus

Mga is a DNA-binding protein that activates expression of several important virulence genes in the group A streptococcus (GAS), including those encoding M protein (emm), C5a peptidase (scpA) and Mga (mga). To determine the functionality of four potential helix-turn-helix DNA-binding motifs (HTH1-HTH4) identified within the amino-terminus of Mga, alanine substitutions were introduced within each domain in a MBP-Mga fusion allele and purified proteins were assayed for binding to Mga-specific promoter fragments (Pmga, PscpA and Pemm) in vitro. Although HTH-1 and HTH-2 mutations showed wild type DNA-binding activity, an altered HTH-3 domain resulted in reduced binding to the three promoters and an HTH-4 mutant was devoid of detectable binding activity. Plasmid-encoded expression of the HTH-3 and HTH-4 alleles from a constitutive promoter (Pspac) in the mga-deleted GAS strain JRS519 demonstrated that Mga-regulated emm expression correlated directly to the DNA-binding activity observed for each mutant protein in vitro. Single-copy expression of HTH-3 and HTH-4 from their native Pmga resulted in a dramatic reduction in autoregulated mga expression in both mutant strains. Thus, Mga appears to contain two DNA-binding domains (HTH-3 and HTH-4) that are required for direct activation of the Mga virulence regulon in vivo.     

M-related protein (Mrp) contributes to group A streptococcal resistance to phagocytosis by human granulocytes

The M protein has been postulated to be a major group A streptococcal (GAS) virulence factor because of its contribution to the bacterial resistance to opsono-phagocytosis. Direct evidence of this was only provided for GAS strains which expressed a single M protein. The majority of GAS express additional, structurally similar M-related proteins, Mrp and Enn, which have been described as IgG- and IgA-binding proteins. To determine the involvement of Mrp and M protein in phagocytosis resistance, the mrp and emm genes from serotypes M2, M4, and M49 as well as from M-untypeable strain 64/14 were insertionally inactivated. The mrp and emm mutants were subjected to direct bactericidal assays. As judged by numbers of surviving colony-forming units, all mutant strains with the exception of the mrp 4 mutant exhibited reduced multiplication factors as compared to the isogenic wild-type strains. Subsequent analysis of phagocytosis by flow cytometry, measuring association of BCECF/AM-labelled bacteria and granulocytes, paralleled the results from direct bactericidal assays regardless of whether isolated granulocytes or whole blood were utilized. Resistant wild-type GAS strains bound to less than 24% of granulocytes, whereas phagocytosis-sensitive controls attached to more than 90% of the white blood cells. 40 to 60% of the granulocytes associated with the mrp and emm mutants within 1 h of co-incubation. Kinetic data suggested that attachment to granulocytes proceeds faster for emm mutants than for corresponding mrp mutants. By adding a dihydro-rhodamine123 stain and measuring fluorescence induced by oxidative burst, the experimental data suggested that bacteria bound to granulocytes were also engulfed and integrated into phagolysosomes. Thus, these data indicated that, if present, both mrp and emm gene products contribute to phagocytosis resistance by decreasing bacterial binding to granulocytes.     

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.     

Regulation of virulence by a two-component system in group B streptococcus

Group B Streptococcus (GBS) is frequently carried in the gastrointestinal or genitourinary tract as a commensal organism, yet it has the potential to cause life-threatening infection in newborn infants, pregnant women, and individuals with chronic illness. Regulation of virulence factor expression may affect whether GBS behaves as an asymptomatic colonizer or an invasive pathogen, but little is known about how such factors are controlled in GBS. We now report the characterization of a GBS locus that encodes a two-component regulatory system similar to CsrRS (or CovRS) in Streptococcus pyogenes. Inactivation of csrR, encoding the putative response regulator, in two unrelated wild-type strains of GBS resulted in a marked increase in production of beta-hemolysin/cytolysin and a striking decrease in production of CAMP factor, an unrelated cytolytic toxin. Quantitative RNA hybridization experiments revealed that these two phenotypes were associated with a marked increase and decrease in expression of the corresponding genes, cylE and cfb, respectively. The CsrR mutant strains also displayed increased expression of scpB encoding C5a peptidase. Similar, but less marked, changes in gene expression were observed in CsrS (putative sensor component) mutants, evidence that CsrR and CsrS constitute a functional two-component system. Experimental infection studies in mice demonstrated reduced virulence of both CsrR and CsrS mutant strains relative to the wild type. Together, these results indicate that CsrRS regulates expression of multiple GBS virulence determinants and is likely to play an important role in GBS pathogenesis.     

