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.

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.

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.

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.

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.

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.

Interplay between tolerance mechanisms to copper and acid stress in Escherichia coli.

Copper (Cu) is a key antibacterial component of the host innate immune system and almost all bacterial species possess systems that defend against the toxic effects of excess Cu. The Cu tolerance system in Gram-negative bacteria is composed minimally of a Cu sensor (CueR) and a Cu export pump (CopA). The cueR and copA genes are encoded on the chromosome typically as a divergent but contiguous operon. In Escherichia coli, cueR and copA are separated by two additional genes, ybaS and ybaT, which confer glutamine (Gln)-dependent acid tolerance and contribute to the glutamate (Glu)-dependent acid resistance system in this organism. Here we show that Cu strongly inhibits growth of a ∆copA mutant strain in acidic cultures. We further demonstrate that Cu stress impairs the pathway for Glu biosynthesis via glutamate synthase, leading to decreased intracellular levels of Glu. Addition of exogenous Glu rescues the ∆copA mutant from Cu stress in acidic conditions. Gln is also protective but this relies on the activities of YbaS and YbaT. Notably, expression of both enzymes is up-regulated during Cu stress. These results demonstrate a link between Cu stress, acid stress, and Glu/Gln metabolism, establish a role for YbaS and YbaT in Cu tolerance, and suggest that subtle changes in core metabolic pathways may contribute to overcoming host-imposed copper toxicity.

New Insights into the Role of Zinc Acquisition and Zinc Tolerance in Group A Streptococcal Infection.

Zinc plays an important role in host innate immune function. However, the innate immune system also utilizes zinc starvation ("nutritional immunity") to combat infections. Here, we investigate the role of zinc import and export in the protection of Streptococcus pyogenes (group A Streptococcus; GAS), a Gram-positive bacterial pathogen responsible for a wide spectrum of human diseases, against challenge from host innate immune defense. In order to determine the role of GAS zinc import and export during infection, we utilized zinc import (ΔadcA ΔadcAII) and export (ΔczcD) deletion mutants in competition with the wild type in both in vitro and in vivo virulence models. We demonstrate that nutritional immunity is deployed extracellularly, while zinc toxicity is utilized upon phagocytosis of GAS by neutrophils. We also show that lysosomes and azurophilic granules in neutrophils contain zinc stores for use against intracellular pathogens.

Copper Ions and Coordination Complexes as Novel Carbapenem Adjuvants.

Carbapenem-resistant Enterobacteriaceae are urgent threats to global human health. These organisms produce ß-lactamases with carbapenemase activity, such as the metallo-ß-lactamase NDM-1, which is notable due to its association with mobile genetic elements and the lack of a clinically useful inhibitor. Here we examined the ability of copper to inhibit the activity of NDM-1 and explored the potential of a copper coordination complex as a mechanism to efficiently deliver copper as an adjuvant in clinical therapeutics. An NDM-positive Escherichia coli isolate, MS6192, was cultured from the urine of a patient with a urinary tract infection. MS6192 was resistant to antibiotics from multiple classes, including diverse ß-lactams (penicillins, cephalosporins, and carbapenems), aminoglycosides, and fluoroquinolones. In the presence of copper (range, 0 to 2 mM), however, the susceptibility of MS6192 to the carbapenems ertapenem and meropenem increased markedly. In standard checkerboard assays, copper decreased the MICs of ertapenem and meropenem against MS6192 in a dose-dependent manner, suggesting a synergistic mode of action. To examine the inhibitory effect of copper in the absence of other ß-lactamases, the blaNDM-1 gene from MS6192 was cloned and expressed in a recombinant E. coli K-12 strain. Analysis of cell extracts prepared from this strain revealed that copper directly inhibited NDM-1 activity, which was confirmed using purified recombinant NDM-1. Finally, delivery of copper at a low concentration of 10 µM by using the FDA-approved coordination complex copper-pyrithione sensitized MS6192 to ertapenem and meropenem in a synergistic manner. Overall, this work demonstrates the potential use of copper coordination complexes as novel carbapenemase adjuvants.

