Biology subjects

Turner, C. E.

Publications and source records attributed to Turner, C. E..

14 recordsLinked to original sources

bioRxiv2026

M1 protein expression determines niche-specific spread of Streptococcus pyogenes

Invasive infections with Streptococcus pyogenes account for significant mortality. Cell surface M protein is believed to be essential for S. pyogenes virulence, with critical roles in dissemination and immune evasion. In contrast, the role of secreted M protein in infection is less clear. Here, we assessed the role of M protein in streptococcal colonisation, persistence, and invasion in respiratory and soft tissue murine infection models, and report that while cell-surface M protein is required to establish nasopharyngeal infection, it does not drive invasive infection. Cell-surface M protein was important for binding of major host matrix proteins and growth in immune human blood, but soluble M protein was the predominant determinant of lymphatic survival, tissue spread, and systemic infection in mice. A mutant emm1 strain which only expresses secreted M protein exhibited enhanced survival in the lymph node niche and overall competitive advantage over the wildtype parent strain in co-infections. Absence of M protein at the bacterial cell surface led to enhanced hyaluronan binding, providing explanation for homing to the draining lymph node. Together, these findings highlight potential nuance in the role of M protein that may impact vaccine approaches targeting M protein. Author SummaryS. pyogenes is a significant cause of illness, both mild and lethal, worldwide. Despite this, there are currently no approved vaccines. Many vaccine candidates are based on the M protein, a surface expressed anti-phagocytic factor that is thought to be critical for the ability of S. pyogenes to evade the host immune system. The unexpected selection of an M1 protein mutant strain following in vivo soft tissue infection raised many questions for us. If M protein is meant to be the critical factor in avoiding immune killing, how was this strain able to persist? One explanation could be that, although the strain was negative for M1 protein on the bacterial surface, it still secreted a truncated version of the protein with potential inflammatory effects. Importantly, the strain was selected in the draining lymph node pointing to a niche-specific advantage. We showed that cell surface-associated M1 protein, although required to establish nasopharyngeal infection, was not required in soft tissue infection, and that expression of a soluble, truncated form of M1 protein gave a survival advantage over the wild-type strain expressing cell wall-associated M1 protein. Furthermore, the absence of cell surface M protein led to enhanced hyaluronan binding providing explanation for lymphatic niche homing.

microbiology↗

bioRxiv2026

Molecular analysis of Lancefield group C/G streptococci causing human infections in Sheffield, UK.

Lancefield group C/G streptococci (GCS/GGS) are increasingly recognised as significant human pathogens that cause a disease spectrum similar to Streptococcus pyogenes. Despite their high clinical burden in the UK, their genomic diversity remains poorly understood. We performed whole-genome sequencing (WGS) on a prospective collection of 109 consecutive GCS/GGS isolates from all infection types in Sheffield, UK, over five months in 2020. Streptococcus dysgalactiae subsp. equisimilis (SDSE) accounted for 104 isolates, while five were identified as Streptococcus canis. The SDSE population was highly diverse, comprising 15 genomic clusters and 38 unique emm-ST combinations. We identified the presence of the ST20/stG62647 international lineage (24% of isolates), a cluster globally associated with severe invasive disease. Antimicrobial resistance genes were prevalent (49%), predominantly linked to mobile genetic elements carrying tetracycline and macrolide resistance. Furthermore, a variation in the penicillin-binding protein PBP2X (P601L) was linked to reduced penicillin sensitivity (MIC 0.03 mg/L). There were few or no genetic changes in isolates obtained from the same patient, even when they were collected 8-10 weeks apart, indicating long-term persistence within a host. The unexpected detection of S. canis in human infections and the high diversity of SDSE, persistence and virulence-associated regions underscore the need for enhanced national genomic surveillance to track emerging virulent and antibiotic-resistant SDSE lineages. Impact statementLancefield group C and G streptococci, most often the species Streptococcus dysgalactiae subsp. equisimilis (SDSE), are an increasingly significant human pathogen, often mirroring the severity of infections caused by Lancefield group A Streptococcus (S. pyogenes). Despite its clinical importance, we know little about the population of SDSE circulating in the UK. This study provides the first comprehensive genomic analysis of SDSE isolates from a single UK region, identifying a highly diverse population comprising 15 distinct genomic clusters but with evidence of long-term persistence within a single host. Notably, we confirm the presence of the international stG62647/ST20 lineage in the UK, which is globally associated with severe invasive disease. Our findings also reveal a high prevalence of antimicrobial resistance genes ([~]49%), primarily linked to mobile genetic elements, and the presence of a specific variation in the penicillin-binding protein PBP2X that reduces penicillin sensitivity. Additionally, the unexpected detection of S. canis in human infections rather than animals highlights a need for monitoring. By defining the UKs SDSE population structure and its resistance landscape, this research underscores the critical need for enhanced national genomic surveillance to track emerging high-virulence and antibiotic-resistant lineages Data summarySequence files for isolates from Sheffield used for this study have been uploaded to the sequence read archive with project accession number PRJNA1333937 and accession numbers provided in Supplementary Dataset 1. The completed genome for SDE096 been deposited on GenBank with the accession number JBSXMJ000000000.

