Streptolysins are the primary inflammasome activators in macrophages during Streptococcus pyogenes infection.

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)-1ß signaling plays an important role in GAS disease progression. The host regulates the production and secretion of IL-1ß 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-1ß 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-1ß 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.

Detection of Epidemic Scarlet Fever Group A Streptococcus in Australia.

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.

Endopeptidase PepO Regulates the SpeB Cysteine Protease and Is Essential for the Virulence of Invasive M1T1 Streptococcus pyogenes.

Streptococcus pyogenes (group A Streptococcus [GAS]) causes a wide range of human infections. The pathogenesis of GAS infections is dependent on the temporal expression of numerous secreted and surface-associated virulence factors that interact with host proteins. Streptococcal pyrogenic exotoxin B (SpeB) is one of the most extensively studied toxins produced by GAS, and the coordinate growth phase-dependent regulation of speB expression is linked to disease severity phenotypes. Here, we identified the endopeptidase PepO as a novel growth phase-dependent regulator of SpeB in the invasive GAS M1 serotype strain 5448. By using transcriptomics followed by quantitative reverse transcriptase PCR and Western blot analyses, we demonstrate through targeted mutagenesis that PepO influences growth phase-dependent induction of speB gene expression. Compared to wild-type and complemented mutant strains, we demonstrate that the 5448ΔpepO mutant strain is more susceptible to killing by human neutrophils and is attenuated in virulence in a murine model of invasive GAS infection. Our results expand the complex regulatory network that is operating in GAS to control SpeB production and suggest that PepO is a virulence requirement during GAS M1T1 strain 5448 infections.IMPORTANCE Despite the continuing susceptibility of S. pyogenes to penicillin, this bacterial pathogen remains a leading infectious cause of global morbidity and mortality. A particular subclone of the M1 serotype (M1T1) has persisted globally for decades as the most frequently isolated serotype from patients with invasive and noninvasive diseases in Western countries. One of the key GAS pathogenicity factors is the potent broad-spectrum cysteine protease SpeB. Although there has been extensive research interest on the regulatory mechanisms that control speB gene expression, its genetic regulation is not fully understood. Here, we identify the endopeptidase PepO as a new regulator of speB gene expression in the globally disseminated M1T1 clone and as being essential for virulence.

Efficacy of Alum-Adjuvanted Peptide and Carbohydrate Conjugate Vaccine Candidates against Group A Streptococcus Pharyngeal Infection in a Non-Human Primate Model.

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.

Tracing the evolutionary history of the pandemic group A streptococcal M1T1 clone.

The past 50 years has witnessed the emergence of new viral and bacterial pathogens with global effect on human health. The hyperinvasive group A Streptococcus (GAS) M1T1 clone, first detected in the mid-1980s in the United States, has since disseminated worldwide and remains a major cause of severe invasive human infections. Although much is understood regarding the capacity of this pathogen to cause disease, much less is known of the precise evolutionary events selecting for its emergence. We used high-throughput technologies to sequence a World Health Organization strain collection of serotype M1 GAS and reconstructed its phylogeny based on the analysis of core genome single-nucleotide polymorphisms. We demonstrate that acquisition of a 36-kb genome segment from serotype M12 GAS and the bacteriophage-encoded DNase Sda1 led to increased virulence of the M1T1 precursor and occurred relatively early in the molecular evolutionary history of this strain. The more recent acquisition of the phage-encoded superantigen SpeA is likely to have provided selection advantage for the global dissemination of the M1T1 clone. This study provides an exemplar for the evolution and emergence of virulent clones from microbial populations existing commensally or causing only superficial infection.

Detection of Streptococcus pyogenes M1UK in Australia and characterization of the mutation driving enhanced expression of superantigen SpeA.

A new variant of Streptococcus pyogenes serotype M1 (designated 'M1UK') 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 'M1global' and M1UK 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 M1UK in Australia, now isolated from the majority of serious infections caused by serotype M1 S. pyogenes. M1UK 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 M1UK lineage. This represents a previously unappreciated mechanism of toxin expression and urges enhanced international surveillance.

Integrative omics identifies conserved and pathogen-specific responses of sepsis-causing bacteria.

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.

Streptococcus pyogenes Hijacks Host Glutathione for Growth and Innate Immune Evasion.

