Phage 3396 from a Streptococcus dysgalactiae subsp. equisimilis pathovar may have its origins in streptococcus pyogenes

Streptococcus dysgalactiae subsp. equisimilis strains (group G streptococcus [GGS]) are largely defined as commensal organisms, which are closely related to the well-defined human pathogen, the group A streptococcus (GAS). While lateral gene transfers are emerging as a common theme in these species, little is known about the mechanisms and role of these transfers and their effect on the population structure of streptococci in nature. It is now becoming evident that bacteriophages are major contributors to the genotypic diversity of GAS and, consequently, are pivotal to the GAS strain structure. Furthermore, bacteriophages are strongly associated with altering the pathogenic potential of GAS. In contrast, little is know about phages from GGS and their role in the population dynamics of GGS. In this study we report the first complete genome sequence of a GGS phage, Phi3396. Exhibiting high homology to the GAS phage Phi315.1, the chimeric nature of Phi3396 is unraveled to reveal evidence of extensive ongoing genetic diversity and dissemination of streptococcal phages in nature. Furthermore, we expand on our recent findings to identify inducible Phi3396 homologues in GAS from a region of endemicity for GAS and GGS infection. Together, these findings provide new insights into not only the population structure of GGS but also the overall population structure of the streptococcal genus and the emergence of pathogenic variants.     

Superantigen-like gene(s) in human pathogenic Streptococcus dysgalactiae,subsp equisimilis: genomic localisation of the gene encoding streptococcal pyrogenic exotoxin G (speG(dys))

Streptococcus pyogenes (GAS) causes about 90% of streptococcal human infections while group C (GCS) and G (GGS) streptococci can be pathogenic for different mammalians. Especially the human pathogenic GCS and GGS, Streptococcus dysgalactiae, subsp. equisimilis, account for 5-8% of the human streptococcal diseases like wound infections, otitis media, purulent pharyngitis and also streptococcal toxic shock syndrome. A defined superantigen so far was not identified in GCS and GGS strains. In the present investigation we screened DNA of GCS and GGS human isolates for the presence of genes for streptococcal pyrogenic exotoxins (spe) by hybridisation with probes that stand for the GAS genes speA, speC, speZ (smeZ), speH, speG, speI, speJ and ssa. In many GCS and GGS strains we found positive reactions with the probes speG, speJ and ssa, but not with the probes for the remaining genes under investigation. PCR amplification with subsequent sequence analysis of the PCR fragments revealed only the presence of the gene speG in GCS and GGS strains, while no DNA fragments specific for speJ and ssa could be amplified. Additionally, the upstream and downstream regions flanking speG in GGS strain 39072 were sequenced. Remarkable differences were found in the neighbourhood of speG between GAS and GGS sequences. Downstream of speG we identified in strain GGS 39072 two new open reading frames encoding proteins with no similarity to protein sequences accessible in the databases so far. In the compared GAS strains SF370 and MGAS8232, this segment, apart from some small fragments, had been deleted. Our analysis suggests that a gene transfer from GGS to GAS has preceded following deletion of the two genes orf1 and orf2 in GAS.     

Presence of streptococcal pyrogenic exotoxin A and C genes in human isolates of group G streptococci

The bacteriophage-associated genes speA and speC encode streptococcal pyrogenic exotoxins of group A streptococci (GAS). Human isolates of group C and G streptococci (GCS and GGS) are commensals and the closest known genetic relatives of GAS; on occasion, GCS-GGS can cause infection that is clinically similar to GAS disease. Thirty-four human isolates of GCS-GGS were tested for speA and speC. Two GGS isolates harbored speA only, whereas a third GGS had both genes. All spe alleles found in GGS were identical to known spe alleles of GAS, except for one speA allele, which was unique. The presence of shared speA and speC alleles in GAS and GGS is highly suggestive of recent interspecies transfer. Acquisition of GAS-like virulence genes by GGS may lead to enhanced pathogenicity in this usually commensal-like organism.     

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.     

