Inter-species gene flow drives ongoing evolution of Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis.

Streptococcus dysgalactiae subsp. equisimilis (SDSE) is an emerging cause of human infection with invasive disease incidence and clinical manifestations comparable to the closely related species, Streptococcus pyogenes. Through systematic genomic analyses of 501 disseminated SDSE strains, we demonstrate extensive overlap between the genomes of SDSE and S. pyogenes. More than 75% of core genes are shared between the two species with one third demonstrating evidence of cross-species recombination. Twenty-five percent of mobile genetic element (MGE) clusters and 16 of 55 SDSE MGE insertion regions were shared across species. Assessing potential cross-protection from leading S. pyogenes vaccine candidates on SDSE, 12/34 preclinical vaccine antigen genes were shown to be present in >99% of isolates of both species. Relevant to possible vaccine evasion, six vaccine candidate genes demonstrated evidence of inter-species recombination. These findings demonstrate previously unappreciated levels of genomic overlap between these closely related pathogens with implications for streptococcal pathobiology, disease surveillance and prevention.

Author Correction: Atlas of group A streptococcal vaccine candidates compiled using large-scale comparative genomics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Atlas of group A streptococcal vaccine candidates compiled using large-scale comparative genomics.

Group A Streptococcus (GAS; Streptococcus pyogenes) is a bacterial pathogen for which a commercial vaccine for humans is not available. Employing the advantages of high-throughput DNA sequencing technology to vaccine design, we have analyzed 2,083 globally sampled GAS genomes. The global GAS population structure reveals extensive genomic heterogeneity driven by homologous recombination and overlaid with high levels of accessory gene plasticity. We identified the existence of more than 290 clinically associated genomic phylogroups across 22 countries, highlighting challenges in designing vaccines of global utility. To determine vaccine candidate coverage, we investigated all of the previously described GAS candidate antigens for gene carriage and gene sequence heterogeneity. Only 15 of 28 vaccine antigen candidates were found to have both low naturally occurring sequence variation and high (>99%) coverage across this diverse GAS population. This technological platform for vaccine coverage determination is equally applicable to prospective GAS vaccine antigens identified in future studies.

Extensive diversity of Streptococcus pyogenes in a remote human population reflects global-scale transmission rather than localised diversification.

The Indigenous population of the Northern Territory of Australia (NT) suffers from a very high burden of Streptococcus pyogenes disease, including cardiac and renal sequelae. The aim of this study was to determine if S. pyogenes isolated from this population represent NT endemic strains, or conversely reflect strains with global distribution. emm sequence typing data were used to select 460 S. pyogenes isolates representing NT S. pyogenes diversity from 1987-2008. These isolates were genotyped using either multilocus sequence typing (MLST) or a high resolution melting-based MLST surrogate (Minim typing). These data were combined with MLST data from other studies on NT S. pyogenes to yield a set of 731 MLST or Minim typed isolates for analysis. goeBURST analysis of MLST allelic profiles and neighbour-joining trees of the MLST allele sequences revealed that a large proportion of the known global S. pyogenes MLST-defined diversity has now been found in the NT. Specifically, fully sequence typed NT isolates encompass 19% of known S. pyogenes STs and 43% of known S. pyogenes MLST alleles. These analyses provided no evidence for major NT-endemic strains, with many STs and MLST alleles shared between the NT and the rest of the world. The relationship between the number of known Minim types, and the probability that a Minim type identified in a calendar year would be novel was determined. This revealed that Minim types typically persist in the NT for >1 year, and indicate that the majority of NT Minim types have been identified. This study revealed that many diverse S. pyogenes strains exhibit global scale mobility that extends to isolated populations. The burden of S. pyogenes disease in the NT is unlikely to be due to the nature of NT S. pyogenes strains, but is rather a function of social and living conditions.

Acute post-streptococcal glomerulonephritis in the Northern Territory of Australia: a review of 16 years data and comparison with the literature.

