The DmsABC S-oxide reductase is an essential component of a novel, hypochlorite-inducible system of extracellular stress defense in Haemophilus influenzae.

Defenses against oxidative damage to cell components are essential for survival of bacterial pathogens during infection, and here we have uncovered that the DmsABC S-/N-oxide reductase is essential for virulence and in-host survival of the human-adapted pathogen, Haemophilus influenzae. In several different infection models, H. influenzae ΔdmsA strains showed reduced immunogenicity as well as lower levels of survival in contact with host cells. Expression of DmsABC was induced in the presence of hypochlorite and paraquat, closely linking this enzyme to defense against host-produced antimicrobials. In addition to methionine sulfoxide, DmsABC converted nicotinamide- and pyrimidine-N-oxide, precursors of NAD and pyrimidine for which H. influenzae is an auxotroph, at physiologically relevant concentrations, suggesting that these compounds could be natural substrates for DmsABC. Our data show that DmsABC forms part of a novel, periplasmic system for defense against host-induced S- and N-oxide stress that also comprises the functionally related MtsZ S-oxide reductase and the MsrAB peptide methionine sulfoxide reductase. All three enzymes are induced following exposure of the bacteria to hypochlorite. MsrAB is required for physical resistance to HOCl and protein repair. In contrast, DmsABC was required for intracellular colonization of host cells and, together with MtsZ, contributed to resistance to N-Chlorotaurine. Our work expands and redefines the physiological role of DmsABC and highlights the importance of different types of S-oxide reductases for bacterial virulence.

Metalloproteome plasticity - a factor in bacterial pathogen adaptive responses?

Through homeostatic processes, bacterial cells maintain intracytoplasmic metal ions at concentrations which enable the 'correct' metal to be inserted into an enzyme, thereby ensuring function. However, fluctuations in intracytoplasmic metal ion concentrations mean that under different conditions certain enzymes may contain different metals at their active site. This perspective describes examples of such cases and suggests that metalloproteome plasticity may contribute to the dynamic adaptation of pathogens to stresses in the host environment.

Bacterial acetate metabolism and its influence on human epithelia.

Short-chain fatty acids are known modulators of host-microbe interactions and can affect human health, inflammation, and outcomes of microbial infections. Acetate is the most abundant but least well-studied of these modulators, with most studies focusing on propionate and butyrate, which are considered to be more potent. In this mini-review, we summarize current knowledge of acetate as an important anti-inflammatory modulator of interactions between hosts and microorganisms. This includes a summary of the pathways by which acetate is metabolized by bacteria and human cells, the functions of acetate in bacterial cells, and the impact that microbially derived acetate has on human immune function.

The Peptide Methionine Sulfoxide Reductase (MsrAB) of Haemophilus influenzae Repairs Oxidatively Damaged Outer Membrane and Periplasmic Proteins Involved in Nutrient Acquisition and Virulence.

Sulfoxide-damage repair mechanisms are emerging as essential for the virulence of bacterial pathogens, and in the human respiratory pathogen Haemophilus influenzae the periplasmic MsrAB peptide methionine sulfoxide reductase is necessary for resistance to reactive chlorine species such as hypochlorite. Additionally, this enzyme has a role in modulating the host immune response to infection. Here, we have analysed the enzymatic properties of MsrAB, which revealed that both domains of the protein are catalytically active, with the turnover number of the MsrA domain being 50% greater than that for the MsrB domain. MsrAB was active with small molecular sulfoxides as well as oxidised calmodulin, and maximal activity was observed at 30°C, a temperature close to that found in the natural niche of H. influenzae, the nasopharynx. Analyses of differential methionine oxidation identified 29 outer membrane and periplasmic proteins that are likely substrates for MsrAB. These included the LldD lactate dehydrogenase and the lipoprotein eP4 that is involved in NAD and hemin metabolism in H. influenzae. Subsequent experiments showed that H. influenzae MsrAB can repair oxidative damage to methionines in purified eP4 with up to 100% efficiency. Our work links MsrAB to the maintenance of different adhesins and essential metabolic processes in the H. influenzae, such as NAD metabolism and access to L-lactate, which is a key growth substrate for H. influenzae during infection.

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.

Access to highly specialized growth substrates and production of epithelial immunomodulatory metabolites determine survival of Haemophilus influenzae in human airway epithelial cells.