microbiology↗

bioRxiv2025

A novel two-component system controls vancomycin resistance in epidemic Clostridioides difficile

The glycopeptide antibiotic vancomycin is the frontline treatment for C. difficile infection in the UK. There have been only sporadic reports of resistance in the clinic but testing is rare so the true resistance landscape is unclear. We have previously shown that resistance can emerge rapidly in vitro via distinct but complementary pathways. Strain Bc2, characterised here, is an experimentally evolved derivative of C. difficile strain R20291 that displays a 16-fold increase in vancomycin MIC over its parent. Bc2 has point mutations in dacS, bclA3, CDR20291_0794, CDR20291_1871 and CDR20291_3124 (vnrS). By genetically engineering a wild-type vancomycin susceptible strain, we demonstrated that a combination of just two mutations, dacSc.798A>T and vnrSc.692G>T, both of which encode two-component system histidine kinases, was sufficient to recapitulate Bc2 resistance. We have previously shown that mutations in dacS can confer low level resistance via increases in the expression of a D,D-carboxypeptidase DacJ. dacSc.798A>T also led to increased transcription of dacJ and led to a modest increase in vancomycin MIC. Surprisingly vnrSc.692G>T led to overexpression of the vanG cluster, which encodes all of the enzymes needed for resistance via substitution of the terminal D-Ala on peptidoglycan lipid II precursors with D-Ser. Genomic analysis of a large collection of European C. difficile strains showed that a three gene cluster, which includes vnrS, vnrR (encoding the cognate response regulator) and an adjacent gene CDR20291_3123, is unique to the phylogenetic branch that contains strains belonging to epidemic ribotypes 027 and 176. Transcriptomic analysis of the wider VnrS regulon also revealed an additional previously unknown role in regulating the expression of the flagellum, an important virulence factor in C. difficile. Analysis of the response to vancomycin exposure also revealed that dacJ is one of a small set of genes that are upregulated shortly after antibiotic stress, even in the absence of mutations that typically lead to its overexpression. Together these data reveal a new synergistic route, needing only two point mutations, by which epidemic lineages of C. difficile can attain vancomycin resistance. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=147 HEIGHT=200 SRC="FIGDIR/small/671617v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@bba369org.highwire.dtl.DTLVardef@e562acorg.highwire.dtl.DTLVardef@1b112deorg.highwire.dtl.DTLVardef@4dcf8a_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Molecular characterisation of Streptococcus pyogenes (StrepA) non-invasive isolates during the 2022-23 UK upsurge