Incompetence of neutrophils to invasive group A streptococcus is attributed to induction of plural virulence factors by dysfunction of a regulator

Group A streptococcus (GAS) causes variety of diseases ranging from common pharyngitis to life-threatening severe invasive diseases, including necrotizing fasciitis and streptococcal toxic shock-like syndrome. The characteristic of invasive GAS infections has been thought to attribute to genetic changes in bacteria, however, no clear evidence has shown due to lack of an intriguingly study using serotype-matched isolates from clinical severe invasive GAS infections. In addition, rare outbreaks of invasive infections and their distinctive pathology in which infectious foci without neutrophil infiltration hypothesized us invasive GAS could evade host defense, especially neutrophil functions. Herein we report that a panel of serotype-matched GAS, which were clinically isolated from severe invasive but not from non-invaive infections, could abrogate functions of human polymorphnuclear neutrophils (PMN) in at least two independent ways; due to inducing necrosis to PMN by enhanced production of a pore-forming toxin streptolysin O (SLO) and due to impairment of PMN migration via digesting interleukin-8, a PMN attracting chemokine, by increased production of a serine protease ScpC. Expression of genes was upregulated by a loss of repressive function with the mutation of csrS gene in the all emm49 severe invasive GAS isolates. The csrS mutants from clinical severe invasive GAS isolates exhibited high mortality and disseminated infection with paucity of neutrophils, a characteristic pathology seen in human invasive GAS infection, in a mouse model. However, GAS which lack either SLO or ScpC exhibit much less mortality than the csrS-mutated parent invasive GAS isolate to the infected mice. These results suggest that the abilities of GAS to abrogate PMN functions can determine the onset and severity of invasive GAS infection.     

The two faces of Janus: virulence gene regulation by CovR/S in group A streptococci

The group A streptococcus (GAS) causes a variety of human diseases, including toxic shock syndrome and necrotizing fasciitis, which are both associated with significant mortality. Even the superficial self-limiting diseases caused by GAS, such as pharyngitis, impose a significant economic burden on society. GAS can cause a wide spectrum of diseases because it elaborates virulence factors that enable it to spread and survive in different environmental niches within the human host. The production of many of these virulence factors is directly controlled by the activity of the CovR/S two-component regulatory system. CovS acts in one direction as a kinase primarily to activate the response regulator CovR and repress the expression of major virulence factors and in the other direction as a phosphatase to permit gene expression in response to environmental changes that mimic conditions found during human infection. This Janus-like behaviour of the CovR/S system is recapitulated in the binding of CovR to the promoters that it directly regulates. Interactions between different faces of the CovR DNA binding domain appear to depend upon DNA sequence, leading to the potential for differential regulation of virulence gene expression.     

Genetic regulation of host responses to group A streptococcus in mice.

The group A streptococci (GAS, Streptococcus pyogenes) are important human pathogens which can cause a variety of diseases, ranging from mild infections to very severe invasive diseases. In recent years, evidence has been accumulated that host genetic factors have a major influence on the outcome of streptococcal infections. Variability in the degree of susceptibility of different inbred mouse strains to infection with GAS has demonstrated that the host genetic background largely determines the susceptibility of mice to this pathogen. This information is particularly useful for studying the immune mechanisms underlying disease susceptibility in mice, and provides an entry point for the identification of host defence loci. This paper reviews the recent advances in the characterisation of pathogenic mechanisms associated with the development of GAS-induced septic shock in the mouse model and outlines the current knowledge regarding the genetic control of immune responses to Group A streptococcus in mice.     

Variations in the distribution of genes encoding virulence and extracellular proteins in group A streptococcus are largely restricted to 11 genomic loci