Data relating to acute post-streptococcal glomerulonephritis (APSGN) from the notifiable diseases surveillance system in the Northern Territory of Australia was extracted and analyzed. Isolates of Streptococcus pyogenes from confirmed cases were emm sequence typed. From 1991 to July 2008, there were 415 confirmed cases and 23 probable cases of APSGN notified. Four hundred fifteen (94.7%) of these were Indigenous Australians and 428 (97.7%) were people living in remote or very remote locations. The median age of cases was 7 years (range 0-54). The incidence of confirmed cases was 12.5/100,000 person-years, with an incidence in Indigenous Australian children younger than 15 years of age of 94.3 cases/100,000 person-years. The overall rate ratio of confirmed cases in Indigenous Australians to non-Indigenous Australians was 53.6 (95% confidence interval 32.6-94.8). Outbreaks of disease across multiple communities occurred in 1995 (N = 68), 2000 (N = 55), and 2005 (N = 87 [confirmed cases]). Various emm types of S. pyogenes were isolated from cases of APSGN including some types not previously recognized to be nephritogenic. The widespread outbreak in 2005 was caused by emm55.0 S. pyogenes. Acute post-streptococcal glomerulonephritis continues to occur in remote Indigenous communities in Australia at rates comparable to or higher than those estimated in developing countries. Improvements in preventative and outbreak control strategies are needed.

Diversity of emm sequence types in group A beta-haemolytic streptococci in two remote Northern Territory Indigenous communities: implications for vaccine development.

There is a high burden of disease due to group A streptococcus (GAS) in remote Northern Territory (NT) Indigenous communities. A proposed 26-valent GAS M-type vaccine covers 80-90% of pharyngeal and invasive isolates in the US. We examined the diversity and distribution of emm types in two remote Indigenous communities in the NT Top End over a 17-year period and compared them to the proposed vaccine types. Eighty emm types were identified between 1991 and 2007. Diversity in both communities was high (overall Simpson's index 0.976), but varied between communities. Prior to 2004, 71 emm types were identified and an additional 9 emm types were identified during a period of active surveillance in 2004-2005. The proposed 26-valent vaccine would be expected to cover only 20% of emm types recovered in this study. Of the 80 emm types, 16 (20%) were new sequence types identified since the last assignment of M types in 2002. The diversity of streptococcal isolates was higher than that reported from most industrialized countries, and similar to that described in several developing countries. A vaccine based on such a variable antigen is unlikely to provide effective protection in the highest risk populations.

Autoantigens identified by screening a human heart cDNA library with acute rheumatic fever sera.

Acute rheumatic fever (ARF) is an autoimmune sequela of group A streptococcal infection mostly affecting school-aged children. Recurrent episodes of ARF can result in the development of rheumatic heart disease (RHD). One in 40 indigenous Australians in the Northern Territory is affected by RHD. This disease mostly impacts young people; 45% of those who require heart valve surgery in Australia due to RHD are younger than 25 years old. ARF is characterized by autoimmune attack of the heart; therefore, the presence of the autoantibodies involved could potentially be used to diagnose ARF. To this end, a human heart cDNA library was screened with serum from a patient with ARF, and 12 autoreactive human heart antigens were identified. They include five different IgG heavy chains and a range of tissue-specific cell-signaling proteins, species of which have been implicated in other autoimmune diseases. Preliminary ELISA results show that ARF patients have significantly higher levels of antibodies recognizing the cardiac autoantigens than controls. These antigens are promising candidates for the development of a serological assay for the diagnosis of ARF. The nature of the proteins identified has exciting implications for future research into the pathogenesis of ARF.

Epidemiology of Streptococcus dysgalactiae subsp. equisimilis in tropical communities, Northern Australia.

Streptococcus dysgalactiae subsp. equisimilis (groups C and G streptococci [GCS/GGS]) is an increasingly recognized human pathogen, although it may follow indirect pathways. Prospective surveillance of selected households in 3 remote Aboriginal communities in Australia provided 337 GCS/GGS isolates that were emm sequence-typed. Lancefield group C isolates (GCS) were localized to specific households and group G isolates (GGS) were more evenly distributed. GCS/GGS was more frequently recovered from the throat than group A streptococci (GAS [S. pyogenes]) but rarely recovered from skin sores, and then only with Staphylococcus aureus or GAS. Symptomatic GGS/GGC pharyngitis was also rare. Specific emm sequence types of GCS/GGS did not appear to cycle through the communities (sequential strain replacement) in a manner suggesting acquisition of type-specific immunity. These communities already have high levels of streptococcal and poststreptococcal disease. GCS/GGS may increase in importance as it acquires key virulence factors from GAS by lateral gene transfer.

Low rates of streptococcal pharyngitis and high rates of pyoderma in Australian aboriginal communities where acute rheumatic fever is hyperendemic.