Haemophilus influenzae (Hi) infections are associated with recurring acute exacerbations of chronic respiratory diseases in children and adults including otitis media, pneumonia, chronic obstructive pulmonary disease and asthma. Here, we show that persistence and recurrence of Hi infections are closely linked to Hi metabolic properties, where preferred growth substrates are aligned to the metabolome of human airway epithelial surfaces and include lactate, pentoses, and nucleosides, but not glucose that is typically used for studies of Hi growth in vitro. Enzymatic and physiological investigations revealed that utilization of lactate, the preferred Hi carbon source, required the LldD L-lactate dehydrogenase (conservation: 98.8% of strains), but not the two redox-balancing D-lactate dehydrogenases Dld and LdhA. Utilization of preferred substrates was directly linked to Hi infection and persistence. When unable to utilize L-lactate or forced to rely on salvaged guanine, Hi showed reduced extra- and intra-cellular persistence in a murine model of lung infection and in primary normal human nasal epithelia, with up to 3000-fold attenuation observed in competitive infections. In contrast, D-lactate dehydrogenase mutants only showed a very slight reduction compared to the wild-type strain. Interestingly, acetate, the major Hi metabolic end-product, had anti-inflammatory effects on cultured human tissue cells in the presence of live but not heat-killed Hi, suggesting that metabolic endproducts also influence HI-host interactions. Our work provides significant new insights into the critical role of metabolism for Hi persistence in contact with host cells and reveals for the first time the immunomodulatory potential of Hi metabolites.

Dysregulation of Streptococcus pneumoniae zinc homeostasis breaks ampicillin resistance in a pneumonia infection model.

Streptococcus pneumoniae is the primary cause of community-acquired bacterial pneumonia with rates of penicillin and multidrug-resistance exceeding 80% and 40%, respectively. The innate immune response generates a variety of antimicrobial agents to control infection, including zinc stress. Here, we characterize the impact of zinc intoxication on S. pneumoniae, observing disruptions in central carbon metabolism, lipid biogenesis, and peptidoglycan biosynthesis. Characterization of the pivotal peptidoglycan biosynthetic enzyme GlmU indicates a sensitivity to zinc inhibition. Disruption of the sole zinc efflux pathway, czcD, renders S. pneumoniae highly susceptible to ß-lactam antibiotics. To dysregulate zinc homeostasis in the wild-type strain, we investigated the safe-for-human-use ionophore 5,7-dichloro-2-[(dimethylamino)methyl]quinolin-8-ol (PBT2). PBT2 rendered wild-type S. pneumoniae strains sensitive to a range of antibiotics. Using an invasive ampicillin-resistant strain, we demonstrate in a murine pneumonia infection model the efficacy of PBT2 + ampicillin treatment. These findings present a therapeutic modality to break antibiotic resistance in multidrug-resistant S. pneumoniae.

The DmsABC Sulfoxide Reductase Supports Virulence in Non-typeable Haemophilus influenzae.

Although molybdenum-containing enzymes are well-established as having a key role in bacterial respiration, it is increasingly recognized that some may also support bacterial virulence. Here, we show that DmsABC, a putative dimethylsulfoxide (DMSO) reductase, is required for fitness of the respiratory pathogen Haemophilus influenzae (Hi) in different models of infection. Expression of the dmsABC operon increased with decreasing oxygen availability, but despite this, a Hi2019Δd msA strain did not show any defects in anaerobic growth on chemically defined medium (CDM), and viability was also unaffected. Although Hi2019Δd msA exhibited increased biofilm formation in vitro and greater resistance to hypochlorite killing compared to the isogenic wild-type strain, its survival in contact with primary human neutrophils, in infections of cultured tissue cells, or in a mouse model of lung infection was reduced compared to Hi2019WT. The tissue cell infection model revealed a two-fold decrease in intracellular survival, while in the mouse model of lung infection Hi2019Δd msA was strongly attenuated and below detection levels at 48 h post-inoculation. While Hi2019WT was recovered in approximately equal numbers from bronchoalveolar lavage fluid (BALF) and lung tissue, survival of Hi2019Δd msA was reduced in lung tissue compared to BALF samples, indicating that Hi2019Δd msA had reduced access to or survival in the intracellular niche. Our data clearly indicate for the first time a role for DmsABC in H. influenzae infection and that the conditions under which DmsABC is required in this bacterium are closely linked to interactions with the host.

Active site architecture reveals coordination sphere flexibility and specificity determinants in a group of closely related molybdoenzymes.

