Conservation of C4BP-binding sequence patterns in Streptococcus pyogenes M and Enn proteins.

Antigenically sequence variable M proteins of the major bacterial pathogen Streptococcus pyogenes (Strep A) are responsible for recruiting human C4b-binding protein (C4BP) to the bacterial surface, which enables Strep A to evade destruction by the immune system. The most sequence divergent portion of M proteins, the hypervariable region (HVR), is responsible for binding C4BP. Structural evidence points to the conservation of two C4BP-binding sequence patterns (M2 and M22) in the HVR of numerous M proteins, with this conservation applicable to vaccine immunogen design. These two patterns, however, only partially explain C4BP binding by Strep A. Here, we identified several M proteins that lack these patterns but still bind C4BP and determined the structures of two, M68 and M87 HVRs, in complex with a C4BP fragment. Mutagenesis of these M proteins led to the identification of amino acids that are crucial for C4BP binding, enabling formulation of new C4BP-binding patterns. Mutagenesis was also carried out on M2 and M22 proteins to refine or generate experimentally grounded C4BP-binding patterns. The M22 pattern was the most prevalent among M proteins, followed by the M87 and M2 patterns, while the M68 pattern was rare. These patterns, except for M68, were also evident in numerous M-like Enn proteins. Binding of C4BP via these patterns to Enn proteins was verified. We conclude that C4BP-binding patterns occur frequently in Strep A strains of differing M types, being present in their M or Enn proteins, or frequently both, providing further impetus for their use as vaccine immunogens.

Molecular characterization of the interaction between human IgG and the M-related proteins from Streptococcus pyogenes.

Group A Streptococcal M-related proteins (Mrps) are dimeric α-helical-coiled-coil cell membrane-bound surface proteins. During infection, Mrp recruit the fragment crystallizable region of human immunoglobulin G via their A-repeat regions to the bacterial surface, conferring upon the bacteria enhanced phagocytosis resistance and augmented growth in human blood. However, Mrps show a high degree of sequence diversity, and it is currently not known whether this diversity affects the Mrp-IgG interaction. Herein, we report that diverse Mrps all bind human IgG subclasses with nanomolar affinity, with differences in affinity which ranged from 3.7 to 11.1 nM for mixed IgG. Using surface plasmon resonance, we confirmed Mrps display preferential IgG-subclass binding. All Mrps were found to have a significantly weaker affinity for IgG3 (p < 0.05) compared to all other IgG subclasses. Furthermore, plasma pulldown assays analyzed via Western blotting revealed that all Mrp were able to bind IgG in the presence of other serum proteins at both 25 °C and 37 °C. Finally, we report that dimeric Mrps bind to IgG with a 1:1 stoichiometry, enhancing our understanding of this important host-pathogen interaction.

Molecular methods enhance the detection of pyoderma-related Streptococcus pyogenes and emm-type distribution in children.

BACKGROUND: Streptococcus pyogenes-related skin infections are increasingly implicated in the development of rheumatic heart disease (RHD) in lower-resourced settings, where they are often associated with scabies. The true prevalence of S. pyogenes-related pyoderma may be underestimated by bacterial culture. METHODS: A multiplex qPCR for S. pyogenes, Staphylococcus aureus and Sarcoptes scabiei was applied to 250 pyoderma swabs from a cross-sectional study of children <5 years in The Gambia. Direct PCR-based emm-typing was used to supplement previous whole genome sequencing (WGS) of cultured isolates. RESULTS: Pyoderma lesions with S. pyogenes increased from 51% (127/250) using culture to 80% (199/250) with qPCR. Compared to qPCR, the sensitivity of culture was 95.4% for S. pyogenes (95% CI 77.2-99.9) in samples with S. pyogenes alone (22/250, 9%), but 59.9% (95% CI 52.3-67.2) for samples with S. aureus co-infection (177/250, 71%). Direct PCR-based emm-typing was successful in 50% (46/92) of cases, identifying 27 emm-types, including six not identified by WGS (total 52 emm-types). CONCLUSIONS: Bacterial culture significantly underestimates the burden of S. pyogenes in pyoderma, particularly when co-infected with S. aureus. Molecular methods should be used to enhance the detection of S. pyogenes in surveillance studies and clinical trials of preventative measures in RHD-endemic settings.