At the end of 2022 into early 2023 the UK Health Security Agency reported unusually high levels of scarlet fever and invasive disease caused by Streptococcus pyogenes (StrepA or group A Streptococcus). During this time, we collected and genome sequenced 341 non-invasive throat and skin S. pyogenes isolates identified during routine clinical diagnostic testing in Sheffield, a large UK city. We compared the data with that obtained from a similar collection of 165 isolates from 2016-17. Numbers of throat-associated isolates collected peaked in early December 2022, reflecting the national scarlet fever upsurge, while skin infections peaked later in December. The most common emm-types in 2022-23 were emm1 (28.7%), emm12 (24.9%), and emm22 (7.7%) in throat; and emm1 (22%), emm12 (10%), emm76 (18%), and emm49 (7%) in skin. Whilst all emm1 isolates were the M1UK lineage, comparison with 2016-17 revealed diverse lineages in other emm-types, including emm12, and emergent lineages within other types including a new acapsular emm75 lineage, demonstrating that the upsurge was not completely driven by a single genotype. Analysis of the capsule locus predicted only 51% of throat isolates would produce capsule compared to 78% of skin isolates. 90% of throat isolates were also predicted to have high NADase and Streptolysin O (SLO) expression, based on the promoter sequence, compared to only 56% of skin isolates. Our study has highlighted the value in analysis of non-invasive isolates to characterise tissue tropisms, as well as changing strain diversity and emerging genomic features which may have implications for spillover into invasive disease and future S. pyogenes upsurges. Data summaryAll new genome sequence data is available on the NCBI short read archive under the bioproject PRJNA1062601 and individual accession numbers are listed in Supplementary Table 1 and Table 2. Impact statementThe human bacterial pathogen Streptococcus pyogenes, also known as group A Streptococcus or StrepA, caused a dramatic and sudden upsurge in scarlet fever in the UK at the end of 2022 into early 2023. We present molecular characterisation of this upsurge, through genome sequence analysis of throat, skin and other types of non-severe infection isolates collected by the microbiology diagnostic lab at the Northern General Hospital in Sheffield, England. We found that, whilst two strain types were the predominant cause of infections during the upsurge, other types had emerged or changed when compared to a similar collection from 2016-17. We also identified differences between throat-associated isolates and skin-associated isolates and highlighted important bacterial factors that might influence infection types. Isolates from non-severe throat/skin types of infections are rarely saved and therefore our knowledge of them is limited. However, here we demonstrate that study of such isolates may be key to understanding upsurges of more severe infections.

microbiology↗

bioRxiv2023

Multiple evolutionary pathways lead to vancomycin resistance in Clostridioides difficile

Clostridioides difficile is an important human pathogen, for which there are very limited treatment options, primarily the glycopeptide antibiotic vancomycin. In recent years vancomycin resistance has emerged as a serious problem in several Gram positive pathogens, but high level resistance has yet to be reported for C. difficile, although it is not known if this is due to constraints upon resistance evolution in this species. Here we show that resistance to vancomycin can evolve rapidly under ramping selection but is accompanied by severe fitness costs and pleiotropic trade-offs, including sporulation defects that would be expected to severely impact transmission. We identified two distinct pathways to resistance, both of which are predicted to result in changes to the muropeptide terminal D-Ala-D-Ala that is the primary target of vancomycin. One of these pathways involves a previously uncharacterised D,D-carboxypeptidase, expression of which is controlled by a dedicated two-component signal transduction system. Our findings suggest that while C. difficile is capable of evolving high-level vancomycin resistance, this outcome may be limited clinically due to pleiotropic effects on key pathogenicity trains. Moreover, our data provide a mutational roadmap to inform genomic surveillance.

microbiology↗

bioRxiv2023

CD9 co-operation with syndecan-1 is required for a major staphylococcal adhesion pathway

ObjectivesEpithelial colonisation is a critical first step in bacterial pathogenesis. Staphylococcus aureus can utilise several host factors to associate with cells, including 5{beta}l integrin and heparan sulphate proteoglycans, such as the syndecans. Here, we demonstrate that a partner protein of both integrins and syndecans, the host membrane adapter protein tetraspanin CD9, is essential for syndecan-mediated staphylococcal adhesion. Fibronectin is also essential in this process while integrins are only critical for post-adhesion entry into human epithelial cells. Methods and ResultsTreatment of epithelial cells with CD9-derived peptide or heparin caused significant reductions in staphylococcal adherence, dependent on both CD9 and syndecan-1. Exogenous fibronectin caused a CD9-dependent increase in staphylococcal adhesion whereas blockade of {beta}1 integrins did not affect adhesion but did reduce the subsequent internalisation of adhered bacteria. CD9 disruption or deletion increased {beta}1 integrin-mediated internalisation, suggesting that CD9 coordinates sequential staphylococcal adhesion and internalisation. ConclusionsCD9 controls staphylococcal adhesion through syndecan-1, using a mechanism that likely requires CD9-mediated syndecan organisation to correctly display fibronectin at the host cell surface. We propose that CD9-derived peptides or heparin analogues could be developed as anti-adhesion treatments to inhibit the initial stages of staphylococcal pathogenesis.

