Bjorn Vergauwen

Characterization of the Bifunctional γ-Glutamate-cysteine Ligase/Glutathione Synthetase (GshF) of Pasteurella multocida

Glutamate-cysteine ligase (γ-ECL) and glutathione synthetase (GS) are the two unrelated ligases that constitute the glutathione biosynthesis pathway in most eukaryotes, purple bacteria, and cyanobacteria. γ-ECL is a member of the glutamine synthetase family, whereas GS enzymes group together with highly diverse carboxyl-to-amine/thiol ligases, all characterized by the so-called two-domain ATP-grasp fold. This generalized scheme toward the formation of glutathione, however, is incomplete, as functional steady-state levels of intracellular glutathione may also accumulate solely by import, as has been reported for the Pasteurellaceae member Haemophilus influenzae, as well as for certain Gram-positive enterococci and streptococci, or by the action of a bifunctional fusion protein (termed GshF), as has been reported recently for the Gram-positive firmicutes Streptococcus agalactiae and Listeria monocytogenes. Here, we show that yet another member of the Pasteurellaceae family, Pasteurella multocida, acquires glutathione both by import and GshF-driven biosynthesis. Domain architecture analysis shows that this P. multocida GshF bifunctional ligase contains an N-terminal γ-proteobacterial γ-ECL-like domain followed by a typical ATP-grasp domain, which most closely resembles that of cyanophycin synthetases, although it has no significant homology with known GS ligases. Recombinant P. multocida GshF overexpresses as an ∼85-kDa protein, which, on the basis of gel-sizing chromatography, forms dimers in solution. The γ-ECL activity of GshF is regulated by an allosteric type of glutathione feedback inhibition (Ki = 13.6 mm). Furthermore, steady-state kinetics, on the basis of which we present a novel variant of half-of-the-sites reactivity, indicate intimate domain-domain interactions, which may explain the bifunctionality of GshF proteins.

Exogenous Glutathione Completes the Defense against Oxidative Stress in Haemophilus influenzae

Since they are equipped with several strategies by which they evade the antimicrobial defense of host macrophages, it is surprising that members of the genus Haemophilus appear to be deficient in common antioxidant systems that are well established to protect prokaryotes against oxidative stress. Among others, no genetic evidence for glutathione (gamma-Glu-Cys-Gly) (GSH) biosynthesis or for alkyl hydroperoxide reduction (e.g., the Ahp system characteristic or enteric bacteria) is apparent from the Haemophilus influenzae Rd genome sequence, suggesting that the organism relies on alternative systems to maintain redox homeostasis or to reduce small alkyl hydroperoxides. In this report we address this apparent paradox for the nontypeable H. influenzae type strain NCTC 8143. Instead of biosynthesis, we could show that this strain acquires GSH by importing the thiol tripeptide from the growth medium. Although such GSH accumulation had no effect on growth rates, the presence of cellular GSH protected against methylglyoxal, tert-butyl hydroperoxide (t-BuOOH), and S-nitrosoglutathione toxicity and regulated the activity of certain antioxidant enzymes. H. influenzae NCTC 8143 extracts were shown to contain GSH-dependent peroxidase activity with t-BuOOH as the peroxide substrate. The GSH-mediated protection against t-BuOOH stress is most probably catalyzed by the product of open reading frame HI0572 (Prx/Grx), which we isolated from a genomic DNA fragment that confers wild-type resistance to t-BuOOH toxicity in the Ahp-negative Escherichia coli strain TA4315 and that introduces GSH-dependent alkyl hydroperoxide reductase activity into naturally GSH peroxidase-negative E. coli. Finally, we demonstrated that cysteine is an essential amino acid for growth and that cystine, GSH, glutathione amide, and cysteinylglycine can be catabolized in order to complement cysteine deficiency.