Group A Streptococcus adaptation to diverse niches: lessons from transcriptomic studies.

Group A Streptococcus (GAS) is a major human pathogen, causing diseases ranging from mild superficial infections of the skin and pharyngeal epithelium to severe systemic and invasive diseases. Moreover, post infection auto-immune sequelae arise by a yet not fully understood mechanism. The ability of GAS to cause a wide variety of infections is linked to the expression of a large set of virulence factors and their transcriptional regulation in response to various physiological environments. The use of transcriptomics, among others -omics technologies, in addition to traditional molecular methods, has led to a better understanding of GAS pathogenesis and host adaptation mechanisms. This review focusing on bacterial transcriptomic provides new insight into gene-expression patterns in vitro, ex vivo and in vivo with an emphasis on metabolic shifts, virulence genes expression and transcriptional regulators role.

Conservation of C4BP-binding Sequence Patterns in Streptococcus pyogenes M and Enn Proteins.

Antigenically sequence variable M proteins of the major bacterial pathogen Streptococcus pyogenes (Strep A) are responsible for recruiting human C4b-binding protein (C4BP) to the bacterial surface, which enables Strep A to evade destruction by the immune system. The most sequence divergent portion of M proteins, the hypervariable region (HVR), is responsible for binding C4BP. Structural evidence points to the conservation of two C4BP-binding sequence patterns (M2 and M22) in the HVR of numerous M proteins, with this conservation applicable to vaccine immunogen design. These two patterns, however, only partially explain C4BP-binding by Strep A. Here, we identified several M proteins that lack these patterns but still bind C4BP, and determined the structures of two, M68 and M87 HVRs, in complex with a C4BP fragment. Mutagenesis of these M proteins led to identification of amino acids that are crucial for C4BP-binding, enabling formulation of new C4BP-binding patterns. Mutagenesis was also carried out on M2 and M22 proteins to refine or generate experimentally grounded C4BP-binding patterns. The M22 pattern was the most populated among M proteins, followed by the M87 and M2 patterns, while the M68 pattern was rare. These patterns, except for M68, were also evident in numerous M-like Enn proteins. Binding of C4BP via these patterns to Enn proteins was verified. We conclude that C4BP-binding patterns occur frequently in Strep A strains of differing M types, being present in their M or Enn proteins, or frequently both, providing further impetus for their use as vaccine immunogens.

Use of the "Sepsis Risk Calculator" in Belgian Newborns: A Retrospective Cohort Study.

BACKGROUND: Prevention of early-onset neonatal sepsis (EONS) is a frequent reason why many newborns receive unnecessary antibiotics. The Sepsis Risk Calculator (SRC) was developed by the Kaiser Permanente Institute as a multivariate risk assessment of EONS, aiming to reduce laboratory testing and empiric neonatal antibiotic therapy. Our objective was to assess the potential of the SRC in reducing antibiotic use in our setting. METHODS: Late preterm and term newborns who received antibiotics from 2019 to 2020 in a tertiary Belgian hospital were included. Newborn-specific data were collected and entered into the online SRC, retrospectively calculating a sepsis risk score and providing recommendations for antibiotic administration. False-positive indications for treatment by the SRC were estimated based on previously published data. Antibiotic therapy rates according to the SRC recommendations were compared to the actual rate of antibiotic therapy. RESULTS: Of 5891 births, 414 newborns received antibiotics and were eligible for this study, representing a rate of 7.6% of newborns receiving antibiotics following our current guidelines. The SRC would have recommended antibiotic administration for 2.7%, reducing antibiotic therapy by 64.5%. Of 5 possible cases of EONS, 3 would have received antibiotics in the first 24 hours according to the SRC. CONCLUSIONS: In this Belgian cohort, use of the SRC has the potential to significantly decrease by 64.5% the newborns that receive antibiotics. This reduction would primarily concern asymptomatic newborns. If use of the SRC was to be implemented in Belgian maternities, strict clinical surveillance practices should be ensured.

Global epidemiological comparison of Streptococcus pyogenes emm-types associated with pharyngitis and pharyngeal carriage.

