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Subunit or inactivated vaccines comprise the majority of vaccines used against viral and bacterial pathogens. However, compared to their live/attenuated counterparts, these vaccines often demonstrate reduced immunogenicity, requiring multiple boosters and or adjuvants to elicit protective immune responses. For this reason, studies of adjuvants and the mechanism through which they can improve inactivated vaccine responses are critical for the development of vaccines with increased efficacy. Studies have shown that the direct conjugation of adjuvant to antigen promotes vaccine immunogenicity, with the advantage of both the adjuvant and antigen targeting the same cell. Using this strategy of direct linkage, we developed an inactivated influenza A (IAV) vaccine that is directly conjugated with the Toll-like receptor 7/8 agonist resiquimod (R848) through a heterobifunctional crosslinker. Previously, we showed that this vaccine resulted in improved protection and viral clearance in newborn nonhuman primates compared to a non-adjuvanted vaccine. We subsequently discovered that the choice of linker used to conjugate R848 to the virus alters the stimulatory activity of the vaccine, promoting increased maturation and proinflammatory cytokine production from DC differentiated in vitro. With this knowledge, we explored how the choice of crosslinker impacts the stimulatory activity of these vaccines. We found that the linker choice alters signaling through the NF-κB pathway in human monocyte-derived dendritic cells (moDCs). Further, we extended our analyses to in vivo differentiated APC present in human peripheral blood, replicating the linker-dependent differences found in in vitro differentiated cells. Finally, we demonstrated in a mouse model that the choice of linker impacts the amount of IAV-specific IgG antibody produced in response to vaccination. These data enhance our understanding of conjugation approaches for improving vaccine immunogenicity. [ABSTRACT FROM AUTHOR]

Entamoeba histolytica, a protozoan intestinal parasite, is the causative agent of human amebiasis. Amebiasis is the fourth leading cause of death and the third leading cause of morbidity due to protozoan infections worldwide1, resulting in ~70,000 deaths annually. E. histolytica has been listed by the National Institutes of Health as a category B priority biodefense pathogen in the United States. Treatment relies on metronidazole (2), which has adverse effects3, and potential resistance of E. histolytica to the drug is an increasing concern (4)(5),. To facilitate drug screening for this anaerobic protozoan, we developed and validated an automated, high-throughput screen (HTS). This screen identified auranofin, a US Food and Drug Administration (FDA)-approved drug used therapeutically for rheumatoid arthritis, as active against E. histolytica in culture. Auranofin was ten times more potent against E. histolytica than metronidazole. Transcriptional profiling and thioredoxin reductase assays suggested that auranofin targets the E. histolytica thioredoxin reductase, preventing the reduction of thioredoxin and enhancing sensitivity of trophozoites to reactive oxygen-mediated killing. In a mouse model of amebic colitis and a hamster model of amebic liver abscess, oral auranofin markedly decreased the number of parasites, the detrimental host inflammatory response and hepatic damage. This new use of auranofin represents a promising therapy for amebiasis, and the drug has been granted orphan-drug status from the FDA., Screening large chemical libraries to identify amebicidals has been hindered by the throughput of labor-intensive traditional assays that rely on microscopic visualization6, radioisotopes7 and/or extensive staining methods (8). We developed [...]

An extended bioinformatic analysis has described the genome-based link for the absence of GHS biosynthesis, the presence of the type III pantothenate kinase (PanK) and the presence of the coenzyme A-disulfide reductase (CoADR)-like enzymes in a number of bacteria and spirochetes. The results have indicated that the type III PanK in the spore-forming Bacillus anthracis plays a vital role in the novel thiol/disulfide redox biology of this category A biodefense pathogen.

The first two enzymes are characterized for the bacillithiol (Cys-GlcN-malate (BSH)) biosynthetic pathway in Bacillus anthracis, which is combined to produce [alpha]-D-glucosaminyl L-malate (GlcN-malate) from UDP-GlcNAc and L-malate. The Bacillus anthracis bshA locus is identified in a transposon-site hybridization study as needed for growth, sporulation or germination, which has shown that the biosynthesis of BSH has represented a target for the development of novel antimicrobials with broad-spectrum activity against Gram-positive pathogens like Bacillus anthracis.