microbiology↗

bioRxiv2022

Characterisation of emergent toxigenic M1UK Streptococcus pyogenes and associated sublineages

Emm1 Streptococcus pyogenes is a successful, globally-distributed epidemic clone that is regarded as inherently invasive. An emm1 sublineage, M1UK, that expresses increased SpeA toxin, was associated with increased scarlet fever and invasive infections in England in 2015/2016. Defined by 27 SNPs in the core genome, M1UK is now dominant in England. To more fully characterise M1UK, we undertook comparative transcriptomic and proteomic analyses of M1UK and contemporary non-M1UK emm1 strains (M1global). Just seven genes were differentially expressed by M1UK compared with contemporary M1global strains. In addition to speA, five genes in the operon that includes glycerol dehydrogenase were upregulated in M1UK (gldA, mipB/talC, pflD, and pts system IIC and IIB components), while aquaporin (glpF2) was downregulated. M1UK strains have a stop codon in gldA. Deletion of the gldA gene in M1global abrogated glycerol dehydrogenase activity, and recapitulated upregulation of gene expression within the operon that includes gldA, consistent with a feedback effect. Phylogenetic analysis identified two intermediate emm1 sublineages in England comprising 13/27 (M113SNPs) and 23/27 SNPs (M123SNPs) respectively, that had failed to expand in the population. Proteomic analysis of these four major phylogenetic emm1 groups highlighted sublineage-specific changes in carbohydrate metabolism, protein synthesis and protein processing; upregulation of SpeA was not observed in chemically-defined medium. In rich broth however, transcription and secretion of SpeA was upregulated ~10-fold in both M123SNPs and M1UK sublineages, compared with M113SNPs and M1global. We conclude that stepwise accumulation of SNPs led to the emergence of M1UK. While increased expression of SpeA is a key indicator of M1UK and undoubtedly important, M1UK strains have outcompeted M123SNPs and other emm types that produce similar or more superantigen toxin. We speculate that an accumulation of adaptive SNPs has contributed to a wider fitness advantage in M1UK on an inherently successful emm1 streptococcal background. Data availabilityRNAseq. All new RNAseq data are uploaded to the European Nucleotide Archive under project reference PRJEB58303 Genomic data. All genomes listed are available on the European Nucleotide Archive using accession numbers as listed in the appendix, Proteomes. Proteomic data are available on FigShare 10.6084/m9.figshare.21777809 and will be uploaded to PRIDE Impact SummaryAlthough the major Streptococcus pyogenes reservoir is in children with pharyngitis and skin infections, S. pyogenes can lead to rarer, invasive infections that are rapidly progressive and associated with high mortality and morbidity. Emm1 S. pyogenes strains are the single most frequent genotype to cause invasive infections in high income countries and are established worldwide as an epidemic clone. The M1UK S. pyogenes emm1 sublineage which is defined by 27 new SNPs in the core genome, and characterised by increased scarlet fever toxin SpeA production, emerged and rose to dominance over a period of 5-6 years since initial recognition, outcompeting other emm1 strains in England. Increased dominance of emm1 among invasive infections this winter, on a background of already-increased numbers of S. pyogenes infections, points to a key shift in host-pathogen interaction. We hypothesize that a combination of pathogen fitness, virulence, and host susceptibility have coalesced to account for the excess of circulating S. pyogenes and emm1 invasive infections. In this paper we undertake a systems-based evaluation of M1UK in comparison to older non-M1UK emm1 strains, and identify a number of pathways that are altered in addition to the previously-reported increased SpeA expression. The emergence of a new sublineage within an already virulent clone requires ongoing surveillance, and more detailed investigation of the likely mechanisms leading to increased fitness. The capacity of S. pyogenes to cause outbreaks at national scale highlights a potential need to consider strain-specific public health guidance, underlining the inherent virulence of this exclusively human pathogen.

microbiology↗

bioRxiv2022

A histone methyltransferase point mutation demonstrates a mechanism for translocation-driven acute leukemias

