ABSTRACT
The pore-forming cytotoxin α-hemolysin, or Hla, is a critical Staphylococcus aureus virulence factor that promotes infection by causing tissue damage, excessive inflammation, and lysis of both innate and adaptive immune cells, among other cellular targets. In this study, we asked whether a virus-like particle (VLP)-based vaccine targeting Hla could attenuate S. aureus Hla-mediated pathogenesis. VLPs are versatile vaccine platforms that can be used to display target antigens in a multivalent array, typically resulting in the induction of high titer, long-lasting antibody responses. In the present study, we describe the first VLP-based vaccines that target Hla. Vaccination with either of two VLPs displaying a 21 amino-acid linear neutralizing domain (LND) of Hla protected both male and female mice from subcutaneous Hla challenge, evident by reduction in lesion size and neutrophil influx to the site of intoxication. Antibodies elicited by VLP-LND vaccination bound both the LND peptide and the native toxin, effectively neutralizing Hla and preventing toxin-mediated lysis of target cells. We anticipate these novel and promising vaccines being part of a multi-component S. aureus vaccine to reduce severity of S. aureus infection.
Subject(s)
Bacterial Toxins/pharmacology , Bacterial Vaccines/pharmacology , Hemolysin Proteins/pharmacology , Skin/drug effects , Staphylococcal Skin Infections/prevention & control , Staphylococcus aureus/drug effects , Vaccines, Virus-Like Particle/pharmacology , Animals , Antibodies, Bacterial/blood , Bacterial Toxins/immunology , Bacterial Vaccines/immunology , Disease Models, Animal , Epitopes , Female , Hemolysin Proteins/immunology , Humans , Immunogenicity, Vaccine , Jurkat Cells , Male , Mice, Inbred BALB C , Neutralization Tests , Skin/immunology , Skin/microbiology , Skin/pathology , Staphylococcal Skin Infections/immunology , Staphylococcal Skin Infections/microbiology , Staphylococcal Skin Infections/pathology , Staphylococcus aureus/immunology , Staphylococcus aureus/pathogenicity , Vaccination , Vaccines, Virus-Like Particle/immunologyABSTRACT
A defining characteristic of bacterial cytochromes (cyt's) in the P460 family is an unusual cross-link connecting the heme porphyrin to the side chain of a lysyl residue in the protein backbone. Here, via proteomics of the periplasmic fraction of the ammonia-oxidizing bacterium (AOB) Nitrosomonas europaea, we report the identification of a variant member of the P460 family that contains a methionyl residue in place of the cross-linking lysine. We formally designate this protein cytochrome "c'ß-Met" to distinguish it from other members bearing different residues at this position (e.g., cyt c'ß-Phe from the methane-oxidizing Methylococcus capsulatus Bath). As isolated, the monoheme cyt c'ß-Met is high-spin (S = 5/2). Optical spectroscopy suggests that a cross-link is absent. Hydroxylamine, the substrate for the cross-linked cyt P460 from N. europaea, did not appreciably alter the optical spectrum of cyt c'ß with up to 1000-fold excess at pH 7.5. Cyt c'ß-Met did however bind 1 equiv of H2O2, and with a slight excess, Mössbauer spectroscopy indicated the formation of a semistable ferryl (FeIVâO) Compound II-like species. The corresponding electron paramagnetic resonance showed a very low intensity signal indicative of a radical at g = 2.0. Furthermore, cyt c'ß-Met exhibited guaiacol-dependent peroxidase activity (kcat = 20.0 ± 1.2 s-1; KM = 2.6 ± 0.4 mM). Unlike cyt c'ß-Met, cyt P460 showed evidence of heme inactivation in the presence of 2 equiv of H2O2 with no appreciable guaiacol-dependent peroxidase activity. Mutagenesis of the cross-linking lysyl residue to an alanine in cyt P460, however, reversed this lack of activity.