OBJECTIVES: To test the prevailing dogma that Streptococcus pyogenes emm-types that cause pharyngitis are the same as those associated with the carriage, using a global dataset. METHODS: Drawing on our systematic review of the global distribution of S. pyogenes emm-types and emm-clusters from 1990 to 2023, we compared the distribution and diversity of strains associated with pharyngitis and pharyngeal carriage, in the context of local United Nations Development Programme Human Development Index (HDI) values. RESULTS: We included 20 222 isolates from 71 studies done in 34 countries, with the vast majority of carriage strain data from studies in 'Low HDI' settings (550/1293; 43%). There was higher emm-type diversity for carriage than pharyngitis strains (Simpson Reciprocal Index of diversity 28.9 vs. 11.4). Compared with pharyngitis strains, carriage emm-types were disproportionately from emm-clusters E and D, usually described as 'generalist' or 'skin' strains. DISCUSSION: A limited number of studies have compared S. pyogenes strains from cases of pharyngitis compared with carriage. Our understanding of strains associated with carriage is the poorest for high-income settings. In low and medium HDI countries, we found greater strain associated with pharyngeal carriage than pharyngitis. Improving our understanding of S. pyogenes carriage epidemiology in the pre-vaccine era will help to decipher the direct and potential indirect effects of vaccines.

Efficient and rapid one-step method to generate gene deletions in Streptococcus pyogenes.

Streptococcus pyogenes or Group A Streptococcus (GAS) remains a significant infectious problem around the world, particularly in low- and middle-income settings. Moreover, a recent invasive GAS infection (iGAS) upsurge has been observed in high-income settings. However, to date, no vaccine is available. Finding a good vaccine antigen and understanding the role of virulence factors in GAS infections have been hampered, in part, by technical difficulties to transform the many different strains and generate knockout mutants. Using colE1-type plasmid as a suicide vector, we have set up a method allowing the generation of non-polar mutants of GAS in 3 days. IMPORTANCE: Group A Streptococcus (GAS) is a major human pathogen, causing diseases ranging from mild and superficial infections of the skin and pharyngeal epithelium to severe systemic and invasive diseases. Since June 2022, several European countries, the US, and Australia are facing an upsurge of invasive life-threatening GAS infections. Finding a good vaccine antigen and understanding the role of virulence factors in GAS infections have been hampered, in part, by technical difficulties to transform the many different GAS strains and generate knockout mutants. Moreover, these tools must be adapted to a large range of different strains, since GAS are divided into more than 260 emm-types (M-type). We have set up a method allowing the generation of non-polar mutants of GAS in 3 days and in diverse backgrounds, which contrasts with previously published protocols.

Streptococcus pyogenes carriage and infection within households in The Gambia: a longitudinal cohort study.