Group A streptococcus (GAS) is a human pathogen associated with a wide range of human diseases that vary in symptoms and clinical severity. In this report we describe the use of a targeted low density array representing genes encoding classical virulence factors, purported virulence factors and other extracellular proteins to examine differences in the genetic profiles of 68 clinical GAS isolates. Of the 226 genes on the array (encoding 217 virulence factors or putative extracellular proteins and nine positive control house-keeping proteins) 62 had distributions that were statistically associated with specific GAS M-types. While 32 of these genes were bacteriophage related, the remaining 30 have not previously been described as bacteriophage associated. We show that these 'non-bacteriophage related' genes are found in 11 loci located in five greater chromosomal regions, often near classical GAS virulence factors, and often accompanied by genes associated with mobile genetic elements (MGEs). Many of these loci also demonstrated genetic variation within strains of the same M-type, suggesting these regions to be recombinatorial and mutational hotspots. Evidence for acquisition of genes from other species is also apparent in these loci. Our data suggests that imprecise recombination events involving MGEs not only result in acquisition of new genes, but can also result in deletion of flanking chromosomal genes. Thus MGE related events would appear to be the major contributor to variation of discrete virulence loci, which could account for the disease causing propensity of individual strains. We believe that profiling of the 11 loci could be a meaningful tool in epidemiological GAS typing studies.     

D-alanylation of teichoic acids promotes group a streptococcus antimicrobial peptide resistance, neutrophil survival, and epithelial cell invasion

Group A streptococcus (GAS) is a leading cause of severe, invasive human infections, including necrotizing fasciitis and toxic shock syndrome. An important element of the mammalian innate defense system against invasive bacterial infections such as GAS is the production of antimicrobial peptides (AMPs) such as cathelicidins. In this study, we identify a specific GAS phenotype that confers resistance to host AMPs. Allelic replacement of the dltA gene encoding d-alanine-d-alanyl carrier protein ligase in an invasive serotype M1 GAS isolate led to loss of teichoic acid d-alanylation and an increase in net negative charge on the bacterial surface. Compared to the wild-type (WT) parent strain, the GAS DeltadltA mutant exhibited increased susceptibility to AMP and lysozyme killing and to acidic pH. While phagocytic uptake of WT and DeltadltA mutants by human neutrophils was equivalent, neutrophil-mediated killing of the DeltadltA strain was greatly accelerated. Furthermore, we observed the DeltadltA mutant to be diminished in its ability to adhere to and invade cultured human pharyngeal epithelial cells, a likely proximal step in the pathogenesis of invasive infection. Thus, teichoic acid d-alanylation may contribute in multiple ways to the propensity of invasive GAS to bypass mucosal defenses and produce systemic infection.     

M protein of a Streptococcus dysgalactiae human wound isolate shows multiple binding to different plasma proteins and shares epitopes with keratin and human cartilage

Besides group A (GAS), Lancefield group C beta-haemolytic streptococci (GCS) have been implicated as a causative agent in outbreaks of purulent pharyngitis. In this study we have investigated a class CI M protein of a Streptococcus dysgalactiae1:256, revealed that 26% of these sera showed serological cross-reactivity between a 68-kDa cartilage protein and the N-terminal part of MC. Only 8% of the sera of healthy patients showed this property. In additional, MC also cross-reacted with antibodies recognising epidermal keratins. The cross-reacting 68-kDa protein from cartilage was different from human serum albumin, but was recognised with anti-vimentin immune serum. The MC was cloned and the gene sequenced. By using PCR, recombinant gene fragments encoding characteristic peptide fragments of MC were expressed in Escherichia coli. The peptides were used to map the binding sites for plasma proteins and to locate the cross-reacting epitopes on the MC molecule. In consequence, sequence alignments revealed that MC shared homologous regions with vimentin and different keratins. Our data, obtained with MC, suggest that not only infections with GAS but also infections with GCS and possibly GGS (the latter species can also produce class CI M-like proteins) may be responsible for the formation of streptococcal-associated sequel diseases.     

Streptococcal protein FOG, a novel matrix adhesin interacting with collagen I in vivo

Group G streptococcus (GGS) is a human pathogen of emerging clinical significance. It causes skin and soft tissue infections, occasionally resulting in life-threatening conditions such as sepsis and necrotizing fasciitis. We recently identified FOG, a novel surface protein of GGS with fibrinogen binding and immune evasion properties. Here we investigated the role of FOG in streptococcal primary adhesion to host tissue. A FOG-expressing clinical isolate adhered more efficiently to human skin biopsies ex vivo and to the murine dermis in vivo than a FOG-deficient strain. Scanning and transmission electron microscopy of skin specimens exhibited that this property was assigned to the ability of FOG to interact with collagen I, a major interstitial component of the dermis. Overlay experiments with human skin extracts and radiolabeled FOG followed by matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis identified both the alpha1- and alpha2-chains of collagen I as targets for FOG. Transmission electron microscopy of the molecular complexes revealed thread-like FOG molecules binding via their NH2 termini to distinct sites on collagen I monomers and fibrils. The results demonstrate that FOG is important for GGS adhesion in vivo, implying a pathogenic role for this surface protein.     