Our understanding of acute leukemia pathology is heavily dependent on 11q23 chromosomal translocations involving the mixed lineage leukemia-1 (MLL1) gene, a key player in histone H3 lysine 4 (H3K4) methylation. These translocations result in distinct MLL1-fusion (MLL1F) proteins that are thought to drive leukemogenesis. However, the mechanism behind increased H3K4 trimethylation in MLL1F-leukemic stem cells (MLL1F-LSCs), following loss of catalytic SET domain of MLL1 (known for H3K4 mono- and dimethylation), remains unclear. In our investigation, we introduced a homozygous loss-of-function point mutation in MLL1 within human induced pluripotent stem cells. Remarkably, this mutation mimics the histone methylation, gene expression, and EMT phenotypes of MLL1F-LSCs- without the need for a translocation or functional wild-type MLL1. This observation underscores the essential role of MLL1s enzymatic activity in restraining the cascade of epigenetic changes associated with the gene activating H3K4 trimethylation mark, which we show is catalyzed by mislocalized SETd1a H3K4 trimethyltransferase in the absence of MLL1s enzymatic activity. Challenging existing models, our findings imply that MLL1F-induced leukemias arise from a dominant-negative impact on MLL1s histone methyltransferase activity. We advocate for a therapeutic paradigm shift, targeting SETd1a for precision medicine. This work opens new avenues for addressing the complexities of MLL1-associated leukemias and improving targeted therapies. SummaryEpigenomic and gene expression changes in iPS cells with a mutated MLL1 histone methyltransferase suggest an oncogenic mechanism for MLL1-fusion leukemias.

cancer biology↗

bioRxiv2021

Comparative genomic analysis of skin and soft tissue Streptococcus pyogenes isolates from low- and high-income settings

Streptococcus pyogenes is a leading cause of human morbidity and mortality, especially in resource limited settings. The World Health Organisation has recently made a vaccine for S. pyogenes a global health priority to reduce the burden of the post-infection rheumatic heart disease. For a vaccine to be active against all relevant strains in each region, molecular characterisation of circulating S. pyogenes isolates is needed. We performed extensive comparative whole genome analyses of S. pyogenes isolates from skin and soft tissue infections in The Gambia, West Africa, where there is a high burden of such infections. To act as a comparator to this low-income country (LIC) collection of isolates, we performed genome sequencing of isolates from skin infections in Sheffield, UK, as representative high-income country (HIC) isolates. LIC isolates from The Gambia were genetically more diverse (46 emm-types in 107 isolates) compared to HIC isolates from Sheffield (23 emm-types in 142 isolates), with only 7 overlapping emm-types and with diverse genetic backgrounds. Characterisation of other molecular markers indicated some shared features, including a high prevalence of the skin infection-associated emm-pattern D and the variable fibronectin-collagen-T antigen (FCT) types FCT-3 and FCT-4. A previously unidentified FCT (FCT-10) was identified in the LIC isolates, belonging to two different emm-types. A high proportion (79/107; 73.8%) of LIC isolates carried genes for tetracycline resistance, compared to 53/142 (37.3%) HIC isolates. There was also evidence of different circulating prophages, as very few prophage-associated DNases and lower numbers of superantigens were detected in LIC isolates. Our study provides much needed insight into the genetics of circulating isolates in a LIC (The Gambia), and how they differ from those circulating in HICs (Sheffield, UK). Common molecular features may act as bacterial drivers for specific infection types, regardless of the diverse genetic background.

microbiology↗

bioRxiv2021

Antibody Activities in Hyperimmune Plasma Against the Rhodococcus equi Virulence -Associated Protein A or Poly-N-Acetyl Glucosamine are Associated with Protection of Foals Against Rhodococcal Pneumonia