The crystal structure characterization at 2.10 [Angstrome] resolution for the Bacillus anthracis coenzyme A-disulfide reductase isoform (BaCoADR-RHD) containing a C-terminal rhodanese homology domain (RHD) which is the first structural representative of the multidomain proteins class of the rhodanese superfamily is reported. The results indicated that the BaCoADR-RHD isoform does not catalyze the reduction of coenzyme A-disulfide, but both enzymes conserved the Cys-SSCoA redox center.

The characterization of the stability, the [super 15]N backbone relaxation, and the hydrogen- deuterium exchange properties of reduced [NTD-[(SH).sub.2]] and oxidized (NTD-[S.sub.2]) N-terminal domain of AhpF (NTD) from Salmonella typhimurium. The significantly increased folded-state dynamic fluctuations observed in the catalytic 'a' fold for residues in NTD-[S.sub.2] compared to NTD-[(SH).sub.2] demonstrated the oxidation of the active site disulfide which resulted in minor increase in stability but dramatic increase in conformational heterogeneity in residues primarily in the redox active 'a' fold.

The p[K.sub.a] of the cysteinyl residues of Salmonella typhimurium AhpC (StAhpC) is assessed by using different approaches. The studies have shown that the p[K.sub.a] value of AhpC has balanced the need for a deprotonated thiol with the fact that thiolates with higher p[K.sub.a] values are stronger nucleophiles.

The structural and functional divergence between [alpha]-glycerophosphate oxidase (GlpO) from Streptococcus sp. and the bacterial and mitochondrial [alpha]-glycerophosphate dehydrogenases (GlpDs) is demonstrated.

Mutations of Thr77 at the decamer-building surface, which was successfully disrupted by the two substitutions T77I and T77D, while it is enhanced by the third substitute T77V, are studied. It is observed that the reduced sensitivity of the T77I and T77D but not the T77, Ahpc muteins to inactivation gives way to the fact that the unfolding of the propensity of the active site is increased by mutations thus leading to disulphide bond formation.

Streptococcus pneumoniae (Spn) causes a variety of disease states including fatal bacterial pneumonia. Our previous finding that introduction of Spn into an animal with ongoing influenza virus infection resulted in a CD8+ T cell population with reduced effector function gave rise to the possibility of direct regulation by pneumococcal components. Here, we show that treatment of effector T cells with lysate derived from Spn resulted in inhibition of IFNγ and tumor necrosis factor α production as well as of cytolytic granule release. Spn aminopeptidase N (PepN) was identified as the inhibitory bacterial component and surprisingly, this property was independent of the peptidase activity found in this family of proteins. Inhibitory activity was associated with reduced activation of ZAP-70, ERK1/2, c-Jun N-terminal kinase, and p38, demonstrating the ability of PepN to negatively regulate TCR signaling at multiple points in the cascade. These results reveal a novel immune regulatory function for a bacterial aminopeptidase. [ABSTRACT FROM AUTHOR]

Summary Peroxiredoxins (Prxs) are ubiquitous cysteine-based peroxidases that guard cells against oxidative damage, are virulence factors for pathogens, and are involved in eukaryotic redox regulatory pathways. We have analyzed catalytically active crystals to capture atomic resolution snapshots of a PrxQ subfamily enzyme (from Xanthomonas campestris ) proceeding through thiolate, sulfenate, and sulfinate species. These analyses provide structures of unprecedented accuracy for seeding theoretical studies, and reveal conformational intermediates giving insight into the reaction pathway. Based on a highly non-standard geometry seen for the sulfenate intermediate, we infer that the sulfenate formation itself can strongly promote local unfolding of the active site to enhance productive catalysis. Further, these structures reveal that preventing local unfolding, in this case via crystal contacts, results in facile hyperoxidative inactivation even for Prxs normally resistant to such inactivation. This supports previous proposals that conformation-specific inhibitors may be useful for achieving selective inhibition of Prxs that are drug targets. [ABSTRACT FROM AUTHOR]