Glutathione import in Haemophilus influenzae Rd is primed by the periplasmic heme-binding protein HbpA

Glutathione (GSH) is a vital intracellular cysteine-containing tripeptide across all kingdoms of life and assumes a plethora of cellular roles. Such pleiotropic behavior relies on a finely tuned spatiotemporal distribution of glutathione and its conjugates, which is not only controlled by synthesis and breakdown, but also by transport. Here, we show that import of glutathione in the obligate human pathogen Haemophilus influenzae, a glutathione auxotrophe, is mediated by the ATP-binding cassette (ABC)-like dipeptide transporter DppBCDF, which is primed for glutathione transport by a dedicated periplasmic-binding protein (PBP). We have identified the periplasmic lipoprotein HbpA, a protein hitherto implicated in heme acquisition, as the cognate PBP that specifically binds reduced (GSH) and oxidized glutathione (GSSG) forms of glutathione with physiologically relevant affinity, while it exhibits marginal binding to hemin. Dissection of the ligand preferences of HbpA showed that HbpA does not recognize bulky glutathione S conjugates or glutathione derivatives with C-terminal modifications, consistent with the need for selective import of useful forms of glutathione and the concomitant exclusion of potentially toxic glutathione adducts. Structural studies of the highly homologous HbpA from Haemophilus parasuis in complex with GSSG have revealed the structural basis of the proposed novel function for HbpA-like proteins, thus allowing a delineation of highly conserved structure-sequence fingerprints for the entire family of HbpA proteins. Taken together, our studies unmask the main physiological role of HbpA and establish a paradigm for glutathione import in bacteria. Accordingly, we propose a name change for HbpA to glutathione-binding protein A.

A kinetic approach to the dependence of dissimilatory metal reduction by Shewanella oneidensis MR-1 on the outer membrane cytochromes c OmcA and OmcB

The Gram‐negative bacterium Shewanella oneidensis MR‐1 shows a remarkably versatile anaerobic respiratory metabolism. One of its hallmarks is its ability to grow and survive through the reduction of metallic compounds. Among other proteins, outer membrane decaheme cytochromes c OmcA and OmcB have been identified as key players in metal reduction. In fact, both of these cytochromes have been proposed to be terminal Fe(III) and Mn(IV) reductases, although their role in the reduction of other metals is less well understood. To obtain more insight into this, we constructed and analyzed omcA, omcB and omcA/omcB insertion mutants of S. oneidensis MR‐1. Anaerobic growth on Fe(III), V(V), Se(VI) and U(VI) revealed a requirement for both OmcA and OmcB in Fe(III) reduction, a redundant function in V(V) reduction, and no apparent involvement in Se(VI) and U(VI) reduction. Growth of the omcB– mutant on Fe(III) was more affected than growth of the omcA– mutant, suggesting OmcB to be the principal Fe(III) reductase. This result was corroborated through the examination of whole cell kinetics of OmcA‐ and OmcB‐dependent Fe(III)‐nitrilotriacetic acid reduction, showing that OmcB is ∼ 11.5 and ∼ 6.3 times faster than OmcA at saturating and low nonsaturating concentrations of Fe(III)‐nitrilotriacetic acid, respectively, whereas the omcA– omcB– double mutant was devoid of Fe(III)‐nitrilotriacetic acid reduction activity. These experiments reveal, for the first time, that OmcA and OmcB are the sole terminal Fe(III) reductases present in S. oneidensis MR‐1. Kinetic inhibition experiments further revealed vanadate (V2O5) to be a competitive and mixed‐type inhibitor of OmcA and OmcB, respectively, showing similar affinities relative to Fe(III)‐nitrilotriacetic acid. Neither sodium selenate nor uranyl acetate were found to inhibit OmcA‐ and OmcB‐dependent Fe(III)‐nitrilotriacetic acid reduction. Taken together with our growth experiments, this suggests that proteins other than OmcA and OmcB play key roles in anaerobic Se(VI) and U(VI) respiration.