The nasopharynx and the skin are the major oxygen-rich anatomical sites for colonization by the human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]). To establish infection, GAS must survive oxidative stress generated during aerobic metabolism and the release of reactive oxygen species (ROS) by host innate immune cells. Glutathione is the major host antioxidant molecule, while GAS is glutathione auxotrophic. Here, we report the molecular characterization of the ABC transporter substrate binding protein GshT in the GAS glutathione salvage pathway. We demonstrate that glutathione uptake is critical for aerobic growth of GAS and that impaired import of glutathione induces oxidative stress that triggers enhanced production of the reducing equivalent NADPH. Our results highlight the interrelationship between glutathione assimilation, carbohydrate metabolism, virulence factor production, and innate immune evasion. Together, these findings suggest an adaptive strategy employed by extracellular bacterial pathogens to exploit host glutathione stores for their own benefit. IMPORTANCE During infection, microbes must escape host immune responses and survive exposure to reactive oxygen species produced by immune cells. Here, we identify the ABC transporter substrate binding protein GshT as a key component of the glutathione salvage pathway in glutathione-auxotrophic GAS. Host-acquired glutathione is crucial to the GAS antioxidant defense system, facilitating escape from the host innate immune response. This study demonstrates a direct link between glutathione assimilation, aerobic metabolism, and virulence factor production in an important human pathogen. Our findings provide mechanistic insight into host adaptation that enables extracellular bacterial pathogens such as GAS to exploit the abundance of glutathione in the host cytosol for their own benefit.

Neurodegenerative Disease Treatment Drug PBT2 Breaks Intrinsic Polymyxin Resistance in Gram-Positive Bacteria.

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.

Rescuing Tetracycline Class Antibiotics for the Treatment of Multidrug-Resistant Acinetobacter baumannii Pulmonary Infection.

Acinetobacter baumannii causes high mortality in ventilator-associated pneumonia patients, and antibiotic treatment is compromised by multidrug-resistant strains resistant to ß-lactams, carbapenems, cephalosporins, polymyxins, and tetracyclines. Among COVID-19 patients receiving ventilator support, a multidrug-resistant A. baumannii secondary infection is associated with a 2-fold increase in mortality. Here, we investigated the use of the 8-hydroxyquinoline ionophore PBT2 to break the resistance of A. baumannii to tetracycline class antibiotics. In vitro, the combination of PBT2 and zinc with either tetracycline, doxycycline, or tigecycline was shown to be bactericidal against multidrug-resistant A. baumannii, and any resistance that did arise imposed a fitness cost. PBT2 and zinc disrupted metal ion homeostasis in A. baumannii, increasing cellular zinc and copper while decreasing magnesium accumulation. Using a murine model of pulmonary infection, treatment with PBT2 in combination with tetracycline or tigecycline proved efficacious against multidrug-resistant A. baumannii. These findings suggest that PBT2 may find utility as a resistance breaker to rescue the efficacy of tetracycline-class antibiotics commonly employed to treat multidrug-resistant A. baumannii infections. IMPORTANCE Within intensive care unit settings, multidrug-resistant (MDR) Acinetobacter baumannii is a major cause of ventilator-associated pneumonia, and hospital-associated outbreaks are becoming increasingly widespread. Antibiotic treatment of A. baumannii infection is often compromised by MDR strains resistant to last-resort ß-lactam (e.g., carbapenems), polymyxin, and tetracycline class antibiotics. During the on-going COVID-19 pandemic, secondary bacterial infection by A. baumannii has been associated with a 2-fold increase in COVID-19-related mortality. With a rise in antibiotic resistance and a reduction in new antibiotic discovery, it is imperative to investigate alternative therapeutic regimens that complement the use of current antibiotic treatment strategies. Rescuing the efficacy of existing therapies for the treatment of MDR A. baumannii infection represents a financially viable pathway, reducing time, cost, and risk associated with drug innovation.

A multivalent T-antigen-based vaccine for Group A Streptococcus.

Pili of Group A Streptococcus (GAS) are surface-exposed structures involved in adhesion and colonisation of the host during infection. The major protein component of the GAS pilus is the T-antigen, which multimerises to form the pilus shaft. There are currently no licenced vaccines against GAS infections and the T-antigen represents an attractive target for vaccination. We have generated a multivalent vaccine called TeeVax1, a recombinant protein that consists of a fusion of six T-antigen domains. Vaccination with TeeVax1 produces opsonophagocytic antibodies in rabbits and confers protective efficacy in mice against invasive disease. Two further recombinant proteins, TeeVax2 and TeeVax3 were constructed to cover 12 additional T-antigens. Combining TeeVax1-3 produced a robust antibody response in rabbits that was cross-reactive to a full panel of 21 T-antigens, expected to provide over 95% vaccine coverage. These results demonstrate the potential for a T-antigen-based vaccine to prevent GAS infections.

Defining the structural basis of human plasminogen binding by streptococcal surface enolase.