Structural determination of Streptococcus pyogenes M1 protein interactions with human immunoglobulin G using integrative structural biology

Streptococcus pyogenes (Group A streptococcus; GAS) is an important human pathogen responsible for mild to severe, life-threatening infections. GAS expresses a wide range of virulence factors, including the M family proteins. The M proteins allow the bacteria to evade parts of the human immune defenses by triggering the formation of a dense coat of plasma proteins surrounding the bacteria, including IgGs. However, the molecular level details of the M1-IgG interaction have remained unclear. Here, we characterized the structure and dynamics of this interaction interface in human plasma on the surface of live bacteria using integrative structural biology, combining cross-linking mass spectrometry and molecular dynamics (MD) simulations. We show that the primary interaction is formed between the S-domain of M1 and the conserved IgG Fc-domain. In addition, we show evidence for a so far uncharacterized interaction between the A-domain and the IgG Fc-domain. Both these interactions mimic the protein G-IgG interface of group C and G streptococcus. These findings underline a conserved scavenging mechanism used by GAS surface proteins that block the IgG-receptor (FcγR) to inhibit phagocytic killing. We additionally show that we can capture Fab-bound IgGs in a complex background and identify XLs between the constant region of the Fab-domain and certain regions of the M1 protein engaged in the Fab-mediated binding. Our results elucidate the M1-IgG interaction network involved in inhibition of phagocytosis and reveal important M1 peptides that can be further investigated as future vaccine targets.     

Population genomic analysis of strain variation in Leptospirillum group II bacteria involved in acid mine drainage formation

Deeply sampled community genomic (metagenomic) datasets enable comprehensive analysis of heterogeneity in natural microbial populations. In this study, we used sequence data obtained from the dominant member of a low-diversity natural chemoautotrophic microbial community to determine how coexisting closely related individuals differ from each other in terms of gene sequence and gene content, and to uncover evidence of evolutionary processes that occur over short timescales. DNA sequence obtained from an acid mine drainage biofilm was reconstructed, taking into account the effects of strain variation, to generate a nearly complete genome tiling path for a Leptospirillum group II species closely related to L. ferriphilum (sampling depth approximately 20x). The population is dominated by one sequence type, yet we detected evidence for relatively abundant variants (>99.5% sequence identity to the dominant type) at multiple loci, and a few rare variants. Blocks of other Leptospirillum group II types ( approximately 94% sequence identity) have recombined into one or more variants. Variant blocks of both types are more numerous near the origin of replication. Heterogeneity in genetic potential within the population arises from localized variation in gene content, typically focused in integrated plasmid/phage-like regions. Some laterally transferred gene blocks encode physiologically important genes, including quorum-sensing genes of the LuxIR system. Overall, results suggest inter- and intrapopulation genetic exchange involving distinct parental genome types and implicate gain and loss of phage and plasmid genes in recent evolution of this Leptospirillum group II population. Population genetic analyses of single nucleotide polymorphisms indicate variation between closely related strains is not maintained by positive selection, suggesting that these regions do not represent adaptive differences between strains. Thus, the most likely explanation for the observed patterns of polymorphism is divergence of ancestral strains due to geographic isolation, followed by mixing and subsequent recombination.     

Type I Polyketide Synthases May Have Evolved Through Horizontal Gene Transfer

Type I polyketide synthases (PKSI) are modular multidomain enzymes involved in the biosynthesis of many natural products of industrial interest. PKSI modules are minimally organized in three domains: ketosynthase (KS), acyltransferase (AT), and acyl carrier protein. The KS domain phylogeny of 23 PKSI clusters was determined. The results obtained suggest that many horizontal transfers of PKSI genes have occurred between actinomycetales species. Such gene transfers may explain the homogeneity and the robustness of the actinomycetales group since gene transfers between closely related species could mimic patterns generated by vertical inheritance. We suggest that the linearity and instability of actinomycetales chromosomes associated with their large quantity of genetic mobile elements have favored such horizontal gene transfers.Reviewing Editor : Dr. Nicolas Galtier     

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.     