Glutathione and Catalase Provide Overlapping Defenses for Protection against Respiration-Generated Hydrogen Peroxide in Haemophilus influenzae

Glutathione is an abundant and ubiquitous low-molecular-weight thiol that may play a role in many cellular processes, including protection against the deleterious effects of reactive oxygen species. We address here the role of glutathione in protection against hydrogen peroxide (H2O2) in Haemophilus influenzae and show that glutathione and catalase provide overlapping defense systems. H. influenzae is naturally glutathione deficient and imports glutathione from the growth medium. Mutant H. influenzae lacking catalase and cultured in glutathione-deficient minimal medium is completely devoid of H2O2 scavenging activity and, accordingly, substantial amounts of H2O2 accumulate in the growth medium. H. influenzae generates H2O2 at rates similar to those reported for Escherichia coli, but the toxicity of this harmful metabolite is averted by glutathione-based H2O2 removal, which appears to be the primary system for protection against H2O2 endogenously generated during aerobic respiration. When H2O2 concentrations exceed low micromolar levels, the hktE gene-encoded catalase becomes the predominant scavenger. The requirement for glutathione in protection against oxidative stress is analogous to that in higher and lower eukaryotes but is unlike the situation in other bacteria in which glutathione is dispensable for aerobic growth during both normal and oxidative stress conditions.

Allosteric competitive inactivation of hematopoietic CSF-1 signaling by the viral decoy receptor BARF1

Hematopoietic human colony-stimulating factor 1 (hCSF-1) is essential for innate and adaptive immunity against viral and microbial infections and cancer. The human pathogen Epstein-Barr virus secretes the lytic-cycle protein BARF1 that neutralizes hCSF-1 to achieve immunomodulation. Here we show that BARF1 binds the dimer interface of hCSF-1 with picomolar affinity, away from the cognate receptor–binding site, to establish a long-lived complex featuring three hCSF-1 at the periphery of the BARF1 toroid. BARF1 locks dimeric hCSF-1 into an inactive conformation, rendering it unable to signal via its cognate receptor on human monocytes. This reveals a new functional role for hCSF-1 cooperativity in signaling. We propose a new viral strategy paradigm featuring an allosteric decoy receptor of the competitive type, which couples efficient sequestration and inactivation of the host growth factor to abrogate cooperative assembly of the cognate signaling complex.

The 285 kDa Bap/RTX hybrid cell surface protein (SO4317) of Shewanella oneidensis MR-1 is a key mediator of biofilm formation.

Shewanella oneidensis, a Gram-negative bacterium with unusual respiratory versatility, is found in soil and sediment environments, and sporadically as an opportunistic pathogen in humans and aquatic animals. The ability to form biofilms is a critical factor in the environmental spread and survival of this bacterium. We subjected S. oneidensis MR-1 to random transposon insertion mutagenesis to identify genes contributing to the ability of the organism to form biofilms on polystyrene surfaces. Follow-up of the clone that was most heavily impaired in biofilm formation led to the identification of a novel 285 kDa multi-domain protein which we have termed biofilm-promoting factor A (BpfA). BpfA is secreted by a type I secretion system to the cell surface, where it is a requisite for biofilm development. The BpfA-dependent biofilm phenotype is positively modulated by sub to low millimolar amounts of calcium. Intriguingly, BpfA features structural motifs and sequence fingerprints that can be traced back to bacterial Bap-family and RTX family proteins, two protein families harboring putative and established calcium binding sites.

Extracellular Complexes of the Hematopoietic Human and Mouse CSF-1 Receptor Are Driven by Common Assembly Principles

The hematopoietic colony stimulating factor-1 receptor (CSF-1R or FMS) is essential for the cellular repertoire of the mammalian immune system. Here, we report a structural and mechanistic consensus for the assembly of human and mouse CSF-1:CSF-1R complexes. The EM structure of the complete extracellular assembly of the human CSF-1:CSF-1R complex reveals how receptor dimerization by CSF-1 invokes a ternary complex featuring extensive homotypic receptor contacts and striking structural plasticity at the extremities of the complex. Studies by small-angle X-ray scattering of unliganded hCSF-1R point to large domain rearrangements upon CSF-1 binding, and provide structural evidence for the relevance of receptor predimerization at the cell surface. Comparative structural and binding studies aiming to dissect the assembly principles of human and mouse CSF-1R complexes, including a quantification of the CSF-1/CSF-1R species cross-reactivity, show that bivalent cytokine binding to receptor coupled to ensuing receptor-receptor interactions are common denominators in extracellular complex formation.