Subject(s)
Cytochromes c/metabolism , Heme/metabolism , Iron Compounds/metabolism , Lysine/metabolism , Nitrosomonas/chemistry , Peroxidase/metabolism , Cytochromes c/chemistry , Cytochromes c/genetics , Electron Spin Resonance Spectroscopy , Heme/chemistry , Iron Compounds/chemistry , Lysine/chemistry , Models, Molecular , Nitrosomonas/cytology , Nitrosomonas/metabolism , Peroxidase/chemistry , Proteomics , Spectroscopy, MossbauerABSTRACT
Sex bias in innate defense against Staphylococcus aureus skin and soft tissue infection (SSTI) is dependent on both estrogen production by the host and S. aureus secretion of the virulence factor, α-hemolysin (Hla). The impact of estrogen signaling on the immune system is most often studied in terms of the nuclear estrogen receptors ERα and ERß. However, the potential contribution of the G protein-coupled estrogen receptor (GPER) to innate defense against infectious disease, particularly with respect to skin infection, has not been addressed. Using a murine model of SSTI, we found that GPER activation with the highly selective agonist G-1 limits S. aureus SSTI and Hla-mediated pathogenesis, effects that were absent in GPER knockout mice. Specifically, G-1 reduced Hla-mediated skin lesion formation and pro-inflammatory cytokine production, while increasing bacterial clearance. In vitro, G-1 reduced surface expression of the Hla receptor, ADAM10, in a human keratinocyte cell line and increased resistance to Hla-mediated permeability barrier disruption. This novel role for GPER activation in skin innate defense against infectious disease suggests that G-1 may have clinical utility in patients with epithelial permeability barrier dysfunction or who are otherwise at increased risk of S. aureus infection, including those with atopic dermatitis or cancer.
Subject(s)
Bacterial Toxins/genetics , Estrogen Receptor alpha/genetics , Estrogen Receptor beta/genetics , Hemolysin Proteins/genetics , Receptors, Estrogen/genetics , Receptors, G-Protein-Coupled/genetics , Staphylococcal Infections/genetics , ADAM10 Protein/genetics , Animals , Bacterial Toxins/metabolism , Epithelial Cells/microbiology , Epithelial Cells/pathology , Hemolysin Proteins/metabolism , Host-Pathogen Interactions/genetics , Humans , Immunity, Innate/genetics , Keratinocytes/microbiology , Mice , Mice, Knockout , Signal Transduction/genetics , Skin/immunology , Skin/microbiology , Staphylococcal Infections/microbiology , Staphylococcal Infections/pathology , Staphylococcus aureus/genetics , Staphylococcus aureus/pathogenicityABSTRACT
Numerous studies have reported sex bias in infectious diseases, with bias direction dependent on pathogen and site of infection. Staphylococcus aureus is the most common cause of skin and soft tissue infections (SSTIs), yet sex bias in susceptibility to S. aureus SSTI has not been described. A search of electronic health records revealed an odds ratio of 2.4 for S. aureus SSTI in males versus females. To investigate the physiological basis of this bias, we compared outcomes between male and female mice in a model of S. aureus dermonecrosis. Consistent with the epidemiological data, female mice were better protected against SSTI, with reduced dermonecrosis followed later by increased bacterial clearance. Protection in females was disrupted by ovariectomy and restored by short-term estrogen administration. Importantly, this sex bias was mediated by a sex-specific response to the S. aureus-secreted virulence factor α-hemolysin (Hla). Infection with wild-type S. aureus suppressed inflammatory cytokine production in the skin of female, but not male, mice when compared with infection with an isogenic hla deletion mutant. This differential response was conserved following injection with Hla alone, demonstrating a direct response to Hla independent of bacterial burden. Additionally, neutrophils, essential for clearing S. aureus, demonstrated sex-specific S. aureus bactericidal capacity ex vivo. This work suggests that sex-specific skin innate responsiveness to Hla and neutrophil bactericidal capacity play important roles in limiting S. aureus SSTI in females. Understanding the molecular mechanisms controlling this sex bias may reveal novel targets to promote host innate defense against S. aureus skin infection.