Distribution of three genes involved in the PJM1 iron-sequestering system in various Vibrio anguillarum serogroups

Gene sequences with high homologies to the two genes fatA and fatD, associated with the pJM1 virulence plasmid-encoded iron-sequestering system (anguibactin) in Vibrio anguillarum serogroup O1 strains, were found in a range of serogroup O2a strains and one NT 4 strain that either contained no plasmids or only small-sized plasmids. None of these strains contained the angR gene responsible for regulation of the anguibactin synthesis. DNA sequences of presumed fatA and fatD fragments from one O2a strain and one NT4 strain were identical to each other. The fatA sequence of these strains included 35 nucleotide substitutions and 7 amino acid substitutions out of 555 nucleotides when compared with the fatA sequence of the O1 strain 76775. The fatD sequence included 10 nucleotide substitutions and 1 amino acid substitution out of 543 nucleotides when compared with 76775. The fact that the majority of nucleotide substitutes in the fatA and fatD fragments did not result in amino acid substitutes suggest that these sequences may to some degree be preserved by selective pressure. The functional role of these genes in the O2a serogroup is most likely different from their role in the pJM1 system.     

高毒力A组链球菌致病机制研究进展

A组链球菌(Group A Streptococcus,GAS)常导致咽炎和皮肤感染,也能引起严重侵袭性感染.根据其表面M蛋白编码基因emm可将GAS分为200多型,严重侵袭性感染多由高毒力株引起,以emm1、emm3、emm12、emm28和emm89型常见.研究发现高毒力GAS株中CovRS基因突变可导致细菌逃逸固...     

Vancomycin-resistant enterococci in humans and imported chickens in Japan

The phenotypes and genotypes of 22 VanA-type vancomycin-resistant enterococci that had been isolated in Japan were examined. The VanA resistance determinant was plasmid mediated in each of the 22 strains. Of the 22 strains, 8 were isolated from different patients and 11 and 3 were obtained from different samples of chickens imported from Thailand and France, respectively. Three of the strains that were isolated from patients and the 11 strains isolated from the Thai chickens showed high-level vancomycin resistance (MICs, 512 to 1,024 micro g/ml) and low-level teicoplanin resistance (MICs, 0.5 to 4 micro g/ml). Each of these strains had three amino acid substitutions in the N-terminal region of the deduced VanS sequence. L50 was converted to V, E54 was converted to Q, and Q69 was converted to H compared to the vanS gene sequence of Tn1546.     

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.     

Comparative analysis of cytotoxin, hemolysin, hemagglutinin and exocellular enzymes among clinical and environmental isolates of Vibrio cholerae O139 and non-O1, non-O139

The presence of three major virulence genes toxR, tcpA and ctxA as well as expression of several putative virulence factors were compared in 12 Vibrio cholerae O139 and non-O1,non-O139 strains of clinical and environmental origin. All the strains possessed the gene encoding the regulatory protein TOXR. None of the non-O1, non-O139 strains as well as one of the O139 environmental strains carried the genes for ctxA and tcpA. Statistically significant differences in hemagglutinin and hemolysin production were observed amongst the strains depending on the source of their isolation. Expression of extracellular enzymes such as protease, elastase, neuraminidase, phospholipase A and phospholipase C, however, did not vary significantly from the groups of strains isolated from different sources.     