The anti-arthritic gold-containing drug Auranofin is lethal to the protozoan intestinal parasite Entamoeba histolytica , the causative agent of human amebiasis, in both culture and animal models of the disease. A putative mechanism of Auranofin action proposes that monovalent gold, Au(I), released from the drug, can bind to the redox-active dithiol group of thioredoxin reductase (TrxR). Au(I) binding in the active site is expected to prevent electron transfer to the downstream substrate thioredoxin (Trx), thus interfering with redox homeostasis in the parasite. To clarify the molecular mechanism of Auranofin action in more detail, we determined a series of atomic resolution X-ray structures for E. histolytica thioredoxin ( Eh Trx) and thioredoxin reductase ( Eh TrxR), the latter with and without Auranofin. Only the disulfide-bonded form of the active site dithiol (Cys 140 –Cys 143 ) was invariably observed in crystals of Eh TrxR in spite of the addition of reductants in various crystallization trials, and no gold was found associated with these cysteines. Non-catalytic Cys 286 was identified as the only site of modification, but further mutagenesis studies using the C286Q mutant demonstrated that this site was not responsible for inhibition of Eh TrxR by Auranofin. Interestingly, we obtained both of the catalytically-relevant conformations of this bacterial-like, low molecular weight TrxR in crystals without requiring an engineered disulfide linkage between Cys mutants of TrxR and Trx (as was originally done with Escherichia coli TrxR and Trx). We note that the –CXXC– catalytic motif, even if reduced, would likely not provide space sufficient to bind Au(I) by both cysteines of the dithiol group. [ABSTRACT FROM AUTHOR]

The primary hyperoxalurias (PH), types 1-3, are disorders of glyoxylate metabolism that result in increased oxalate production and calcium oxalate stone formation. The breakdown of trans-4-hydroxy-L-proline (Hyp) from endogenous and dietary sources of collagen makes a significant contribution to the cellular glyoxylate pool. Proline dehydrogenase 2 (PRODH2), historically known as hydroxyproline oxidase, is the first step in the hydroxyproline catabolic pathway and represents a drug target to reduce the glyoxylate and oxalate burden of PH patients. This study is the first report of the expression, purification, and biochemical characterization of human PRODH2. Evaluation of a panel of N-terminal and C-terminal truncation variants indicated that residues 157-515 contain the catalytic core with one FAD molecule. The 12-fold higher kcat/Km value of 0.93 M-1·s-1 for Hyp over Pro demonstrates the preference for Hyp as substrate. Moreover, an anaerobic titration determined a Kd value of 125 µM for Hyp, a value ~1600-fold lower than the Km value. A survey of ubiquinone analogues revealed that menadione, duroquinone, and CoQ1 reacted more efficiently than oxygen as the terminal electron acceptor during catalysis. Taken together, these data and the slow reactivity with sodium sulfite support that PRODH2 functions as a dehydrogenase and most likely utilizes CoQ10 as the terminal electron acceptor in vivo. Thus, we propose that the name of PRODH2 be changed to hydroxyproline dehydrogenase (HYPDH). Three Hyp analogues were also identified to inhibit the activity of HYPDH, representing the first steps toward the development of a novel approach to treat all forms of PH. [ABSTRACT FROM AUTHOR]