Subject(s)
Bacterial Toxins/metabolism , Hemolysin Proteins/metabolism , Staphylococcal Skin Infections/microbiology , Staphylococcus aureus/pathogenicity , Animals , Cytokines/metabolism , Disease Models, Animal , Disease Resistance , Estrogens/metabolism , Female , Gene Expression , Immunity, Innate , Inflammasomes/metabolism , Inflammation Mediators , Male , Mice , Microbial Viability/immunology , Neutrophils/immunology , Neutrophils/metabolism , Neutrophils/microbiology , Sex Factors , Staphylococcal Skin Infections/genetics , Staphylococcal Skin Infections/immunology , Staphylococcal Skin Infections/metabolism , Virulence , Virulence FactorsABSTRACT
Staphylococcus aureus is the leading cause of skin and soft tissue infections (SSTIs) and mounting antibiotic resistance requires innovative treatment strategies. S. aureus uses secreted cyclic autoinducing peptides (AIPs) and the accessory gene regulator (agr) operon to coordinate expression of virulence factors required for invasive infection. Of the four agr alleles (agr types I-IV and corresponding AIPs1-4), agr type I isolates are most frequently associated with invasive infection. Cyclization via a thiolactone bond is essential for AIP function; therefore, recognition of the cyclic form of AIP1 may be necessary for antibody-mediated neutralization. However, the small sizes of AIPs and labile thiolactone bond have hindered vaccine development. To overcome this, we used a virus-like particle (VLP) vaccine platform (PP7) for conformationally-restricted presentation of a modified AIP1 amino acid sequence (AIP1S). Vaccination with PP7-AIP1S elicited AIP1-specific antibodies and limited agr-activation in vivo. Importantly, in a murine SSTI challenge model with a highly virulent agr type I S. aureus isolate, PP7-AIP1S vaccination reduced pathogenesis and increased bacterial clearance compared to controls, demonstrating vaccine efficacy. Given the contribution of MRSA agr type I isolates to human disease, vaccine targeting of AIP1-regulated virulence could have a major clinical impact in the fight against antibiotic resistance.
Subject(s)
Staphylococcal Infections/immunology , Staphylococcal Infections/prevention & control , Staphylococcal Vaccines/immunology , Staphylococcus aureus/immunology , Staphylococcus aureus/pathogenicity , Vaccines, Virus-Like Particle/immunology , Virulence/immunology , Animals , Antibodies, Bacterial , Bacterial Proteins/chemistry , Bacterial Proteins/immunology , Disease Models, Animal , Immunization , Mice , Models, Molecular , Peptides/chemistry , Peptides/immunology , Peptides, Cyclic/chemistry , Peptides, Cyclic/immunology , Protein ConformationABSTRACT
Second coordination sphere (SCS) effects in proteins are modulated by active site residues and include hydrogen bonding, electrostatic/dipole interactions, steric interactions, and π-stacking of aromatic residues. In Cyt P450s, extended H-bonding networks are located around the proximal cysteinate ligand of the heme, referred to as the 'Cys pocket'. These hydrogen bonding networks are generally believed to regulate the Fe-S interaction. Previous work identified the S(Cys) â Fe σ CT transition in the high-spin (hs) ferric form of Cyt P450cam and corresponding Cys pocket mutants by low-temperature (LT) MCD spectroscopy [Biochemistry 50:1053, 2011]. In this work, we have investigated the effect of the hydrogen bond from W409 to the axial Cys ligand of the heme in the hs ferric state (with H4B and L-Arg bound) of rat neuronal nitric oxide synthase oxygenase construct (nNOSoxy) using MCD spectroscopy. For this purpose, wt enzyme and W409 mutants were investigated where the H-bonding network with the axial Cys ligand is perturbed. Overall, the results are similar to Cyt P450cam and show the intense S(Cys) â Fe σ CT band in the LT MCD spectrum at about 27,800 cm-1, indicating that this feature is a hallmark of {heme-thiolate} active sites. The discovery of this MCD feature could constitute a new approach to classify {heme-thiolate} sites in hs ferric proteins. Finally, the W409 mutants show that the hydrogen bond from this group only has a small effect on the Fe-S(Cys) bond strength, at least in the hs ferric form of the protein studied here. Low-temperature MCD spectroscopy is used to investigate the effect of the hydrogen bond from W409 to the axial Cys ligand of the heme in neuronal nitric oxide synthase. The intense S(Cys) â Fe σ-CT band is monitored to identify changes in the Fe-S(Cys) bond in wild-type protein and W409 mutants.
