Amanda J. Cork

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.

Group A Streptococcus (GAS) is a human-adapted pathogen responsible for a variety of diseases. The GAS M1 _UK 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 M1 _UK isolates. The resulting CovS ^Ala318Val 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 CovS ^Ala318Val 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 M1 _UK lineage. IMPORTANCE: The M1 _UK 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 M1 _UK lineage. This study identified a novel covS mutation in invasive Australian M1 _UK 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 M1 _UK virulence and persistence.

Vaccine development against group A Streptococcus (GAS) has gained traction in the last decade, fuelled by recognition of the significant worldwide burden of the disease. Several vaccine candidates are currently being evaluated in preclinical and early clinical studies. Here, we investigate two conjugate vaccine candidates that have shown promise in mouse models of infection. Two antigens, the J8 peptide from the conserved C-terminal end of the M protein, and the group A carbohydrate lacking N-acetylglucosamine side chain (ΔGAC) were each conjugated to arginine deiminase (ADI), an anchorless surface protein from GAS. Both conjugate vaccine candidates combined with alum adjuvant were tested in a non-human primate (NHP) model of pharyngeal infection. High antibody titres were detected against J8 and ADI antigens, while high background antibody titres in NHP sera hindered accurate quantification of ΔGAC-specific antibodies. The severity of pharyngitis and tonsillitis signs, as well as the level of GAS colonisation, showed no significant differences in NHPs immunised with either conjugate vaccine candidate compared to NHPs in the negative control group.

Even in the setting of optimal resuscitation in high-income countries severe sepsis and septic shock have a mortality of 20–40%, with antibiotic resistance dramatically increasing this mortality risk. To develop a reference dataset enabling the identification of common bacterial targets for therapeutic intervention, we applied a standardized genomic, transcriptomic, proteomic and metabolomic technological framework to multiple clinical isolates of four sepsis-causing pathogens: Escherichia coli, Klebsiella pneumoniae species complex, Staphylococcus aureus and Streptococcus pyogenes. Exposure to human serum generated a sepsis molecular signature containing global increases in fatty acid and lipid biosynthesis and metabolism, consistent with cell envelope remodelling and nutrient adaptation for osmoprotection. In addition, acquisition of cholesterol was identified across the bacterial species. This detailed reference dataset has been established as an open resource to support discovery and translational research.

A new variant of Streptococcus pyogenes serotype M1 (designated ‘M1_UK’) has been reported in the United Kingdom, linked with seasonal scarlet fever surges, marked increase in invasive infections, and exhibiting enhanced expression of the superantigen SpeA. The progenitor S. pyogenes ‘M1_global’ and M1_UK clones can be differentiated by 27 SNPs and 4 indels, yet the mechanism for speA upregulation is unknown. Here we investigate the previously unappreciated expansion of M1_UK in Australia, now isolated from the majority of serious infections caused by serotype M1 S. pyogenes. M1_UK sub-lineages circulating in Australia also contain a novel toxin repertoire associated with epidemic scarlet fever causing S. pyogenes in Asia. A single SNP in the 5’ transcriptional leader sequence of the transfer-messenger RNA gene ssrA drives enhanced SpeA superantigen expression as a result of ssrA terminator read-through in the M1_UK lineage. This represents a previously unappreciated mechanism of toxin expression and urges enhanced international surveillance.

Gram-positive bacteria do not produce lipopolysaccharide as a cell wall component. As such, the polymyxin class of antibiotics, which exert bactericidal activity against Gram-negative pathogens, are ineffective against Gram-positive bacteria. The safe-for-human-use hydroxyquinoline analog ionophore PBT2 has been previously shown to break polymyxin resistance in Gram-negative bacteria, independent of the lipopolysaccharide modification pathways that confer polymyxin resistance. Here, in combination with zinc, PBT2 was shown to break intrinsic polymyxin resistance in Streptococcus pyogenes (Group A Streptococcus; GAS), Staphylococcus aureus (including methicillin-resistant S. aureus), and vancomycin-resistant Enterococcus faecium. Using the globally disseminated M1T1 GAS strain 5448 as a proof of principle model, colistin in the presence of PBT2 + zinc was shown to be bactericidal in activity. Any resistance that did arise imposed a substantial fitness cost. PBT2 + zinc dysregulated GAS metal ion homeostasis, notably decreasing the cellular manganese content. Using a murine model of wound infection, PBT2 in combination with zinc and colistin proved an efficacious treatment against streptococcal skin infection. These findings provide a foundation from which to investigate the utility of PBT2 and next-generation polymyxin antibiotics for the treatment of Gram-positive bacterial infections.