BACKGROUND: Streptococcus pyogenes causes more than 500 000 deaths per year globally, which occur disproportionately in low-income and middle-income countries. The roles of S pyogenes skin and pharyngeal carriage in transmission are unclear. We aimed to investigate the clinical epidemiology and household transmission dynamics of both S pyogenes asymptomatic carriage and infection in a high-burden setting. METHODS: We did a 1-year prospective, longitudinal, household cohort study, recruiting healthy participants from households in Sukuta, The Gambia. Households were eligible if they comprised at least three members, including one child younger than 18 years, and were excluded if more than half of household members declined to participate. Households were identified by random GPS coordinates derived from census data. At monthly visits, pharyngeal and normal skin swabs were collected for S pyogenes culture, and sociodemographic data were recorded by interview. Incident pharyngitis and pyoderma infections were captured. Cultured isolates underwent emm genotyping. The primary outcome measures were incidence of S pyogenes carriage and disease. Additional outcomes were prevalence of S pyogenes skin and pharyngeal carriage, S pyogenes skin and pharyngeal clearance time, S pyogenes emm type, risk factors for carriage and disease events, household secondary attack rate, and emm-linked household transmission events. The study is registered on ClinicalTrials.gov, NCT05117528. FINDINGS: Between July 27, 2021, and Sept 28, 2022, 442 participants were enrolled from 44 households. The median age was 15 years (IQR 6-28) and 233 (53%) were female. We identified 17 pharyngitis and 99 pyoderma events and 49 pharyngeal and 39 skin S pyogenes carriage acquisition events. Mean monthly prevalence was 1·4% (95% CI 1·1-1·9) for S pyogenes pharyngeal carriage and 1·2% (0·9-1·6) for S pyogenes skin carriage. Incidence was 120 per 1000 person-years (95% CI 87-166) for S pyogenes pharyngeal carriage, 124 per 1000 person-years (90-170) for S pyogenes skin carriage, 51 per 1000 person-years (31-84) for S pyogenes pharyngitis, and 263 per 1000 person-years (212-327) for S pyogenes pyoderma. Pharyngeal carriage risk was higher during the rainy season (HR 5·67, 95% CI 2·19-14·69) and in larger households (per additional person: 1·03, 1·00-1·05), as was pharyngitis risk (rainy season: 3·00, 1·10-8·22; household size: 1·04, 1·02-1·07). Skin carriage risk was not affected by season or household size, but was lower in female than in male participants (0·45, 0·22-0·92) and highest in children younger than 5 years compared with adults (22·69, 3·08-167·21), with similar findings for pyoderma (female sex: 0·34, 0·19-0·61; age <5 years: 7·00, 2·78-17·64). Median clearance time after carriage acquisition was 4·0 days for both skin (IQR 3·5-7·0) and pharynx (3·5-7·3). The mean household secondary attack rate was 4·9 (95% CI 3·5-6·3) for epidemiologically linked S pyogenes events and 0·74 (0·3-1·2) for emm-linked S pyogenes events. Of the 204 carriage and disease events, emm types were available for 179 (88%). Only 18 emm-linked between-visit household transmission events were identified. Pyoderma was the most common source of S pyogenes household transmissions in 11 (61%) of 18 emm-linked transmissions. Both pharynx to skin and skin to pharynx transmission events were observed. INTERPRETATION: S pyogenes carriage and infection are common in The Gambia, particularly in children. Most events are non-household acquisitions, but skin carriage and pyoderma have an important role in S pyogenes household transmission and bidirectional transmission between skin and pharynx occurs. FUNDING: Wellcome Trust, Chadwick Trust, Fonds National de la Recherche Scientifique (Belgium), European Society for Paediatric Infectious Diseases, and Medical Research Council (UK).

Increase in invasive group A streptococcal disease among Australian children coinciding with northern hemisphere surges.

Background: Increases in invasive group A streptococcal disease (iGAS) have recently been reported in multiple countries in the northern hemisphere, occurring during, and outside of, typical spring peaks. We report the epidemiology of iGAS among children in Australia from 1 July 2018 to 31 December 2022. Methods: The Paediatric Active Enhanced Disease Surveillance (PAEDS) Network prospectively collected iGAS patient notifications for children and young people aged less than 18 years admitted to five major Australian paediatric hospitals in Victoria, Queensland, Western Australia and the Northern Territory. Patients were eligible for inclusion if they had GAS isolated from a normally sterile body site, or met clinical criteria for streptococcal toxic shock syndrome or necrotising fasciitis with GAS isolated from a non-sterile site. We report patients' clinical and demographic characteristics, and estimate minimum incidence rates. Findings: We identified 280 paediatric iGAS patients, median age 4.5 years (interquartile range 1.4-6.4). We observed a pre-pandemic peak annualised incidence of 3.7 per 100,000 (95% CI 3.1-4.4) in the 3rd quarter of 2018, followed by a decline to less than 1.0 per 100,000 per quarter from 2020 to mid-2021. The annualised incidence increased sharply from mid-2022, peaking at 5.2 per 100,000 (95% CI 4.4-6.0) in the 3rd quarter and persisting into the 4th quarter (4.9 per 100,000, 95% CI 4.2-5.7). There were 3 attributable deaths and 84 (32%) patients had severe disease (overall case fatality rate 1%, 95% CI 0.2-3.3). Respiratory virus co-infection, positive in 57 of 119 patients tested, was associated with severe disease (RR 1.9, 95% CI 1.2-3.0). The most common emm-type was emm-1 (60 of 163 isolates that underwent emm-typing, 37%), followed by emm-12 (18%). Interpretation: Australia experienced an increase in the incidence of iGAS among children and young people in 2022 compared to pandemic years 2020-2021. This is similar to northern hemisphere observations, despite differences in seasons and circulating respiratory viruses. Outbreaks of iGAS continue to occur widely. This emphasises the unmet need for a vaccine to prevent significant morbidity associated with iGAS disease. Funding: Murdoch Children's Research Institute funded open access publishing of this manuscript.