Subject(s)
Catalytic Domain , Coordination Complexes/chemistry , Cysteine/chemistry , Iron/chemistry , Nitric Oxide Synthase Type I/chemistry , Animals , Binding Sites/genetics , Circular Dichroism/methods , Coordination Complexes/metabolism , Cysteine/genetics , Cysteine/metabolism , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Heme/chemistry , Heme/metabolism , Hydrogen Bonding , Iron/metabolism , Ligands , Models, Molecular , Mutation , Nitric Oxide/chemistry , Nitric Oxide/metabolism , Nitric Oxide Synthase Type I/genetics , Nitric Oxide Synthase Type I/metabolism , Oxidation-Reduction , Rats , Spectrophotometry , Static Electricity , ThermodynamicsABSTRACT
Hyperlipidemia has been extensively studied in the context of atherosclerosis, whereas the potential health consequences of the opposite extreme, hypolipidemia, remain largely uninvestigated. Circulating lipoproteins are essential carriers of insoluble lipid molecules and are increasingly recognized as innate immune effectors. Importantly, severe hypolipidemia, which may occur with trauma or critical illness, is clinically associated with bacterial pneumonia. To test the hypothesis that circulating lipoproteins are essential for optimal host innate defense in the lung, we used lipoprotein-deficient mice and a mouse model of Staphylococcus aureus pneumonia in which invasive infection requires virulence factor expression controlled by the accessory gene regulator (agr) operon. Activation of agr and subsequent virulence factor expression is inhibited by apolipoprotein B, the structural protein of low-density lipoprotein, which binds and sequesters the secreted agr-signaling peptide (AIP). In this article, we report that lipoprotein deficiency impairs early pulmonary innate defense against S. aureus quorum-sensing-dependent pathogenesis. Specifically, apolipoprotein B levels in the lung early postinfection are significantly reduced with lipoprotein deficiency, coinciding with impaired host control of S. aureus agr-signaling and increased agr-dependent morbidity (weight loss) and inflammation. Given that lipoproteins also inhibit LTA- and LPS-mediated inflammation, these results suggest that hypolipidemia may broadly impact posttrauma pneumonia susceptibility to both Gram-positive and -negative pathogens. Together with previous reports demonstrating that hyperlipidemia also impairs lung innate defense, these results suggest that maintenance of normal serum lipoprotein levels is necessary for optimal host innate defense in the lung.
Subject(s)
Bacterial Proteins/metabolism , Hypolipoproteinemias/immunology , Lipoproteins, LDL/blood , Pneumonia, Staphylococcal/immunology , Quorum Sensing/immunology , Staphylococcus aureus/immunology , Trans-Activators/metabolism , Animals , Apolipoproteins B/immunology , Bacterial Proteins/genetics , Cell Line , Disease Models, Animal , Humans , Hypolipoproteinemias/genetics , Immunity, Innate/immunology , Lipoproteins, LDL/immunology , Lung/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Signal Transduction/genetics , Trans-Activators/geneticsABSTRACT
Oxidation of L-arginine (L-Arg) to nitric oxide (NO) by NO synthase (NOS) takes place at the heme active site. It is of current interest to study structures of the heme species that activates O2 and transforms the substrate. The NOS ferrous-NO complex is a close mimic of the obligatory ferric (hydro)peroxo intermediate in NOS catalysis. In this work, pulsed electron-nuclear double resonance (ENDOR) spectroscopy was used to probe the hydrogen bonding of the NO ligand in the ferrous-NO heme center of neuronal NOS (nNOS) without a substrate and with L-Arg or N-hydroxy-L-arginine (NOHA) substrates. Unexpectedly, no H-bonding interaction connecting the NO ligand to the active site water molecule or the Arg substrate was detected, in contrast to the results obtained by X-ray crystallography for the Arg-bound nNOS heme domain [Li et al. J. Biol. Inorg. Chem. 2006, 11, 753-768]. The nearby exchangeable proton in both the no-substrate and Arg-containing nNOS samples is located outside the H-bonding range and, on the basis of the obtained structural constraints, can belong to the active site water (or OH). On the contrary, in the NOHA-bound sample, the nearby exchangeable hydrogen forms an H-bond with the NO ligand (on the basis of its distance from the NO ligand and a nonzero isotropic hfi constant), but it does not belong to the active site water molecule because the water oxygen atom (detected by (17)O ENDOR) is too far. This hydrogen should therefore come from the NOHA substrate, which is in agreement with the X-ray crystallography work [Li et al. Biochemistry 2009, 48, 10246-10254]. The nearby nonexchangeable hydrogen atom assigned as H(ε) of Phe584 was detected in all three samples. This hydrogen atom may have a stabilizing effect on the NO ligand and probably determines its position.