Group A Streptococcus (GAS) is a Gram‐positive bacterial pathogen that causes an array of infectious diseases in humans. Accumulating clinical evidence suggests that proinflammatory interleukin (IL)‐1β signaling plays an important role in GAS disease progression. The host regulates the production and secretion of IL‐1β via the cytosolic inflammasome pathway. Activation of the NLR family pyrin domain‐containing 3 (NLRP3) inflammasome complex requires two signals: a priming signal that stimulates increased transcription of genes encoding the components of the inflammasome pathway, and an activating signal that induces assembly of the inflammasome complex. Here we show that GAS‐derived lipoteichoic acid can provide a priming signal for NLRP3 inflammasome activation. As only few GAS‐derived proteins have been associated with inflammasome‐dependent IL‐1β signaling, we investigated novel candidates that might play a role in activating the inflammasome pathway by infecting mouse bone marrow‐derived macrophages and human THP‐1 macrophage‐like cells with a panel of isogenic GAS mutant strains. We found that the cytolysins streptolysin O (SLO) and streptolysin S are the main drivers of IL‐1β release in proliferating logarithmic phase GAS. Using a mutant form of recombinant SLO, we confirmed that bacterial pore formation on host cell membranes is a key mechanism required for inflammasome activation. Our results suggest that streptolysins are major determinants of GAS‐induced inflammation and present an attractive target for therapeutic intervention.

The emergence of polymyxin resistance in carbapenem-resistant and extended-spectrum β-lactamase (ESBL)–producing bacteria is a critical threat to human health, and alternative treatment strategies are urgently required. We investigated the ability of the hydroxyquinoline analog ionophore PBT2 to restore antibiotic sensitivity in polymyxin-resistant, ESBL-producing, carbapenem-resistant Gram-negative human pathogens. PBT2 resensitized Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa to last-resort polymyxin class antibiotics, including the less toxic next-generation polymyxin derivative FADDI-287, in vitro. We were unable to select for mutants resistant to PBT2 + FADDI-287 in polymyxin-resistant E. coli containing a plasmid-borne mcr-1 gene or K. pneumoniae carrying a chromosomal mgrB mutation. Using a highly invasive K. pneumoniae strain engineered for polymyxin resistance through mgrB mutation, we successfully demonstrated the efficacy of PBT2 + polymyxin (colistin or FADDI-287) for the treatment of Gram-negative sepsis in immunocompetent mice. In comparison to polymyxin alone, the combination of PBT2 + polymyxin improved survival and reduced bacterial dissemination to the lungs and spleen of infected mice. These data present a treatment modality to break antibiotic resistance in high-priority polymyxin-resistant Gram-negative pathogens.

The re-emergence of scarlet fever poses a new global public health threat. The capacity of North-East Asian serotype M12 ( emm 12) Streptococcus pyogenes (group A Streptococcus , GAS) to cause scarlet fever has been linked epidemiologically to the presence of novel prophages, including prophage ΦHKU.vir encoding the secreted superantigens SSA and SpeC and the DNase Spd1. Here we report the comprehensive molecular characterization of ΦHKU.vir-encoded exotoxins. We demonstrate that streptolysin O (SLO)-induced glutathione efflux from host cellular stores is a previously unappreciated GAS virulence mechanism that promotes SSA release and activity, representing the first description of a thiol-activated bacterial superantigen. Spd1 is required for optimal growth in human blood, confers resistance to neutrophil killing, and degrades neutrophil extracellular traps (NETs). Investigating single, double and triple isogenic knockout mutants of the ΦHKU.vir-encoded exotoxins, we find that SpeC and Spd1 act synergistically to facilitate nasopharyngeal colonization in a mouse model. These results offer insight into the etiology and pathogenesis of scarlet fever-causing GAS mediated by phage ΦHKU.vir exotoxins.

Sentinel hospital surveillance was instituted in Australia to detect the presence of pandemic group A Streptococcus strains causing scarlet fever. Genomic and phylogenetic analyses indicated the presence of an Australian GAS emm12 scarlet fever isolate related to United Kingdom outbreak strains. National surveillance to monitor this pandemic is recommended.