Peroxiredoxins make up a ubiquitous family of cysteine-dependent peroxidases that reduce hydroperoxide or peroxynitrite substrates through formation of a cysteine sulfenic acid (R-SOH) at the active site. In the 2-Cys peroxiredoxins, a second (resolving) cysteine reacts with the sulfenic acid to form a disulfide bond. For all peroxiredoxins, structural rearrangements in the vicinity of the active site cysteine(s) are necessary to allow disulfide bond formation and subsequent reductive recycling. In this study, we evaluated the rate constants for individual steps in the catalytic cycle of Salmonella typhimurium AhpC. Conserved Trp residues situated close to both peroxidatic and resolving cysteines in AhpC give rise to large changes in fluorescence during the catalytic cycle. For recycling, AhpF very efficiently reduces the AhpC disulfide, with a single discernible step and a rate constant of 2.3 × 107 M-1 s-1. Peroxide reduction was more complex and could be modeled as three steps, beginning with a reversible binding of H2O2 to the enzyme (k1 = 1.36 × 108 M-1 s-1, and k-1 = 53 s-1), followed by rapid sulfenic acid generation (620 s-1) and then rate-limiting disulfide bond formation (75 s-1). Using bulkier hydroperoxide substrates with higher Km values, we found that different efficiencies (kcat/Km) for turnover of AhpC with these substrates are primarily caused by their slower rates of binding. Our findings indicate that this bacterial peroxiredoxin exhibits rates for both reducing and oxidizing parts of the catalytic cycle that are among the fastest observed so far for this diverse family of enzymes. [ABSTRACT FROM AUTHOR]

In Escherichia coli, bacterioferritin comigratory protein (BCP) is a peroxiredoxin (Prx) that catalyzes the reduction of H2O2 and organic hydroperoxides. This protein, along with plant PrxQ, is a founding member of one of the least studied subfamilies of Prxs. Recent structural data have suggested that proteins in the BCP/PrxQ group can exist as monomers or dimers; we report here that, by analytical ultracentrifugation, both oxidized and reduced E. coli BCP behave as monomers in solution at concentrations as high as 200 μM. Unexpectedly, thioredoxin (Trx1)-dependent peroxidase assays conducted by stopped-flow spectroscopy demonstrated that Vmax,app increases with increasing Trx1 concentrations, indicating a nonsaturable interaction (Km > 100 μM). At a physiologically reasonable Trx1 concentration of 10 μM, the apparent Km value for H2O2 is ∼80 μM, and overall, the Vmax/Km for H2O2, which remains constant at the various Trx1 concentrations (consistent with a ping-pong mechanism), is ∼1.3 × 104 M-1 s-1. Our kinetic analyses demonstrated that BCP can utilize a variety of reducing substrates, including Trx1, Trx2, Grx1, and Grx3. BCP exhibited a high redox potential of −145.9 ± 3.2 mV, the highest to date observed for a Prx. Moreover, BCP exhibited a broad peroxide specificity, with comparable rates for H2O2 and cumene hydroperoxide. We determined a pKa of 5.8 for the peroxidatic cysteine (Cys45) using both spectroscopic and activity titration data. These findings support an important role for BCP in interacting with multiple substrates and remaining active under highly oxidizing cellular conditions, potentially serving as a defense enzyme of last resort. [ABSTRACT FROM AUTHOR]

Bacillithiol (Cys-GlcN-malate, BSH) has recently been identified as a novel low-molecular weight thiol in Bacillus anthracis, Staphylococcus aureus, and several other Gram-positive bacteria lacking glutathione and mycothiol. We have now characterized the first two enzymes for the BSH biosynthetic pathway in B. anthracis, which combine to produce a-D-glucosaminyl L-malate (GlcN-malate) from UDP- GlcNAc and L-malate. The structure of the GlcNAc-malate intermediate has been determined, as have the kinetic parameters for the BaBshA glycosyltransferase (→GlcNAc-malate) and the BaBshB deacetylase (-GlcN-malate). BSH is one of only two natural products reported to contain a malyl glycoside, and the crystal structure of the BaBshA-UDP-malate ternary complex, determined in this work at 3.3 A resolution, identifies several active-site interactions important for the specific recognition of L-malate, but not other a-hydroxy acids, as the acceptor substrate. In sharp Contrast to the structures reported for the GlcNAc- l-D- myo-inositol-3-phosphate synthase (MshA) apo and ternary complex forms, there is no major conformational change observed in the structures of the Corresponding BaBshA forms. A mutant strain of B. anthracis deficient in the BshA glycosyltransferase fails to produce BSH, as predicted. This B. anthracis bshA locus (BA 1558) has been identified in a transposon-site hybridization study as required for growth, sporulation, or germination [Day, W. A., Jr., Rasmussen, S. L., Carpenter, B. M., Peterson, S. N., and Friedlander, A. M. (2007) J. Bacterial. 189, 3296-3301], suggesting that the biosynthesis of BSH could represent a target for the development of novel antimicrobials with broad-spectrum activity against Gram-positive pathogens like B. anthracis. The metabolites that function in thiol redox buffering and homeostasis in Bacillus are not well understood, and we present a composite picture based on this and other recent work. [ABSTRACT FROM AUTHOR]