Hydrogen peroxide scavenging is not a virulence determinant in the pathogenesis of Haemophilus influenzae type b strain Eagan

BackgroundA potentially lethal flux of hydrogen peroxide (H2O2) is continuously generated during aerobic metabolism. It follows that aerobic organisms have equipped themselves with specific H2O2 dismutases and H2O2 reductases, of which catalase and the alkyl hydroperoxide reductase (AhpR) are the best-studied prokaryotic members. The sequenced Haemophilus influenzae Rd genome reveals one catalase, designated HktE, and no AhpR. However, Haemophilus influenzae type b strain Eagan (Hib), a causative agent of bacterial sepsis and meningitis in young children, disrupted in its hktE gene is not attenuated in virulence, and retains the ability to rapidly scavenge H2O2. This redundancy in H2O2-scavenging is accounted for by peroxidatic activity which specifically uses glutathione as the reducing substrate.ResultsWe show here that inside acatalasaemic H. influenzae all of the residual peroxidatic activity is catalyzed by PGdx, a hybrid peroxiredoxin-glutaredoxin glutathione-dependent peroxidase. In vitro kinetic assays on crude hktE-pgdx-H. influenzae Rd extracts revealed the presence of NAD(P)H:peroxide oxidoreductase activity, which, however, appears to be physiologically insignificant because of its low affinity for H2O2 (Km = 1.1 mM). Hydroperoxidase-deficient hktE-pgdx-H. influenzae Rd showed a slightly affected aerobic growth phenotype in rich broth, while, in chemically defined medium, growth was completely inhibited by aerobic conditions, unless the medium contained an amino acid/vitamin supplement. To study the role of PGdx in virulence and to assess the requirement of H2O2-scavenging during the course of infection, both a pgdx single mutant and a pgdx/hktE double mutant of Hib were assayed for virulence in an infant rat model. The ability of both mutant strains to cause bacteremia was unaffected.ConclusionCatalase (HktE) and a sole peroxidase (PGdx) account for the majority of scavenging of metabolically generated H2O2 in the H. influenzae cytoplasm. Growth experiments with hydroperoxidase-deficient hktE-pgdx-H. influenzae Rd suggest that the cytotoxicity inflicted by the continuous accumulation of H2O2 during aerobic growth brings about bacteriostasis rather than bacterial killing. Finally, H2O2-scavenging is not a determinant of Hib virulence in the infant rat model of infection.

Physiological Characterization of Haemophilus influenzae Rd Deficient in Its Glutathione-dependent Peroxidase PGdx

The chimeric peroxidase PGdx of Haemophilus influenzae Rd belongs to a recently identified family of thiol peroxidases capable of reducing hydrogen peroxide as well as alkylhydroperoxides by means of glutathione redox cycling. In the present study, we constructed a H. influenzae Rd strain, deficient in its PGdx encoding gene (open reading frame HI0572). The mutant was shown by disk inhibition and liquid culture growth assays to exhibit increased susceptibility to organic hydroperoxides. The hampered growth was restored by complementing the interrupted gene on the genome with a replicating plasmid bearing an intact copy of the gene, hereby rejecting the possible influences of polar effects. Elevated levels of hydrogen peroxide scavenging activity, due to the catalase HktE, were measured in the absence of a functional pgdx gene rendering the mutant more resilient against hydrogen peroxide. On the other hand, after initiation of the stationary phase, aerobic cultures of the pgdx mutant were practically devoid of living cells, whereas wild-type counterparts retained viability. This observed feature was alleviated by complementation with the functional gene or with the addition of catalase.