Mark J. Walker

mBio • December 17, 2024

Johanna Richter, Amanda Cork, Yvette Ong, Nadia Keller, Andrew Hayes, Mark Schembri, Amy Jennison, Mark Davies, Kate Schroder, Mark Walker, Stephan Brouwer

Group A Streptococcus (GAS) is a human-adapted pathogen responsible for a variety of diseases. The GAS M1UK lineage has contributed significantly to the recently reported increases in scarlet fever and invasive infections. However, the basis for its evolutionary success is not yet fully understood. During the transition to systemic disease, the M1 serotype is known to give rise to spontaneous mutations in the control of virulence two-component regulatory system (CovRS) that confer a fitness advantage during invasive infections. Mutations that inactivate CovS function result in the de-repression of key GAS virulence factors such as streptolysin O (SLO), a pore-forming toxin and major trigger of inflammasome/interleukin-1β-dependent inflammation. Conversely, expression of the streptococcal cysteine protease SpeB, which is required during initial stages of colonization and onset of invasive disease, is typically lost in such mutants. In this study, we identified and characterized a novel covS single nucleotide polymorphism detected in three separate invasive M1UK isolates. The resulting CovSAla318Val mutation caused a significant upregulation of SLO resulting in increased inflammasome activation in human THP-1 macrophages, indicating an enhanced inflammatory potential. Surprisingly, SpeB production was unaffected. Site-directed mutagenesis was performed to assess the impact of this mutation on virulence and global gene expression. We found that the CovSAla318Val mutation led to subtle, virulence-specific changes of the CovRS regulon compared to previously characterized covS mutations, highlighting an unappreciated level of complexity in CovRS-dependent gene regulation. Continued longitudinal surveillance is warranted to determine whether this novel covS mutation will expand in the M1UK lineage.IMPORTANCEThe M1UK lineage of GAS has contributed to a recent global upsurge in scarlet fever and invasive infections. Understanding how GAS can become more virulent is critical for infection control and identifying new treatment approaches. The two-component CovRS system, comprising the sensor kinase CovS and transcription factor CovR, is a central regulator of GAS virulence genes. In the M1 serotype, covRS mutations are associated with an invasive phenotype. Such mutations have not been fully characterized in the M1UK lineage. This study identified a novel covS mutation in invasive Australian M1UK isolates that resulted in a more nuanced virulence gene regulation compared to previously characterized covS mutations. A representative isolate displayed upregulated SLO production and triggered amplified interleukin-1β secretion in infected human macrophages, indicating an enhanced inflammatory potential. These findings underscore the need for comprehensive analyses of covRS mutants to fully elucidate their contribution to M1UK virulence and persistence.

Nature Communications • January 17, 2025

Nichaela Harbison Price, Ismail Sebina, Rhiannon Bolton, Meredith Finn, Amanda Cork, Isabel Courtney, Steven Hancock, Ruby Pelingon, Johanna Richter, Olivia Ericsson, Shannon Green, Celeste Cuellar, Laura Davis, Brody Pullinger, Jack Na, Gayathiri Elangovan, David M De Oliveira, Bodie Curren, Nia Bickham, Miguel Aguirre, Christina Dold, Stephan Brouwer, Obadiah Plante, Gabrielle Belz, Mark Walker

A commercial vaccine to address the high global burden of Group A Streptococcus (GAS) disease is an urgent and unmet medical need. Messenger RNA (mRNA) lipid-nanoparticle (LNP) vaccines represent a largely untapped platform for targeting bacterial pathogens. Here, we evaluate the immunogenicity and preclinical efficacy of a multicomponent mRNA-LNP vaccine formulation based on the GAS vaccine, Combo#5. Combo#5 mRNA-LNP antigens confer protection from infection in mouse intraperitoneal and subcutaneous challenge models. Combo#5 mRNA-LNP vaccination generates significantly increased frequencies and numbers of effector type CD4+ and CD8 + T cells in the spleen, enhances T follicular helper cells, germinal center B cells and memory B cells in the spleen and draining lymph nodes, and boosts the production of antigen-specific antibodies. These findings demonstrate the potential of the mRNA-LNP platform for the development of vaccines against bacterial pathogens.

The FEBS Journal • November 09, 2024

Tahnee Mcewan, David M De Oliveira, Emily Stares, Lauren Hartley Tassell, Christopher Day, Mark Walker, Michael Jennings, Ronald Sluyter, Martina Sanderson Smith

Glycosaminoglycans (GAGs) are enriched in the cutaneous extracellular matrix and have important roles in bacterial colonisation. Group A Streptococcus (GAS) can be categorised by emm patterning and M-family protein expression. M proteins of GAS are major adhesins with lectin-binding properties. This study aimed to provide a comprehensive specificity and affinity profile of phylogenetically diverse M proteins to a range of sulfated host GAGs and to investigate the physiological relevance of these interactions. Chondroitin sulfate preferentially associated with M proteins of A-C pattern strains, with binding localised to the central variable region of M1 protein. Dermatan sulfate was shown to associate with M proteins of all pattern type strains, with recognition involving multiple sites on M proteins. Heparin and heparan sulfate exclusively interacted with M proteins of A-C and D pattern strains. Multiple sites of M proteins were involved in heparin recognition, as indicated by surface plasmon resonance and site-directed mutagenesis of the heparin-binding XBXBX motif in the hypervariable-central region of M53 protein. In contrast, binding of heparan sulfate was localised to the non-repeat region between the B2 repeat and C1 repeat of M53 proteins. 5448 (M1-expressing GAS, A-C pattern) was shown to bind chondroitin sulfate, dermatan sulfate and heparin in an M protein-dependent manner. Furthermore, recruitment of chondroitin sulfate or dermatan sulfate by M1 proteins, but not heparin, was shown to increase GAS adherence to human HaCaT keratinocytes. This study increases our understanding of the molecular mechanisms underlying GAS adhesion, with key implications for bacterial colonisation and persistence of infection.

Pathogens (Basel, Switzerland) • October 09, 2024

Phoebe Shaw, Andrew Hayes, Maree Langton, Angela Berkhout, Keith Grimwood, Mark Davies, Mark Walker, Stephan Brouwer

Streptococcus pyogenes (Group A Streptococcus, GAS) is a human-restricted pathogen that causes a wide range of diseases from pharyngitis and scarlet fever to more severe, invasive infections such as necrotising fasciitis and streptococcal toxic shock syndrome. There has been a global increase in both scarlet fever and invasive infections during the COVID-19 post-pandemic period. The aim of this study was the molecular characterisation of 17 invasive and non-invasive clinical non-emm1 GAS isolates from an Australian tertiary hospital collected between 2021 and 2022. Whole genome sequencing revealed a total of nine different GAS emm types with the most prevalent being emm22, emm12 and emm3 (each 3/17, 18%). Most isolates (14/17, 82%) carried at least one superantigen gene associated with contemporary scarlet fever outbreaks, and the carriage of these toxin genes was non-emm type specific. Several mutations within key regulatory genes were identified across the different GAS isolates, which may be linked to an increased expression of several virulence factors. This study from a single Australian centre provides a snapshot of non-emm1 GAS clinical isolates that are multiclonal and linked with distinct epidemiological markers commonly observed in high-income settings. These findings highlight the need for continual surveillance to monitor genetic markers that may drive future outbreaks.