Abstract: Peroxiredoxins (Prxs) are important peroxidases associated with both antioxidant protection and redox signaling. They use a conserved Cys residue to reduce peroxide substrates. The Prxs have a remarkably high catalytic efficiency that makes them a dominant player in cell-wide peroxide reduction, but the origins of their high activity have been mysterious. We present here a novel structure of human PrxV at 1.45 Å resolution that has a dithiothreitol bound in the active site with its diol moiety mimicking the two oxygens of a peroxide substrate. This suggests diols and similar di-oxygen compounds as a novel class of competitive inhibitors for the Prxs. Common features of this and other structures containing peroxide, peroxide-mimicking ligands, or peroxide-mimicking water molecules reveal hydrogen bonding and steric factors that promote its high reactivity by creating an oxygen track along which the peroxide oxygens move as the reaction proceeds. Key insights include how the active-site microenvironment activates both the peroxidatic cysteine side chain and the peroxide substrate and how it is exquisitely well suited to stabilize the transition state of the in-line SN2 substitution reaction that is peroxidation. [Copyright &y& Elsevier]

Little is known about the mechanisms by which Treponema pallidum (Tp), the causative agent of syphilis, copes with oxidative stress as it establishes persistent infection within its obligate human host. The Tp genomic sequence indicates that the bacterium's antioxidant defenses do not include glutathione and are limited to just a few proteins, with only one, TP0509, offering direct defense against peroxides. Although this Tp peroxiredoxin (Prx) closely resembles AhpC-like Prxs, Tp lacks AhpF, the typical reductant for such enzymes. Functionally, TpAhpC resembles largely eukaryotic, nonAhpC typical 2-Cys Prx proteins in using thioredoxin (Trx, TP0919) as an efficient electron donor and exhibiting broad specificity toward hydroperoxide substrates. Unlike many of the eukaryotic Prxs, however, TpAhpC is relatively resistant to inactivation during turnover with hydroperoxide substrates. As is often observed in typical 2-Cys Prxs, TpAhpC undergoes redox-sensitive oligomer formation. Quantitative immunoblotting revealed that TpTrx and TpAhpC are present at very high levels (over 100 and 300 riM, respectively) in treponemes infecting rabbit testes; their redox potentials, at -242 ± 1 and -192 ± 2 mV, respectively, are consistent with the role of TpTrx as the cellular reductant of TpAhpC. Transcriptional analysis of select antioxidant genes confirmed the presence of high mRNA levels for ahpC and trx which diminish greatly when spirochetes replicate under in vitro growth conditions. Thus, T. pallidum has evolved an extraordinarily robust, broad-spectrum AhpC as its sole mechanism for peroxide defense to combat this significant threat to treponemal growth and survival during infection. [ABSTRACT FROM AUTHOR]