Structural basis of thiol-based regulation of formaldehyde detoxification in H. influenzae by a MerR regulator with no sensor region.

Pathogenic bacteria such as Haemophilus influenzae, a major cause of lower respiratory tract diseases, must cope with a range of electrophiles generated in the host or by endogenous metabolism. Formaldehyde is one such compound that can irreversibly damage proteins and DNA through alkylation and cross-linking and interfere with redox homeostasis. Its detoxification operates under the control of HiNmlR, a protein from the MerR family that lacks a specific sensor region and does not bind metal ions. We demonstrate that HiNmlR is a thiol-dependent transcription factor that modulates H. influenzae response to formaldehyde, with two cysteine residues (Cys54 and Cys71) identified to be important for its response against a formaldehyde challenge. We obtained crystal structures of HiNmlR in both the DNA-free and two DNA-bound forms, which suggest that HiNmlR enhances target gene transcription by twisting of operator DNA sequences in a two-gene operon containing overlapping promoters. Our work provides the first structural insights into the mechanism of action of MerR regulators that lack sensor regions.

The PerR-Regulated P1B-4-Type ATPase (PmtA) Acts as a Ferrous Iron Efflux Pump in Streptococcus pyogenes.

Streptococcus pyogenes (group A Streptococcus [GAS]) is an obligate human pathogen responsible for a broad spectrum of human disease. GAS has a requirement for metal homeostasis within the human host and, as such, tightly modulates metal uptake and efflux during infection. Metal acquisition systems are required to combat metal sequestration by the host, while metal efflux systems are essential to protect against metal overload poisoning. Here, we investigated the function of PmtA (PerR-regulated metal transporter A), a P1B-4-type ATPase efflux pump, in invasive GAS M1T1 strain 5448. We reveal that PmtA functions as a ferrous iron [Fe(II)] efflux system. In the presence of high Fe(II) concentrations, the 5448ΔpmtA deletion mutant exhibited diminished growth and accumulated 5-fold-higher levels of intracellular Fe(II) than did the wild type and the complemented mutant. The 5448ΔpmtA deletion mutant also showed enhanced susceptibility to killing by the Fe-dependent antibiotic streptonigrin as well as increased sensitivity to hydrogen peroxide and superoxide. We suggest that the PerR-mediated control of Fe(II) efflux by PmtA is important for bacterial defense against oxidative stress. PmtA represents an exemplar for an Fe(II) efflux system in a host-adapted Gram-positive bacterial pathogen.

Salmonella employs multiple mechanisms to subvert the TLR-inducible zinc-mediated antimicrobial response of human macrophages.

We aimed to characterize antimicrobial zinc trafficking within macrophages and to determine whether the professional intramacrophage pathogen Salmonella enterica serovar Typhimurium (S Typhimurium) subverts this pathway. Using both Escherichia coli and S Typhimurium, we show that TLR signaling promotes the accumulation of vesicular zinc within primary human macrophages. Vesicular zinc is delivered to E. coli to promote microbial clearance, whereas S. Typhimurium evades this response via Salmonella pathogenicity island (SPI)-1. Even in the absence of SPI-1 and the zinc exporter ZntA, S Typhimurium resists the innate immune zinc stress response, implying the existence of additional host subversion mechanisms. We also demonstrate the combinatorial antimicrobial effects of zinc and copper, a pathway that S. Typhimurium again evades. Our use of complementary tools and approaches, including confocal microscopy, direct assessment of intramacrophage bacterial zinc stress responses, specific E. coli and S Typhimurium mutants, and inductively coupled plasma mass spectroscopy, has enabled carefully controlled characterization of this novel innate immune antimicrobial pathway. In summary, our study provides new insights at the cellular level into the well-documented effects of zinc in promoting host defense against infectious disease, as well as the complex host subversion strategies employed by S Typhimurium to combat this pathway.-Kapetanovic, R., Bokil, N. J., Achard, M. E. S., Ong, C.-L. Y., Peters, K. M., Stocks, C. J., Phan, M.-D., Monteleone, M., Schroder, K., Irvine, K. M., Saunders, B. M., Walker, M. J., Stacey, K. J., McEwan, A. G., Schembri, M. A., Sweet, M. J. Salmonella employs multiple mechanisms to subvert the TLR-inducible zinc-mediated antimicrobial response of human macrophages.