The flesh-eating bacterium group A Streptococcus (GAS) binds and activates human plasminogen, promoting invasive disease. Streptococcal surface enolase (SEN), a glycolytic pathway enzyme, is an identified plasminogen receptor of GAS. Here we used mass spectrometry (MS) to confirm that GAS SEN is octameric, thereby validating in silico modeling based on the crystal structure of Streptococcus pneumoniae alpha-enolase. Site-directed mutagenesis of surface-located lysine residues (SEN(K252 + 255A), SEN(K304A), SEN(K334A), SEN(K344E), SEN(K435L), and SEN(Delta434-435)) was used to examine their roles in maintaining structural integrity, enzymatic function, and plasminogen binding. Structural integrity of the GAS SEN octamer was retained for all mutants except SEN(K344E), as determined by circular dichroism spectroscopy and MS. However, ion mobility MS revealed distinct differences in the stability of several mutant octamers in comparison with wild type. Enzymatic analysis indicated that SEN(K344E) had lost alpha-enolase activity, which was also reduced in SEN(K334A) and SEN(Delta434-435). Surface plasmon resonance demonstrated that the capacity to bind human plasminogen was abolished in SEN(K252 + 255A), SEN(K435L), and SEN(Delta434-435). The lysine residues at positions 252, 255, 434, and 435 therefore play a concerted role in plasminogen acquisition. This study demonstrates the ability of combining in silico structural modeling with ion mobility-MS validation for undertaking functional studies on complex protein structures.

Vaccine-Induced Th1-Type Response Protects against Invasive Group A Streptococcus Infection in the Absence of Opsonizing Antibodies.

Recent global advocacy efforts have highlighted the importance of development of a vaccine against group A Streptococcus (GAS). Combo5 is a non-M protein-based vaccine that provides protection against GAS skin infection in mice and reduces the severity of pharyngitis in nonhuman primates. However, Combo5 with the addition of aluminum hydroxide (alum) as an adjuvant failed to protect against invasive GAS infection of mice. Here, we show that formulation of Combo5 with adjuvants containing saponin QS21 significantly improves protective efficacy, even though all 7 adjuvants tested generated high antigen-specific IgG antibody titers, including alum. Detailed characterization of Combo5 formulated with SMQ adjuvant, a squalene-in-water emulsion containing a TLR4 agonist and QS21, showed significant differences from the results obtained with alum in IgG subclasses generated following immunization, with an absence of GAS opsonizing antibodies. SMQ, but not alum, generated strong interleukin-6 (IL-6), gamma interferon (IFN-γ), and tumor necrosis alpha (TNF-α) responses. This work highlights the importance of adjuvant selection for non-M protein-based GAS vaccines to optimize immune responses and protective efficacy.IMPORTANCE Availability of a group A Streptococcus vaccine remains an unmet public health need. Here, we tested different adjuvant formulations to improve the protective efficacy of non-M protein vaccine Combo5 in an invasive disease model. We show that novel adjuvants can dramatically shape the type of immune response developed following immunization with Combo5 and significantly improve protection. In addition, protection afforded by Combo5 is not mediated by opsonizing antibodies, believed to be the main correlate of protection against GAS infections. Overall, this report highlights the importance of adjuvant selection in raising protective immune responses against GAS invasive infection. Adjuvants that can provide a more balanced Th1/Th2-type response may be required to optimize protection of GAS vaccines, particularly those based on non-M protein antigens.

Prophage exotoxins enhance colonization fitness in epidemic scarlet fever-causing Streptococcus pyogenes.

The re-emergence of scarlet fever poses a new global public health threat. The capacity of North-East Asian serotype M12 (emm12) 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 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 resistance to neutrophil killing. 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 pathogenesis of scarlet fever-causing GAS mediated by prophage ΦHKU.vir exotoxins.

Repurposing a neurodegenerative disease drug to treat Gram-negative antibiotic-resistant bacterial sepsis.

The emergence of polymyxin resistance in carbapenem-resistant and extended-spectrum ß-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.

Allelic variants of streptokinase from Streptococcus pyogenes display functional differences in plasminogen activation.

A common mammalian defense mechanism employed to prevent systemic dissemination of invasive bacteria involves occlusion of local microvasculature and encapsulation of bacteria within fibrin networks. Acquisition of plasmin activity at the bacterial cell surface circumvents this defense mechanism, allowing invasive disease initiation. To facilitate this process, S. pyogenes secretes streptokinase, a plasminogen-activating protein. Streptokinase polymorphism exhibited by S. pyogenes isolates is well characterized. However, the functional differences displayed by these variants and the biological significance of this variation has not been elucidated. Phylogenetic analysis of ska sequences from 28 S. pyogenes isolates revealed 2 main sequence clusters (clusters 1 and 2). All strains secreted streptokinase, as determined by Western blotting, and were capable of acquiring cell surface plasmin activity after incubation in human plasma. Whereas culture supernatants from strains containing cluster 1 ska alleles also displayed soluble plasminogen activation activity, supernatants from strains containing cluster 2 ska alleles did not. Furthermore, plasminogen activation activity in culture supernatants from strains containing cluster 2 ska alleles could only be detected when plasminogen was prebound with fibrinogen. This study indicates that variant streptokinase proteins secreted by S. pyogenes isolates display differing plasminogen activation characteristics and may therefore play distinct roles in disease pathogenesis.