BACKGROUND: Acute rheumatic fever is a major cause of heart disease in Aboriginal Australians. The epidemiology differs from that observed in regions with temperate climates; streptococcal pharyngitis is reportedly rare, and pyoderma is highly prevalent. A link between pyoderma and acute rheumatic fever has been proposed but is yet to be proven. Group C beta-hemolytic streptococci and group G beta-hemolytic streptococci have also been also implicated in the pathogenesis. METHODS: Monthly, prospective surveillance of selected households was conducted in 3 remote Aboriginal communities. People were questioned about sore throat and pyoderma; swab specimens were obtained from all throats and any pyoderma lesions. Household population density was determined. RESULTS: From data collected during 531 household visits, the childhood incidence of sore throat was calculated to be 8 cases per 100 person-years, with no cases of symptomatic group A beta-hemolytic streptococci pharyngitis. The median point prevalence for throat carriage was 3.7% for group A beta-hemolytic streptococci, 0.7% for group C beta-hemolytic streptococci, and 5.1% for group G beta-hemolytic streptococci. Group A beta-hemolytic streptococci were recovered from the throats of 19.5% of children at some time during the study. There was no seasonal trend or correlation with overcrowding. Almost 40% of children had pyoderma at least once, and the prevalence was greatest during the dry season. In community 1, the prevalence of pyoderma correlated with household crowding. Group C and G beta-hemolytic streptococci were rarely recovered from pyoderma lesions. CONCLUSIONS: These data are consistent with the hypothesis that recurrent skin infections immunize against throat colonization and infection. High rates of acute rheumatic fever were not driven by symptomatic group A beta-hemolytic streptococci throat infection. Group G and C beta-hemolytic streptococci were found in the throat but rarely in pyoderma lesions.

Fibronectin-binding protein gene recombination and horizontal transfer between group A and G streptococci.

We report evidence of interspecies gene transfer between the important virulence factor genes sfbI and gfbA. Because the identified group G streptococcus gfbA types possess DNA cassettes that can be identified in a number of group A streptococcus strains, it appears that homologous recombination is occurring between these species.

Inhibition of Streptococcus pyogenes adherence to HaCaT cells by a peptide corresponding to the streptococcal fibronectin-binding protein, SfbI, is strain dependent.

Streptococcus pyogenes (group A streptococcus, GAS) is a human-specific pathogen, which employs a large number of adhesins for colonization. Fibronectin-binding proteins (FBPs) play a major role in GAS adhesion to host cells. SfbI, a major streptococcal FBP, has been well studied. A peptide (peptide-MSG) based on this adhesin inhibits fibronectin (Fn)-binding by the pathogen. To test whether this peptide also inhibits adherence of GAS to host cells, adhesion assays were performed with strains possessing different combinations of genes for three distinct FBPs. Peptide-MSG inhibited GAS adherence to human keratinocytes (HaCaT) in a strain dependent manner. There is no consistent pattern between the effect and the ability to express one or more of the FBPs. A single peptide may be insufficient to prevent GAS adherence to host cells.

Plasminogen binding by group A streptococcal isolates from a region of hyperendemicity for streptococcal skin infection and a high incidence of invasive infection.

Reports of resurgence in invasive group A streptococcal (GAS) infections come mainly from affluent populations with infrequent exposure to GAS. In the Northern Territory (NT) of Australia, high incidence of invasive GAS disease is secondary to endemic skin infection, serotype M1 clones are rare in invasive infection, the diversity and level of exposure to GAS strains are high, and no particular strains dominate. Expression of a plasminogen-binding GAS M-like protein (PAM) has been associated with skin infection in isolates elsewhere (D. Bessen, C. M. Sotir, T. M. Readdy, and S. K. Hollingshead, J. Infect. Dis. 173:896-900, 1996), and subversion of the host plasminogen system by GAS is thought to contribute to invasion in animal models. Here, we describe the relationship between plasminogen-binding capacity of GAS isolates, PAM genotype, and invasive capacity in 29 GAS isolates belonging to 25 distinct strains from the NT. In the presence of fibrinogen and streptokinase, invasive isolates bound more plasminogen than isolates from uncomplicated infections (P < or = 0.004). Only PAM-positive isolates bound substantial levels of plasminogen by a fibrinogen-streptokinase-independent pathway (direct binding). Despite considerable amino acid sequence variation within the A1 repeat region of PAM where the plasminogen-binding domain maps, the critical lysine residue was conserved.

Evolution of sfbI encoding streptococcal fibronectin-binding protein I: horizontal genetic transfer and gene mosaic structure.