Bacillithiol (BSH), the α-anomeric glycoside of L-cysteinyl-D-glucosamine with 1-malic acid, is a major low-molecular-weight thiol in Bacillus subtills and related bacteria. Here, we identify genes required for BSH biosynthesis and provide evidence that the synthetic pathway has similarities to that established for the related thiol (mycothiol) in the Actinobacteria. Consistent with a key role for BSH in detoxification of electroph lIes, the BshA glycosyltransferase and BshBl deacetylase are encoded in an operon with methylglyoxal synthase. BshBl is partially redundant in function with BshB2, a deacetylase of the LmbE family. Phylogenomic profiling identif led a conserved unknown function protein (COG4365) as a candidate cysteine-adding enzyme (BshC) that cooccurs in genomes also encoding BshA, BshB1, and BshB2. Additional evolutionarily linked proteins include a thioredoxin reductase homolog and two thiol:disulfide oxidoreductases of the DUF1094 (CxC motif) family. Mutants lacking BshA, BshC, or both BshBl and BshB2 are devoid of BSH. BSH is at least partially redundant in function with other low-molecular-weight thiols: redox proteomics indicates that protein thiols are largely reduced even in the absence of BSH. At the transcriptional level, the induction of genes controlled by two thiol-based regulators (OhrR, Spx) occurs normally. However, BSH null cells are significantly altered in acid and salt resistance, sporulation, and resistance to electrophiles and thiol reactive compounds. Moreover, cells lacking BSH are highly sensitive to fosfomycin, an epoxidecontaining antibiotic detoxified by FosB, a prototype for bacillithiol-Stransferase enzymes. [ABSTRACT FROM AUTHOR]

The FAD-dependent α-glycerophosphate oxidase (GIpO) from Enterococcus casseliflavus and Streptococcus sp. was originally studied as a soluble flavoprotein oxidase; surprisingly, the GlpO sequence is 30-43% identical to those of the α-glycerophosphate dehydrogenases (GlpDs) from mitochondrial and bacterial sources. The structure of a deletion mutant of Streptococcus sp. GIpO (GIpOΔ, lacking a 50-residue insert that includes a flexible surface region) has been determined using multiwavelength anomalous dispersion data and refined at 2.3 Å resolution. Using the GIpOΔ structure as a search model, we have also determined the intact GIpO structure, as refined at 2.4 Å resolution. The first two domains of the GIpO fold are most closely related to those of the flavoprotein glycine oxidase, where they function in FAD binding and substrate binding, respectively; the GIpO C-terminal domain consists of two helix bundles and is not closely related to any known structure. The flexible surface region in intact GIpO corresponds tp a segment of missing electron density that links the substrate-binding domain to a ββα element of the FAD-binding domain. In accordance with earlier biochemical studies (stabilizations of the covalent FAD-N5-sulfite adduct and p-quinonoid form of 8-mercapto-FAD), Ile430-N, Thr431-N, and Thr431 -OG are hydrogen bonded to FAD-O2α. in GlpOΔ, stabilizing the negative charge in these two modified flavins and facilitating transfer of a hydride to FAD-N5 (from Glp) as well. Active-site overlays with the glycine oxidase-N-acetylglycine and D-amino acid oxidase-D-alanine complexes demonstrate that Arg346 of GlpOΔ is structurally equivalent to Arg302 and Arg285, respectively; in both cases, these residues interact directly with the amino acid substrate or inhibitor carboxylate. The structural and functional divergence between GlpO and the bacterial and mitochondrial GlpDs is also discussed. [ABSTRACT FROM AUTHOR]

Uses DNase I protection assays to further investigate the interaction of the RofA protein of Streptococcus pyogenes with its target promoters. Analysis of mutants using a novel vector for examination of transcriptional regulation in gram-positive bacteria; Indication that RofA recognizes two binding sites in the region between prtF and rofA.

Explores the equilibrium analysis of the active-site asymmetry in enterococcal NADH oxidase. Thermodynamic inequivalence between the two active sites per dimer in recombinant wild-type oxidase; Role of the cystein-sulfenic acid redox center; Pyridine nucleotides with both recombinant wild-type and C42S NADH oxidase under equilibrium conditions.

Reports that in order to test the proposal that the active-site His10 of NADH peroxidase, functions as an essential acid-base catalyst, an analysis of mutants in which this residue was replaced by Gln or Ala was done. Basis of study; Findings.