Role for a secreted cysteine proteinase in the establishment of host tissue tropism by group A streptococci

Primary infection of the human host by group A streptococci (GAS) most often involves either the epidermis of the skin or the oropharyngeal mucosa. A humanized in vivo model for impetigo was used to investigate the basis for host tissue tropism among GAS. Disruption of the speB gene (encoding for a secreted cysteine proteinase) led to a loss of virulence for two impetigo-derived strains (M-types 33 and 53), as evidenced by a diminution in tissue damage and a lack of reproductive growth. The level of cysteine proteinase activity in overnight cultures was associated with the extent of gross pathological changes induced by strains displaying varied degrees of virulence in the impetigo model. Moreover, high levels of secreted cysteine proteinase activity correlated with a genetic marker for preferred tissue site of infection at the skin (emm pattern D). The addition of exogenous SpeB to a speB mutant (emm pattern D) or to an avirulent throat-like strain (emm pattern A) led to increased bacterial reproduction at the skin. The data provide both experimental and epidemiological evidence for a critical role of a secreted bacterial protease in promoting host tissue-specific infection.     

含ICE-emm12和ΦHKU.vir化脓链球菌引起的猩红热1例

2011年中国开始出现猩红热疫情,病例以普通型为主,外科型较少报道。ICE-emm12和ΦHKU.vir是中国香港地区猩红热暴发株中首次发现的2个可移动遗传元件,尚不清楚其是否存在于中国内地的猩红热致病株中。本文报道1例猩红热病例,其咽部和皮肤伤口皆分离出同一克隆株的化脓链球菌,该菌株携带ICE-emm12和ΦHKU.vir,从而证实这2个新元件存在于中国内地的化脓链球菌中。因此,国内有必要对猩红热分离株进行ICE-emm12和ΦHKU.vir检测,以监测致病株在基因水平的变化。     

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.     

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.     

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.     

Population structure and genetic differentiation associated with breeding history and selection in tomato (Solanum lycopersicum L.)

Tomato (Solanum lycopersicum L.) has undergone intensive selection during and following domestication. We investigated population structure and genetic differentiation within a collection of 70 tomato lines representing contemporary (processing and fresh-market) varieties, vintage varieties and landraces. The model-based Bayesian clustering software, STRUCTURE, was used to detect subpopulations. Six independent analyses were conducted using all marker data (173 markers) and five subsets of markers based on marker type (single-nucleotide polymorphisms, simple sequence repeats and insertion/deletions) and location (exon and intron sequences) within genes. All of these analyses consistently separated four groups predefined by market niche and age into distinct subpopulations. Furthermore, we detected at least two subpopulations within the processing varieties. These subpopulations correspond to historical patterns of breeding conducted for specific production environments. We found no subpopulation within fresh-market varieties, vintage varieties and landraces when using all marker data. High levels of admixture were shown in several varieties representing a transition in the demarcation between processing and fresh-market breeding. The genetic clustering detected by using the STRUCTURE software was confirmed by two statistics, pairwise F(st) (θ) and Nei's standard genetic distance. We also identified a total of 19 loci under positive selection between processing, fresh-market and vintage germplasm by using an F(st)-outlier method based on the deviation from the expected distribution of F(st) and heterozygosity. The markers and genome locations we identified are consistent with known patterns of selection and linkage to traits that differentiate the market classes. These results demonstrate how human selection through breeding has shaped genetic variation within cultivated tomato.     

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.     

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.     

Antimicrobial susceptibility patterns,emm type distribution and genetic diversity of Streptococcus pyogenes recovered in Brazil

Streptococcus pyogenes is responsible for a variety of infectious diseases and immunological complications. In this study, 91 isolates of S. pyogenes recovered from oropharynx secretions were submitted to antimicrobial susceptibility testing, emm typing and pulsed-field gel electrophoresis (PFGE) analysis. All isolates were susceptible to ceftriaxone, levofloxacin, penicillin G and vancomycin. Resistance to erythromycin and clindamycin was 15.4%, which is higher than previous reports from this area, while 20.9% of the isolates were not susceptible to tetracycline. The macrolide resistance phenotypes were cMLSB (10) and iMLSB (4). The ermB gene was predominant, followed by the ermA gene. Thirty-two emm types and subtypes were found, but five (emm1, emm4, emm12, emm22, emm81) were detected in 48% of the isolates. Three new emm subtypes were identified (emm1.74, emm58.14, emm76.7). There was a strong association between emm type and PFGE clustering. A variety of PFGE profiles as well as emm types were found among tetracycline and erythromycin-resistant isolates, demonstrating that antimicrobial resistant strains do not result from the expansion of one or a few clones. This study provides epidemiological data that contribute to the development of suitable strategies for the prevention and treatment of such infections in a poorly studied area.     

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.     

Whole-genome association study on tissue tropism phenotypes in group A Streptococcus

Group A Streptococcus (GAS) has a rich evolutionary history of horizontal transfer among its core genes. Yet, despite extensive genetic mixing, GAS strains have discrete ecological phenotypes. To further our understanding of the molecular basis for ecological phenotypes, comparative genomic hybridization of a set of 97 diverse strains to a GAS pangenome microarray was undertaken, and the association of accessory genes with emm genotypes that define tissue tropisms for infection was determined. Of the 22 nonprophage accessory gene regions (AGRs) identified, only 3 account for all statistically significant linkage disequilibrium among strains having the genotypic biomarkers for throat versus skin infection specialists. Networked evolution and population structure analyses of loci representing each of the AGRs reveal that most strains with the skin specialist and generalist biomarkers form discrete clusters, whereas strains with the throat specialist biomarker are highly diverse. To identify coinherited and coselected accessory genes, the strength of genetic associations was determined for all possible pairwise combinations of accessory genes among the 97 GAS strains. Accessory genes showing very strong associations provide the basis for an evolutionary model, which reveals that a major transition between many throat and skin specialist haplotypes correlates with the gain or loss of genes encoding fibronectin-binding proteins. This study employs a novel synthesis of tools to help delineate the major genetic changes associated with key adaptive shifts in an extensively recombined bacterial species.     