The Role of Copper and Zinc Toxicity in Innate Immune Defense against Bacterial Pathogens.

Zinc (Zn) and copper (Cu) are essential for optimal innate immune function, and nutritional deficiency in either metal leads to increased susceptibility to bacterial infection. Recently, the decreased survival of bacterial pathogens with impaired Cu and/or Zn detoxification systems in phagocytes and animal models of infection has been reported. Consequently, a model has emerged in which the host utilizes Cu and/or Zn intoxication to reduce the intracellular survival of pathogens. This review describes and assesses the potential role for Cu and Zn intoxication in innate immune function and their direct bactericidal function.

Imperfect coordination chemistry facilitates metal ion release in the Psa permease.

The relative stability of divalent first-row transition metal ion complexes, as defined by the Irving-Williams series, poses a fundamental chemical challenge for selectivity in bacterial metal ion acquisition. Here we show that although the substrate-binding protein of Streptococcus pneumoniae, PsaA, is finely attuned to bind its physiological substrate manganese, it can also bind a broad range of other divalent transition metal cations. By combining high-resolution structural data, metal-binding assays and mutational analyses, we show that the inability of open-state PsaA to satisfy the preferred coordination chemistry of manganese enables the protein to undergo the conformational changes required for cargo release to the Psa permease. This is specific for manganese ions, whereas zinc ions remain bound to PsaA. Collectively, these findings suggest a new ligand binding and release mechanism for PsaA and related substrate-binding proteins that facilitate specificity for divalent cations during competition from zinc ions, which are more abundant in biological systems.

A genetic screen reveals a periplasmic copper chaperone required for nitrite reductase activity in pathogenic Neisseria.

Under conditions of low oxygen availability, Neisseria meningitidis and Neisseria gonorrhoeae are able to respire via a partial denitrification pathway in which nitrite is converted to nitrous oxide. In this process, nitrite reductase (AniA), a copper (Cu)-containing protein converts nitrite to NO, and this product is converted to nitrous oxide by nitric oxide reductase (NorB). NorB also confers protection against toxic NO, and so we devised a conditional lethal screen, using a norB mutant, to identify mutants that were resistant to nitrite-dependent killing. After random-deletion mutagenesis of N. meningitidis, this genetic screen identified a gene encoding a Cu chaperone that is essential for AniA function, AccA. Purified AccA binds one Cu (I) ion and also possesses a second binding site for Cu (II). This novel periplasmic Cu chaperone (AccA) appears to be essential for provision of Cu ions to AniA of pathogenic Neisseria to generate an active nitrite reductase. Apart from the Neisseria genus, AccA is distributed across a wide range of environmental Proteobacteria species.

Functional analysis of an feoB mutant in Clostridium perfringens strain 13.

Bacterial pathogens have adopted numerous mechanisms for acquiring iron from host proteins during an infection, including the direct acquisition of ferric iron from heme-associated proteins or from iron-scavenging siderophores. Ferric iron then is transported into the cytosol, where it can be utilized by the bacterial pathogen. Under anaerobic conditions bacteria can also transport ferrous iron using the transmembrane complex FeoAB, but little is known about iron transport systems in anaerobic bacteria such as the pathogenic clostridia. In this study we sought to characterize the iron acquisition process in Clostridium perfringens. Bioinformatic analysis of the Clostridium perfringens strain 13 genome sequence revealed that it has seven potential iron acquisition systems: three siderophore-mediated systems, one ferric citrate uptake system, two heme-associated acquisition systems and one ferrous iron uptake system (FeoAB). The relative level of expression of these systems was determined using quantitative real-time RT-PCR assays that were specific for one gene from each system. Each of these genes was expressed, with the feoAB genes generating the most abundant iron-uptake related transcripts. To further examine the role of this system in the growth of C. perfringens, insertional inactivation was used to isolate a chromosomal feoB mutant. Growth of this mutant in the presence and absence of iron revealed that it had altered growth properties and a markedly reduced total iron and manganese content compared to the wild type; effects that were reversed upon complementation with the wild-type feoB gene. These studies suggest that under anaerobic conditions FeoB is the major protein required for the uptake of iron into the cell and that it may play an important role in the pathogenesis of C. perfringens infections.