Prevalence of a Streptococcal Inhibitor of a Complement-Mediated Cell lysis-like Gene (sicG) in Streptococcus Dysgalactiae subsp. Equisimilis

Streptococcus dysgalactiae subsp. equisimilis isolates (n = 110) were analyzed by PCR to determine whether the gene encoding SICG, a homolog of Streptococcus pyogenes SIC, was present. Nineteen strains (17%) had this gene of which 11 (55%) were isolated from patients with invasive disease. All 19 strains possessed group G carbohydrate. Molecular characterization of emm type revealed that the majority of emm sequences were stG643 and stG2078. Only the N-terminal sequence of SICG was similar to that of SIC in S. pyogenes. Although we found no significant relationship between pathogenic severity and sicG possession, further investigation into the mechanism of SICG may elucidate the virulence in S. dysgalactiae subsp. equisimilis infection.     

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.     

Role of biased hypermutation in evolution of subacute sclerosing panencephalitis virus from progenitor acute measles virus.

We identified an acute measles virus (Nagahata strain) closely related to a defective virus (Biken strain) isolated from a patient with subacute sclerosing panencephalitis (SSPE). The proteins of Nagahata strain measles virus are antigenically and electrophoretically similar to the proteins of Edmonston strain measles virus. However, the nucleotide sequence of the Nagahata matrix (M) gene is significantly different from the M genes of all the acute measles virus strains studied to date. The Nagahata M gene is strikingly similar to the M gene of Biken strain SSPE virus isolated several years later in the same locale. Eighty percent of the nucleotide differences between the Nagahata and Biken M genes are uridine-to-cytosine transitions known as biased hypermutation, which has been postulated to be caused by a cellular RNA-modifying activity. These biased mutations account for all but one of the numerous missense genetic changes predicted to cause amino acid substitutions. As a result, the Biken virus M protein loses conformation-specific epitopes that are conserved in the M proteins of Nagahata and Edmonston strain acute measles viruses. These conformation-specific epitopes are also absent in the cryptic M proteins encoded by the hypermutated M genes of two other defective SSPE viruses (Niigata and Yamagata strains). Nagahata-like sequences are found in the M genes of at least five other SSPE viruses isolated from three continents. These data indicate that Biken strain SSPE virus is derived from a progenitor closely resembling Nagahata strain acute measles virus and that biased hypermutation is largely responsible for the structural defects in the Biken virus M protein.     

Unique Genomic Arrangements in an Invasive Serotype M23 Strain of Streptococcus pyogenes Identify Genes That Induce Hypervirulence

The first genome sequence of a group A Streptococcus pyogenes serotype M23 (emm23) strain (M23ND), isolated from an invasive human infection, has been completed. The genome of this opacity factor-negative (SOF⁻) strain is composed of a circular chromosome of 1,846,477 bp. Gene profiling showed that this strain contained six phage-encoded and 24 chromosomally inherited well-known virulence factors, as well as 11 pseudogenes. The bacterium has acquired four large prophage elements, ΦM23ND.1 to ΦM23ND.4, harboring genes encoding streptococcal superantigen (ssa), streptococcal pyrogenic exotoxins (speC, speH, and speI), and DNases (spd1 and spd3), with phage integrase genes being present at one flank of each phage insertion, suggesting that the phages were integrated by horizontal gene transfer. Comparative analyses revealed unique large-scale genomic rearrangements that result in genomic rearrangements that differ from those of previously sequenced GAS strains. These rearrangements resulted in an imbalanced genomic architecture and translocations of chromosomal virulence genes. The covS sensor in M23ND was identified as a pseudogene, resulting in the attenuation of speB function and increased expression of the genes for the chromosomal virulence factors multiple-gene activator (mga), M protein (emm23), C5a peptidase (scpA), fibronectin-binding proteins (sfbI and fbp54), streptolysin O (slo), hyaluronic acid capsule (hasA), streptokinase (ska), and DNases (spd and spd3), which were verified by PCR. These genes are responsible for facilitating host epithelial cell binding and and/or immune evasion, thus further contributing to the virulence of M23ND. In conclusion, strain M23ND has become highly pathogenic as the result of a combination of multiple genetic factors, particularly gene composition and mutations, prophage integrations, unique genomic rearrangements, and regulated expression of critical virulence factors.