The efficacy of transfusion with hyperimmune plasma (HIP) for preventing pneumonia caused by Rhodococcus equi remains ill-defined. Quarter Horse foals at 2 large breeding farms were randomly assigned to be transfused with 2 L of HIP from adult donors hyperimmunized either with R. equi (RE HIP) or a conjugate vaccine eliciting antibody to the surface polysaccharide {beta}-1[->]6-poly-N-acetyl glucosamine (PNAG HIP) within 24 hours of birth. Antibody activities against PNAG and the rhodococcal virulence-associated protein A (VapA), and to deposition of complement component 1q (C1q) onto PNAG were determined by ELISA, and then associated with either clinical pneumonia at Farm A (n=119) or subclinical pneumonia at Farm B (n=114). Data were analyzed using multivariable logistic regression. Among RE HIP-transfused foals, the odds of pneumonia were approximately 6-fold higher (P = 0.0005) among foals with VapA antibody activity [≤] the population median. Among PNAG HIP-transfused foals, the odds of pneumonia were approximately 3-fold (P = 0.0347) and 11-fold (P = 0.0034) higher for foals with antibody activities [≤] the population median for PNAG or C1q deposition, respectively. Results indicated that levels of activity of antibodies against R. equi antigens are correlates of protection against both subclinical and clinical R. equi pneumonia in field settings. Among PNAG HIP-transfused foals, activity of antibodies with C1q deposition (an indicator of functional antibodies) were a stronger predictor of protection than was PNAG antibody activity alone. Collectively, these findings suggest that the amount and activity of antibodies in HIP (i.e., plasma volume and/or antibody activity) is positively associated with protection against R. equi pneumonia in foals.

immunology↗

bioRxiv2020

Vaccine-induced, but not natural immunity, against the Streptococcal Inhibitor of Complement protects against invasive disease

Highly pathogenic emm1 Streptococcus pyogenes strains secrete the multidomain Streptococcal inhibitor of complement (SIC) that binds and inactivates components of the innate immune response. We aimed to determine if naturally occurring or vaccine-induced antibodies to SIC are protective against invasive S. pyogenes infection. Immunisation with full length SIC protected mice against systemic bacterial dissemination following intranasal or intramuscular infection with emm1 S. pyogenes. Vaccine-induced rabbit anti-SIC antibodies, but not naturally occurring human anti-SIC antibodies, enhanced bacterial clearance in an ex vivo whole blood assay. SIC vaccination of both mice and rabbits resulted in antibody recognition of all domains of SIC, whereas naturally occurring human anti-SIC antibodies recognised the proline-rich region of SIC only. We therefore propose a model whereby natural infection with S. pyogenes generates non-protective antibodies against the proline-rich region of SIC, while vaccination with full length SIC permits development of protective antibodies against all SIC domains.

microbiology↗

bioRxiv2020

Cryptic prophages within a Streptococcus pyogenes genotype emm4 lineage

The major human pathogen Streptococcus pyogenes shares an intimate evolutionary history with mobile genetic elements, which in many cases, carry genes encoding bacterial virulence factors. During recent whole genome sequencing of a longitudinal sample of S. pyogenes isolates in the United Kingdom, we identified a lineage within emm4 that clustered with the reference genome MEW427. Like MEW427, this lineage was characterised by substantial gene loss within all three prophage regions, compared to MGAS10750 and isolates outside of the MEW427-like lineage. Gene loss primarily affected lysogeny, replicatory and regulatory modules, and to a lesser and more variable extent, structural genes. Importantly, prophage-encoded superantigen and DNase genes were retained in all isolates. In isolates where the prophage elements were complete, like MGAS10750, they could be induced experimentally, but not in MEW427-like isolates with degraded prophages. We also found gene loss within the chromosomal island SpyCIM4 of MEW427-like isolates, although surprisingly, the SpyCIM4 element could not be experimentally induced in either MGAS10750-like or MEW427-like isolates. This did not, however, appear to abolish expression of the mismatch repair operon, within which this element resides. The inclusion of further emm4 genomes in our analyses ratified our observations and revealed an international emm4 lineage characterised by prophage degradation. Intriguingly, the USA population of emm4 S. pyogenes appeared to constitute predominantly MEW427-like isolates, whereas the UK comprised both MEW427-like and MGAS10750-like strains. The degradation and cryptic nature of these elements may have important phenotypic ramifications for emm4 S. pyogenes and the geographical distribution of this lineage raises interesting questions on the population dynamics of the genotype. Data summaryAll raw sequence data used in this study has been previously published and was obtained from NCBI short read archive. Accession numbers and citations for the genome data for each individual isolate is provided in Supplementary Table 1.