Copper(II)-Bis(Thiosemicarbazonato) Complexes as Antibacterial Agents: Insights into Their Mode of Action and Potential as Therapeutics.

There is increasing interest in the use of lipophilic copper (Cu)-containing complexes to combat bacterial infections. In this work, we showed that Cu complexes with bis(thiosemicarbazone) ligands [Cu(btsc)] exert antibacterial activity against a range of medically significant pathogens. Previous work using Neisseria gonorrhoeae showed that Cu(btsc) complexes may act as inhibitors of respiratory dehydrogenases in the electron transport chain. We now show that these complexes are also toxic against pathogens that lack a respiratory chain. Respiration in Escherichia coli was slightly affected by Cu(btsc) complexes, but our results indicate that, in this model bacterium, the complexes act primarily as agents that deliver toxic Cu ions efficiently into the cytoplasm. Although the chemistry of Cu(btsc) complexes may dictate their mechanism of action, their efficacy depends heavily on bacterial physiology. This is linked to the ability of the target bacterium to tolerate Cu and, additionally, the susceptibility of the respiratory chain to direct inhibition by Cu(btsc) complexes. The physiology of N. gonorrhoeae, including multidrug-resistant strains, makes it highly susceptible to damage by Cu ions and Cu(btsc) complexes, highlighting the potential of Cu(btsc) complexes (and Cu-based therapeutics) as a promising treatment against this important bacterial pathogen.

Electrochemically mediated enantioselective reduction of chiral sulfoxides.

The respiratory DMSO reductase from Rhodobacter capsulatus catalyzes the reduction of dimethyl sulfoxide to dimethyl sulfide. Herein, we have utilized this Mo enzyme as an enantioselective catalyst to generate optically pure sulfoxides (methyl p-tolyl sulfoxide, methyl phenyl sulfoxide and phenyl vinyl sulfoxide) from racemic starting materials. A hexaaminecobalt coordination compound in its divalent oxidation state was employed as the mediator of electron transfer between the working electrode and DMSO reductase to continually reactivate the enzyme after turnover. In all cases, chiral HPLC analysis of the reaction mixture revealed that the S-sulfoxide was reduced more rapidly leading to enrichment or isolation of the R isomer.

An antimicrobial role for zinc in innate immune defense against group A streptococcus.

BACKGROUND: Zinc plays an important role in human immunity, and it is known that zinc deficiency in the host is linked to increased susceptibility to bacterial infection. In this study, we investigate the role of zinc efflux in the pathogenesis of Streptococcus pyogenes (group A Streptococcus [GAS]), a human pathogen responsible for superficial infections, such as pharyngitis and impetigo, and severe invasive infections. METHODS: The clinically important M1T1 wild-type strain was used in this study, and isogenic mutants were constructed with deletions in the czcD gene (Spy0653; which encodes a putative zinc efflux pump) and adjacent gczA gene (Spy0654; which encodes a putative zinc-dependent activator of czcD). Wild-type, isogenic mutants and complemented strains were tested for resistance against zinc stress, intracellular zinc accumulation, and virulence. RESULTS: Both czcD and gczA mutants exhibited increased sensitivity to zinc. Transcriptional analyses indicate that GczA upregulates czcD in response to zinc. Both mutants displayed increased susceptibility to human neutrophil killing and reduced virulence in a murine infection model. Furthermore, we showed that neutrophils mobilize zinc in response to GAS. CONCLUSIONS: These data indicate that the innate immune system may use zinc as an antimicrobial agent and that zinc efflux is an important contributor to GAS pathogenesis.