An Experimental Group A Streptococcus Vaccine That Reduces Pharyngitis and Tonsillitis in a Nonhuman Primate Model.

Group A Streptococcus (GAS) infections account for an estimated 500,000 deaths every year. This bacterial pathogen is responsible for a variety of mild and life-threatening infections and the triggering of chronic autoimmune sequelae. Pharyngitis caused by group A Streptococcus (GAS), but not asymptomatic GAS carriage, is a prerequisite for acute rheumatic fever (ARF). Repeated bouts of ARF may trigger rheumatic heart disease (RHD), a major cause of heart failure and stroke accounting for 275,000 deaths annually. A vaccine that prevents pharyngitis would markedly reduce morbidity and mortality from ARF and RHD. Nonhuman primates (NHPs) have been utilized to model GAS diseases, and experimentally infected rhesus macaques develop pharyngitis. Here we use an NHP model of GAS pharyngitis to evaluate the efficacy of an experimental vaccine, Combo5 (arginine deiminase [ADI], C5a peptidase [SCPA], streptolysin O [SLO], interleukin-8 [IL-8] protease [SpyCEP], and trigger factor [TF]), specifically designed to exclude GAS components potentially linked to autoimmune complications. Antibody responses against all Combo5 antigens were detected in NHP serum, and immunized NHPs showed a reduction in pharyngitis and tonsillitis compared to controls. Our work establishes the NHP model as a gold standard for the assessment of GAS vaccines.IMPORTANCE GAS-related diseases disproportionally affect disadvantaged populations (e.g., indigenous populations), and development of a vaccine has been neglected. A recent strong advocacy campaign driven by the World Health Organization and the International Vaccine Institute has highlighted the urgent need for a GAS vaccine. One significant obstacle in GAS vaccine development is the lack of a widely used animal model to assess vaccine efficacy. Researchers in the field use a wide range of murine models of infection and in vitro assays, sometimes yielding conflicting results. Here we present the nonhuman primate pharyngeal infection model as a tool to assess vaccine-induced protection against colonization and clinical symptoms of pharyngitis and tonsillitis. We have tested the efficacy of an experimental vaccine candidate with promising results. We believe that the utilization of this valuable tool by the GAS vaccine research community could significantly accelerate the realization of a safe and effective GAS vaccine for humans.

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.

Chemical Synergy between Ionophore PBT2 and Zinc Reverses Antibiotic Resistance.

The World Health Organization reports that antibiotic-resistant pathogens represent an imminent global health disaster for the 21st century. Gram-positive superbugs threaten to breach last-line antibiotic treatment, and the pharmaceutical industry antibiotic development pipeline is waning. Here we report the synergy between ionophore-induced physiological stress in Gram-positive bacteria and antibiotic treatment. PBT2 is a safe-for-human-use zinc ionophore that has progressed to phase 2 clinical trials for Alzheimer's and Huntington's disease treatment. In combination with zinc, PBT2 exhibits antibacterial activity and disrupts cellular homeostasis in erythromycin-resistant group A Streptococcus (GAS), methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus (VRE). We were unable to select for mutants resistant to PBT2-zinc treatment. While ineffective alone against resistant bacteria, several clinically relevant antibiotics act synergistically with PBT2-zinc to enhance killing of these Gram-positive pathogens. These data represent a new paradigm whereby disruption of bacterial metal homeostasis reverses antibiotic-resistant phenotypes in a number of priority human bacterial pathogens.IMPORTANCE The rise of bacterial antibiotic resistance coupled with a reduction in new antibiotic development has placed significant burdens on global health care. Resistant bacterial pathogens such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus are leading causes of community- and hospital-acquired infection and present a significant clinical challenge. These pathogens have acquired resistance to broad classes of antimicrobials. Furthermore, Streptococcus pyogenes, a significant disease agent among Indigenous Australians, has now acquired resistance to several antibiotic classes. With a rise in antibiotic resistance and reduction in new antibiotic discovery, it is imperative to investigate alternative therapeutic regimens that complement the use of current antibiotic treatment strategies. As stated by the WHO Director-General, "On current trends, common diseases may become untreatable. Doctors facing patients will have to say, Sorry, there is nothing I can do for you."