MtsZ is a molybdenum-containing methionine sulfoxide reductase that supports virulence in the human respiratory pathogen Haemophilus influenzae (Hi). HiMtsZ belongs to a group of structurally and spectroscopically uncharacterized S-/N-oxide reductases, all of which are found in bacterial pathogens. Here, we have solved the crystal structure of HiMtsZ, which reveals that the HiMtsZ substrate-binding site encompasses a previously unrecognized part that accommodates the methionine sulfoxide side chain via interaction with His182 and Arg166. Charge and amino acid composition of this side chain-binding region vary and, as indicated by electrochemical, kinetic, and docking studies, could explain the diverse substrate specificity seen in closely related enzymes of this type. The HiMtsZ Mo active site has an underlying structural flexibility, where dissociation of the central Ser187 ligand affected catalysis at low pH. Unexpectedly, the two main HiMtsZ electron paramagnetic resonance (EPR) species resembled not only a related dimethyl sulfoxide reductase but also a structurally unrelated nitrate reductase that possesses an Asp-Mo ligand. This suggests that contrary to current views, the geometry of the Mo center and its primary ligands, rather than the specific amino acid environment, is the main determinant of the EPR properties of mononuclear Mo enzymes. The flexibility in the electronic structure of the Mo centers is also apparent in two of three HiMtsZ EPR-active Mo(V) species being catalytically incompetent off-pathway forms that could not be fully oxidized.

The Alternative Sigma Factor RpoE2 Is Involved in the Stress Response to Hypochlorite and in vivo Survival of Haemophilus influenzae.

Extracytoplasmic function (ECF) sigma factors underpin the ability of bacteria to adapt to changing environmental conditions, a process that is particularly relevant in human pathogens that inhabit niches where human immune cells contribute to high levels of extracellular stress. Here, we have characterized the previously unstudied RpoE2 ECF sigma factor from the human respiratory pathogen H. influenzae (Hi) and its role in hypochlorite-induced stress. Exposure of H. influenzae to oxidative stress (HOCl, H2O2) increased rpoE2 gene expression, and the activity of RpoE2 was controlled by a cytoplasmic 67-aa anti-sigma factor, HrsE. RpoE2 regulated the expression of the periplasmic MsrAB peptide methionine sulfoxide reductase that, in H. influenzae, is required for HOCl resistance, thus linking RpoE2 to HOCl stress. Interestingly, a HiΔrpoE2 strain had wild-type levels of resistance to oxidative stress in vitro, but HiΔrpoE2 survival was reduced 26-fold in a mouse model of lung infection, demonstrating the relevance of this sigma factor for H. influenzae pathogenesis. The HiRpoE2 system has some similarity to the ECF sigma factors described in Streptomyces and Neisseria sp. that also control the expression of msr genes. However, HiRpoE2 regulation extended to genes encoding other periplasmic damage repair proteins, an operon containing a DoxX-like protein, and also included selected OxyR-controlled genes. Based on our results, we propose that the highly conserved HiRpoE2 sigma factor is a key regulator of H. influenzae responses to oxidative damage in the cell envelope region that controls a variety of target genes required for survival in the host.

Role of Glutathione in Buffering Excess Intracellular Copper in Streptococcus pyogenes.

Copper (Cu) is an essential metal for bacterial physiology but in excess it is bacteriotoxic. To limit Cu levels in the cytoplasm, most bacteria possess a transcriptionally responsive system for Cu export. In the Gram-positive human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]), this system is encoded by the copYAZ operon. This study demonstrates that although the site of GAS infection represents a Cu-rich environment, inactivation of the copA Cu efflux gene does not reduce virulence in a mouse model of invasive disease. In vitro, Cu treatment leads to multiple observable phenotypes, including defects in growth and viability, decreased fermentation, inhibition of glyceraldehyde-3-phosphate dehydrogenase (GapA) activity, and misregulation of metal homeostasis, likely as a consequence of mismetalation of noncognate metal-binding sites by Cu. Surprisingly, the onset of these effects is delayed by ∼4 h even though expression of copZ is upregulated immediately upon exposure to Cu. Further biochemical investigations show that the onset of all phenotypes coincides with depletion of intracellular glutathione (GSH). Supplementation with extracellular GSH replenishes the intracellular pool of this thiol and suppresses all the observable effects of Cu treatment. These results indicate that GSH buffers excess intracellular Cu when the transcriptionally responsive Cu export system is overwhelmed. Thus, while the copYAZ operon is responsible for Cu homeostasis, GSH has a role in Cu tolerance and allows bacteria to maintain metabolism even in the presence of an excess of this metal ion.IMPORTANCE The control of intracellular metal availability is fundamental to bacterial physiology. In the case of copper (Cu), it has been established that rising intracellular Cu levels eventually fill the metal-sensing site of the endogenous Cu-sensing transcriptional regulator, which in turn induces transcription of a copper export pump. This response caps intracellular Cu availability below a well-defined threshold and prevents Cu toxicity. Glutathione, abundant in many bacteria, is known to bind Cu and has long been assumed to contribute to bacterial Cu handling. However, there is some ambiguity since neither its biosynthesis nor uptake is Cu-regulated. Furthermore, there is little experimental support for this physiological role of glutathione beyond measuring growth of glutathione-deficient mutants in the presence of Cu. Our work with group A Streptococcus provides new evidence that glutathione increases the threshold of intracellular Cu availability that can be tolerated by bacteria and thus advances fundamental understanding of bacterial Cu handling.