microbiology↗

bioRxiv2019

The emergence of successful Streptococcus pyogenes lineages through convergent pathways of capsule loss and recombination directing high toxin expression

Gene transfer and homologous recombination in Streptococcus pyogenes has the potential to trigger the emergence of pandemic lineages, as exemplified by lineages of emm1 and emm89 that emerged in the 1980s and 2000s respectively. Although near-identical replacement gene transfer events in the nga (NADase) and slo (Streptolysin O) locus conferring high expression of these toxins underpinned the success of these lineages, extension to other emm-genotype lineages is unreported. The emergent emm89 lineage was characterised by five regions of homologous recombination additional to nga/slo, including complete loss of the hyaluronic acid capsule synthesis locus hasABC, a genetic trait replicated in two other leading emm types and recapitulated by other emm types by inactivating mutations. We hypothesised that other leading genotypes may have undergone a similar recombination events. We analysed a longitudinal dataset of genomes from 344 clinical invasive disease isolates representative of locations across England, dating from 2001 to 2011, and an international collection of S. pyogenes genomes representing 54 different genotypes, and found frequent evidence of recombination events at the nga-slo locus predicted to confer higher toxin expression. We identified multiple associations between recombination at this locus and inactivating mutations within hasA/B, suggesting convergent evolutionary pathways in successful genotypes. This included common genotypes emm28 and emm87. The combination of no or low capsule, and high expression of nga and slo, may underpin the success for many emergent S. pyogenes lineages of different genotypes, triggering new pandemics and could change the way S. pyogenes causes disease.\n\nImportanceStreptococcus pyogenes is a genetically diverse pathogen, with over 200 different genotypes defined by emm typing, but only a minority of these genotypes are responsible for majority of human infection in high income countries. Two prevalent genotypes associated with disease rose to international dominance following recombination of a toxin locus that conferred increased expression. Here, we found that recombination of this locus and promoter has occurred in other diverse genotypes, events that may allow these genotypes to expand in the population. We identified an association between the loss of hyaluronic acid capsule synthesis and high toxin expression, which we propose may be associated with an adaptive advantage. As S. pyogenes pathogenesis depends both on capsule and toxin production, new variants with altered expression may result in abrupt changes in the molecular epidemiology of this pathogen in the human population over time.

microbiology↗

bioRxiv2018

Streptococcal superantigen-induced expansion of human tonsil T cells leads to altered T follicular helper cell phenotype, B cell death, and reduced immunoglobulin release

BackgroundStreptococcal pyrogenic exotoxin (SPE)A expression is epidemiologically linked to streptococcal tonsillo-pharyngitis and outbreaks of scarlet fever, although the mechanisms by which superantigens confer advantage Streptococcus pyogenes are unclear. S. pyogenes is an exclusively human pathogen. As the leukocyte profile of tonsil differs from peripheral blood, the impact of SPEA production on human tonsil cell function was investigated.\n\nMethodsHuman tonsil cells from routine tonsillectomy, were co-incubated with purified streptococcal superantigens or isogenic streptococcal culture supernatants, differing only in superantigen content. Tonsil cell proliferation was quantified by tritium-incorporation, and cell surface characteristics assessed by flow-cytometry. Soluble mediators were measured using ELISA and quantitative (q)RT-PCR was performed for immunoglobulin gene expression.\n\nResultsTonsil T cells proliferated in response to SPEA and demonstrated typical release of pro-inflammatory cytokines. When cultured in the absence of superantigen, tonsil preparations released large quantities of immunoglobulin over 7d. In contrast, marked B cell apoptosis and abrogation of total IgA, IgM, and IgG production occurred in the presence of SPEA and other superantigens. In SPEA-stimulated cultures, T follicular helper (TfH) cells showed a reduction in CXCR5 expression, but up-regulation of CD134 (OX40), CD278 (ICOS) and CD150 (SLAM) expression, indicative of a phenotypic change in the TfH population and associated with impaired chemotactic response to CXCL13.\n\nConclusionsSPEA and other superantigens cause dysregulated tonsil immune function, driving T cells from TFH to a proliferating phenotype, with resultant loss of B cells and immunoglobulin production, providing superantigen-producing bacteria with a likely survival advantage.

microbiology↗