Differences in the aromatic domain of homologous streptococcal fibronectin-binding proteins trigger different cell invasion mechanisms and survival rates

Group A streptococci (GAS, Streptococcus pyogenes) and Group G streptococci (GGS, Streptococcus dysgalactiae ssp. equisimilis) adhere to and invade host cells by binding to fibronectin. The fibronectin-binding protein SfbI from GAS acts as an invasin by using a caveolae-mediated mechanism. In the present study we have identified a fibronectin-binding protein, GfbA, from GGS, which functions as an adhesin and invasin. Although there is a high degree of similarity in the C-terminal sequence of SfbI and GfbA, the invasion mechanisms are different. Unlike caveolae-mediated invasion by SfbI-expressing GAS, the GfbA-expressing GGS isolate trigger cytoskeleton rearrangements. Heterologous expression of GfbA on the surface of a commensal Streptococcus gordonii and purified recombinant protein also triggered actin rearrangements. Expression of a truncated GfbA (lacking the aromatic domain) and chimeric GfbA/SfbI protein (replacing the aromatic domain of SfbI with the GfbA aromatic domain) on S. gordonii or recombinant proteins alone showed that the aromatic domain of GfbA is responsible for different invasion mechanisms. This is the first evidence for a biological function of the aromatic domain of fibronectin-binding proteins. Furthermore, we show that streptococci invading via cytoskeleton rearrangements and intracellular trafficking along the classical endocytic pathway are less persistence than streptococci entering via caveolae.     

The Mga virulence regulon: infection where the grass is greener

Co-ordinate regulation of virulence gene expression in response to different host environments is central to the success of the group A streptococcus (GAS, Streptococcus pyogenes) as an important human pathogen. Mga represents a ubiquitous stand-alone virulence regulator that controls genes (Mga regulon) whose products are necessary for adherence, internalization and host immune evasion. Mga highly activates a core set of virulence genes, including its own gene, by directly binding to their promoters. Yet, Mga also influences expression of over 10% of the GAS genome, primarily genes and operons involved in metabolism and sugar utilization. Expression of the Mga regulon is influenced by conditions that signify favourable growth conditions, presumably allowing GAS to take advantage of promising new niches in the host. The ability of Mga to respond to growth signals clearly involves regulation of mga expression via global regulatory networks such as RALPs, Rgg/RopB and the catabolite control protein CcpA. However, the presence of predicted PTS regulatory domains (PRDs) within Mga suggests an intriguing model whereby phosphorylation of Mga by the PTS phosphorelay might link growth and sugar utilization with virulence in GAS. As Mga homologues have been found in several important Gram-positive pathogens, the Mga regulon could provide a valuable paradigm for increasing our understanding of global virulence networks in bacteria.     

Group A streptococcus cell-associated pathogenic proteins as revealed by growth in hyaluronic acid-enriched media

Group A streptococcus (GAS), also know as Streptococcus pyogenes, is a human pathogen and can cause several fatal invasive diseases such as necrotising fasciitis, the so-called flesh-eating disease, and toxic shock syndrome. The destruction of connective tissue and the hyaluronic acid (HA) therein, is a key element of GAS pathogenesis. We therefore propagated GAS in HA-enriched growth media in an attempt to create a simple biological system that could reflect some elements of GAS pathogenesis. Our results show that several recognised virulence factors were up-regulated in HA-enriched media, including the M1 protein, a collagen-like surface protein and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, which has been shown to play important roles in streptococcal pathogenesis. Interestingly, two hypothetical proteins of unknown function were also up-regulated and detailed bioinformatics analysis showed that at least one of these hypothetical proteins is likely to be involved in pathogenesis. It was therefore concluded that this simple biological system provided a valuable tool for the identification of potential GAS virulence factors.