Streptococcal fibronectin-binding protein is an important virulence factor involved in colonization and invasion of epithelial cells and tissues by Streptococcus pyogenes. In order to investigate the mechanisms involved in the evolution of sfbI, the sfbI genes from 54 strains were sequenced. Thirty-four distinct alleles were identified. Three principal mechanisms appear to have been involved in the evolution of sfbI. The amino-terminal aromatic amino acid-rich domain is the most variable region and is apparently generated by intergenic recombination of horizontally acquired DNA cassettes, resulting in a genetic mosaic in this region. Two distinct and divergent sequence types that shared only 61 to 70% identity were identified in the central proline-rich region, while variation at the 3' end of the gene is due to deletion or duplication of defined repeat units. Potential antigenic and functional variabilities in SfbI imply significant selective pressure in vivo with direct implications for the microbial pathogenesis of S. pyogenes.

The pyruvate dehydrogenase complex of Mycoplasma hyopneumoniae contains a novel lipoyl domain arrangement.

The genes encoding the pyruvate dehydrogenase (PDH) complex (pdhA, pdhB, pdhC and pdhD) from Mycoplasma hyopneumoniae have been cloned and sequenced. The genes are arranged into two operons, designated pdhAB and pdhCD, which are not found together in the chromosome. The pdhA, pdhB, pdhC and pdhD genes encode proteins of predicted molecular masses of 44.2 kDa (pyruvate dehydrogenase major subunit; E1alpha), 36.6 kDa (pyruvate dehydrogenase minor subunit; E1beta), 33.1 kDa (dihydrolipoyl acetyltransferase; E2) and 66.3 kDa (dihydrolipoyl dehydrogenase; E3), respectively. Sequence analysis of the pdhCD operon revealed the presence of a lipoyl-binding domain in pdhD but not in pdhC. The lipoyl domain is believed to act as a "swinging arm" that spans the gaps between the catalytic domains of each of the subunits. Portions of the N-terminal regions of pdhA and pdhD were expressed as 6xHis-tag fusion proteins in Escherichia coli and purified by nickel affinity chromatography. The purified proteins were used to raise antibodies in rabbits, and Western blot analysis was performed with the polyclonal rabbit antiserum. Both the pdhA and pdhD genes were expressed among various strains of M. hyopneumoniae as well as the porcine mycoplasmas, Mycoplasma hyorhinis and Mycoplasma flocculare. Southern hybridisation analysis using probes from pdhA and pdhD detected one copy of each gene in the chromosome of M. hyopneumoniae. Since previous studies have shown pyruvate dehydrogenase activity in M. hyopneumoniae [J. Gen. Microbiol. 134 (1988) 791], it appears likely that a functional lipoyl-binding domain in the N terminus of PdhC is not an absolute prerequisite for pyruvate dehydrogenase enzyme activity. We hypothesise that the lipoyl-binding domain of PdhD is performing the enzymatic function normally attributed to the PdhC lipoyl-binding domain in other organisms. Searches of pyruvate dehydrogenase gene sequences derived from other Mycoplasma species showed that a putative lipoyl domain was absent in the pdhC gene from Mycoplasma pulmonis. However, like other bacterial species, pdhC gene sequences from Mycoplasma capricolum, Mycoplasma genitalium and Mycoplasma pneumoniae contain a putative lipoyl domain.

Immunological response mounted by Aboriginal Australians living in the Northern Territory of Australia against Streptococcus pyogenes serum opacity factor.

Streptococcus pyogenes (Group A streptococcus) interacts with host fibronectin via a number of distinct surface components. The streptococcal serum opacity factor (SOF) is a cell-surface protein of S. pyogenes which causes opalescence of human serum and mediates bacterial binding to fibronectin. In this study, hexahistidyl-tagged fusion proteins encompassing full-length SOF, and domains of SOF encompassing opacity factor activity and fibronectin-binding regions, were used in the characterization of the Aboriginal immune response to SOF. Anti-SOF serum IgG responses were found to be significantly higher (P<0.0001) in Aboriginal adults and children when compared to a non-Aboriginal adult group. The Aboriginal immune response against the fibronectin-binding region of SOF was significantly reduced when compared to the response against the whole SOF protein and N-terminal domains examined in this study (P<0.001). This pattern of immune response was also observed in rabbits immunized with recombinant SOF. Comparison of the deduced amino acid sequence of SOF from a number of common Australian isolates with other SOF sequences revealed that the N-terminus of SOF exhibits sequence similarity values ranging from 42.9% to 96.5%. The C-terminus containing the fibronectin-binding domain and membrane-spanning regions was more highly conserved, exhibiting sequence similarity values ranging from 84.6% to 100% within the fibronectin-binding repeats. These data suggest that the immune response against SOF is directed toward the variable N-terminus of the SOF protein. Phylogenetic analysis indicated that the sof genes of S. pyogenes do not exhibit geographical variation.