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.

Peptide Methionine Sulfoxide Reductase from Haemophilus influenzae Is Required for Protection against HOCl and Affects the Host Response to Infection.

Peptide methionine sulfoxide reductases (Msrs) are enzymes that repair ROS-damage to sulfur-containing amino acids such as methionine, ensuring functional integrity of cellular proteins. Here we have shown that unlike the majority of pro- and eukaryotic Msrs, the peptide methionine sulfoxide reductase (MsrAB) from the human pathobiont Haemophilus influenzae (Hi) is required for the repair of hypochlorite damage to cell envelope proteins, but more importantly, we were able to demonstrate that MsrAB plays a role in modulating the host immune response to Hi infection. Loss of MsrAB resulted in >1000-fold increase in sensitivity of Hi to HOCl-mediated killing, and also reduced biofilm formation and in-biofilm survival. Expression of msrAB was also induced by hydrogen peroxide and paraquat, but a Hi2019ΔmsrAB strain was not susceptible to killing by these ROS in vitro. Hi2019ΔmsrAB fitness in infection models was low, with a 3-fold reduction in intracellular survival in bronchial epithelial cells, increased susceptibility to neutrophil killing, and a 10-fold reduction in survival in a mouse model of lung infection. Interestingly, infection with Hi2019ΔmsrAB led to specific changes in the antibacterial response of human host cells, with genes encoding antimicrobial peptides (BPI, CAMP) upregulated between 4 and 9 fold compared to infection with Hi2019WT, and reduction in expression of two proteins with antiapoptotic functions (BIRC3, XIAP). Modulation of host immune responses is a novel role for an enzyme of this type and provides first insights into mechanisms by which MsrAB supports Hi survival in vivo.

Two Distinct L-Lactate Dehydrogenases Play a Role in the Survival of Neisseria gonorrhoeae in Cervical Epithelial Cells.

L-lactate is an abundant metabolite in a number of niches in host organisms and represents an important carbon source for bacterial pathogens such as Neisseria gonorrhoeae. In this study, we describe an alternative, iron-sulfur cluster-containing L-lactate dehydrogenase (LutACB), that is distinct from the flavoprotein L-lactate dehydrogenase (LldD). Expression of lutACB was found to be positively regulated by iron, whereas lldD was more highly expressed under conditions of iron-limitation. The functional role of LutACB and LldD was reflected in in vitro studies of growth and in the survival of N gonorrhoeae in primary cervical epithelial cells.

Dietary zinc and the control of Streptococcus pneumoniae infection.

Human zinc deficiency increases susceptibility to bacterial infection. Although zinc supplementation therapies can reduce the impact of disease, the molecular basis for protection remains unclear. Streptococcus pneumoniae is a major cause of bacterial pneumonia, which is prevalent in regions of zinc deficiency. We report that dietary zinc levels dictate the outcome of S. pneumoniae infection in a murine model. Dietary zinc restriction impacts murine tissue zinc levels with distribution post-infection altered, and S. pneumoniae virulence and infection enhanced. Although the activation and infiltration of murine phagocytic cells was not affected by zinc restriction, their efficacy of bacterial control was compromised. S. pneumoniae was shown to be highly sensitive to zinc intoxication, with this process impaired in zinc restricted mice and isolated phagocytic cells. Collectively, these data show how dietary zinc deficiency increases sensitivity to S. pneumoniae infection while revealing a role for zinc as a component of host antimicrobial defences.

Discovery of glycerol phosphate modification on streptococcal rhamnose polysaccharides.

Cell wall glycopolymers on the surface of Gram-positive bacteria are fundamental to bacterial physiology and infection biology. Here we identify gacH, a gene in the Streptococcus pyogenes group A carbohydrate (GAC) biosynthetic cluster, in two independent transposon library screens for its ability to confer resistance to zinc and susceptibility to the bactericidal enzyme human group IIA-secreted phospholipase A2. Subsequent structural and phylogenetic analysis of the GacH extracellular domain revealed that GacH represents an alternative class of glycerol phosphate transferase. We detected the presence of glycerol phosphate in the GAC, as well as the serotype c carbohydrate from Streptococcus mutans, which depended on the presence of the respective gacH homologs. Finally, nuclear magnetic resonance analysis of GAC confirmed that glycerol phosphate is attached to approximately 25% of the GAC N-acetylglucosamine side-chains at the C6 hydroxyl group. This previously unrecognized structural modification impacts host-pathogen interaction and has implications for vaccine design.