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.      

Genetic exchange by recombination or reassortment is infrequent in natural populations of a tripartite RNA plant virus.

Two hundred seventeen field isolates of cucumber mosaic cucumovirus (CMV), sampled from 11 natural populations, were typed by RNase protection assay (RPA) using probes from the genomic RNAs of strains in subgroup I and in subgroup II of CMV strains. Most (85%) of the analyzed isolates belonged to subgroup I. For these subgroup I isolates, only two clearly different RPA patterns, A and B, were found for each of four probes representing RNA1, RNA2, and each of the two open reading frames in RNA3. On the basis of these RPA patterns for each probe, different haplotypes were defined. The frequency composition for these haplotypes differed for the various analyzed populations, with no correlation with place or year of sampling. This genetic structure corresponds to a metapopulation with local extinctions and recolonizations. Most subgroup I isolates (73%) belonged to haplotypes with RPA pattern A (type 1) or B (type 2) for all four probes. A significant fraction of subgroup I isolates (16%) gave evidence of mixed infections with these two main types, from which genetic exchange could occur. Genetic exchange by segment reassortment was seen to occur: the fraction of reassortant isolates was 4%, reassortment did not occur at random, and reassortants did not become established in the population. Thus, there is evidence of selection against reassortment between types 1 and 2 of subgroup I isolates. Aphid transmission experiments with plants doubly infected with type 1 and type 2 isolates gave further evidence that reassortment is selected against in CMV. Genetic exchange by recombination was detected for RNA3, for which two RPA probes were used. Recombinant isolates amounted to 7% and also did not become established in CMV populations. Sequence analyses of regions of RNA1, RNA2, and RNA3 showed that there are strong constraints to maintain the encoded sequence and also gave evidence that these constraints may have been different during divergence of types 1 and 2 and, later on, during diversification of these two types. Constraints to the evolution of encoded proteins may be related to selection against genetic exchange. Our data, thus, do not favor current hypotheses that explain the evolution of multipartite viral genomes to promote genetic exchange.     

A new rapid and cost-effective method for detection of phages, ICEs and virulence factors encoded by Streptococcus pyogenes

Streptococcus pyogenes (group A Streptococcus, GAS) is a human pathogen that causes diseases of various intensity, from mild strep throat to life threatening invasive infections and postinfectional sequelae. S. pyogenes encodes multiple, often phage encoded, virulence factors and their presence is related to severity of the disease. Acquisition of mobile genetic elements, carrying virulence factors, as phages or ICEs (integrative and cojugative elements) has been shown previously to promote selection of virulent clones. We designed the system of eight low volume multi- and one singleplex PCR reactions to detect genes encoding twenty virulence factors (spd3, sdc, sdaB, sdaD, speB, spyCEP, scpA, mac, sic, speL, K, M, C, I, A, H, G, J, smeZ and ssa) and twenty one phage and ICE integration sites described so far for S. pyogenes. Classification of strains based on the phage and virulence factors absence or presence, correlates with PFGE MLST and emm typing results. We developed a novel, fast and cost effective system that can be used to detect GAS virulence factors. Moreover, this system may become an alternative and effective system to differentiate between GAS strains.     

emm Typing of group A streptococcus clinical isolates: identification of dominant types for throat and skin isolates

T and emm types were determined for group A streptococci isolated from patients with various infections during 1990-1999 in Toyama Prefecture, Japan. Out of 906 isolates, 872 isolates were divided into 20 T serotypes, and 34 isoltes were T nontypeable (TNT). T12, T1, and T4 were dominant among 699 throat isolates; on the other hand, T11, T28, TB3264, and TNT were dominant among 80 skin isolates. The emm types of 190 isolates were determined following specific PCR amplification and sequencing of the products. Twenty T serotypes were divided into 34 T type/emm type combinations. Thirty-four TNT isolates were divided into 14 emm types, in which emm58 was the most common (38%). Among 82 throat isolates randomly selected, predominant T types T12, T1, and T4 isolates were of the respective same numbers in emm type. T11/emm89, T28/emm28, TB3264/emm13w, and TNT/emm58 were predominant among 80 skin isolates. emm-type distribution observed in the present study was that usually reported in the western world. To our knowledge, 3 T/emm is a novel combination. These results show that emm typing allows the characterization of group A streptococci from various sources.     

Molecular heterogeneity among north Indian isolates of Group A Streptococcus

AIM: To monitor molecular heterogeneity among the clinical isolates of group A Streptococcus (GAS) from north India by Vir and emm typing. METHODS AND RESULTS: GAS isolates, 31 from pharyngitis and nine from rheumatic fever (RF)/rheumatic heart disease (RHD) patients were differentiated into 16 Vir types (VT). These isolates were further discriminated into 23 emm types. Most of emm types were Vir type specific, except few (7.5%), which revealed different Vir types within same emm type. The most prevalent emm type found was emm 49 (15%) followed by 7.5% of emm 69, emm 71 and emm 75 which were different from emm type distribution reported from south India. CONCLUSIONS: Analysis of data revealed 40% heterogeneity by Vir typing and 57.5% by emm typing among GAS isolates which is significant in view of small number of isolates studied. SIGNIFICANCE OF IMPACT OF THE STUDY: The molecular study for the first time demonstrates different emm types prevalent and circulating in northern region of India and such data may help in selection of types for vaccine development.