Metabolic analyses reveal common adaptations in two invasive Haemophilus influenzae strains.

Non-typeable Haemophilus influenzae (NTHi) is a major pathogen in upper and lower respiratory tract infections in humans, and is increasingly also associated with invasive disease. We have examined two unrelated NTHi invasive disease isolates, R2866 and C188, in order to identify metabolic and physiological properties that distinguish them from respiratory tract disease isolates such as Hi2019. While the general use of the Hi metabolic network was similar across all three strains, the two invasive isolates secreted increased amounts of succinate, which can have anti-inflammatory properties. In addition, they showed a common shift in their carbon source utilization patterns, with strongly enhanced metabolism of nucleoside substrates, glucose and sialic acid. The latter two are major compounds present in blood and cerebrospinal fluid (CSF). Interestingly, C188 and R2866 also shared a reduced ability to invade or survive intracellularly in 16HBE14 bronchial epithelial cells relative to Hi2019 (4-fold (4 h), 25-fold (24 h) reduction). Altered metabolic properties, such as the ones observed here, could arise from genomic adaptations that NTHi undergo during infection. Together these data indicate that shifts in substrate preferences in otherwise conserved metabolic pathways may underlie strain niche specificity and thus have the potential to alter the outcomes of host-NTHi interactions.

Uropathogenic Escherichia coli employs both evasion and resistance to subvert innate immune-mediated zinc toxicity for dissemination.

Toll-like receptor (TLR)-inducible zinc toxicity is a recently described macrophage antimicrobial response used against bacterial pathogens. Here we investigated deployment of this pathway against uropathogenic Escherichia coli (UPEC), the major cause of urinary tract infections. Primary human macrophages subjected EC958, a representative strain of the globally disseminated multidrug-resistant UPEC ST131 clone, to zinc stress. We therefore used transposon-directed insertion site sequencing to identify the complete set of UPEC genes conferring protection against zinc toxicity. Surprisingly, zinc-susceptible EC958 mutants were not compromised for intramacrophage survival, whereas corresponding mutants in the nonpathogenic E. coli K-12 strain MG1655 displayed significantly reduced intracellular bacterial loads within human macrophages. To investigate whether the intramacrophage zinc stress response of EC958 reflected the response of only a subpopulation of bacteria, we generated and validated reporter systems as highly specific sensors of zinc stress. Using these tools we show that, in contrast to MG1655, the majority of intramacrophage EC958 evades the zinc toxicity response, enabling survival within these cells. In addition, EC958 has a higher tolerance to zinc than MG1655, with this likely being important for survival of the minor subset of UPEC cells exposed to innate immune-mediated zinc stress. Indeed, analysis of zinc stress reporter strains and zinc-sensitive mutants in an intraperitoneal challenge model in mice revealed that EC958 employs both evasion and resistance against zinc toxicity, enabling its dissemination to the liver and spleen. We thus demonstrate that a pathogen of global significance uses multiple mechanisms to effectively subvert innate immune-mediated zinc poisoning for systemic spread.

Group A Streptococcus co-ordinates manganese import and iron efflux in response to hydrogen peroxide stress.

Bacterial pathogens encounter a variety of adverse physiological conditions during infection, including metal starvation, metal overload and oxidative stress. Here, we demonstrate that group A Streptococcus (GAS) utilises Mn(II) import via MtsABC during conditions of hydrogen peroxide stress to optimally metallate the superoxide dismutase, SodA, with Mn. MtsABC expression is controlled by the DtxR family metalloregulator MtsR, which also regulates the expression of Fe uptake systems in GAS. Our results indicate that the SodA in GAS requires Mn for full activity and has lower activity when it contains Fe. As a consequence, under conditions of hydrogen peroxide stress where Fe is elevated, we observed that the PerR-regulated Fe(II) efflux system PmtA was required to reduce intracellular Fe, thus protecting SodA from becoming mismetallated. Our findings demonstrate the co-ordinate action of MtsR-regulated Mn(II) import by MtsABC and PerR-regulated Fe(II) efflux by PmtA to ensure appropriate Mn(II) metallation of SodA for optimal superoxide dismutase function.