Subject(s)
Heme/chemistry , Nitric Oxide Synthase Type I/chemistry , Nitric Oxide/chemistry , Animals , Arginine/chemistry , Catalysis , Catalytic Domain , Electron Spin Resonance Spectroscopy , Hydrogen/chemistry , Hydrogen Bonding , Protons , Rats , Water/chemistryABSTRACT
Serum lipoproteins (LP) are increasingly being recognized as dual purpose molecules that contribute to both cholesterol homeostasis and host innate defense. In fact, very low LP levels are associated with increased risk of bacterial infection in critically ill patients. In this respect, we reported that apolipoprotein B100 (apoB100), the 4536 amino acid structural protein of very low density lipoprotein (VLDL) produced by the liver, limits Staphylococcus aureus pathogenesis. S. aureus uses quorum-sensing (QS) via the accessory gene regulator (agr) operon and an autoinducing peptide (AIP) to coordinate expression of over 200 virulence genes. ApoB100 prevents agr activation by binding and sequestering secreted AIP. Importantly, human serum LP are produced not only by the liver, but are also produced by enterocytes, in the form of chylomicrons, during uptake of dietary lipids. In contrast to apoB100 in VLDL, human enterocytes use apoB48, the N-terminal 2152 amino acids (48%) of apoB100, as the structural component of chylomicrons. Interestingly, enteral feeding of critically ill patients has been associated with decreased risk of infectious complications, suggesting chylomicrons could contribute to host innate defense in critically ill patients when serum LP production by the liver is limited during the acute phase response. Therefore, we hypothesized that apoB48 would be sufficient to antagonize S. aureus QS. As expected, isolated apoB48-LP bound immobilized AIP and antagonized agr-signaling. ApoB48- and apoB100-LP inhibited agr activation with IC50s of 3.5 and 2.3 nM, respectively, demonstrating a conserved AIP binding site. Importantly, apoB48-LP antagonized QS, limited morbidity and promoted bacterial clearance in a mouse model of S. aureus infection. This work demonstrates that both naturally occurring forms of apolipoprotein B can antagonize S. aureus QS, and may suggest a previously unrecognized role for chylomicrons and enterocytes in host innate defense against S. aureus QS-mediated pathogenesis.
Subject(s)
Apolipoprotein B-48/metabolism , Chylomicrons/metabolism , Quorum Sensing , Staphylococcus aureus/physiology , Animals , Apolipoprotein B-100/metabolism , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Humans , Mice , Mice, Knockout , Models, Biological , Peptides, Cyclic/metabolism , Promoter Regions, Genetic , Protein Binding , Signal Transduction , Staphylococcal Infections/metabolism , Staphylococcal Infections/microbiology , Staphylococcus aureus/pathogenicity , Trans-Activators/antagonists & inhibitors , Trans-Activators/genetics , Trans-Activators/metabolismABSTRACT
Antibiotic-resistant pathogens are a global health threat. Small molecules that inhibit bacterial virulence have been suggested as alternatives or adjuncts to conventional antibiotics, as they may limit pathogenesis and increase bacterial susceptibility to host killing. Staphylococcus aureus is a major cause of invasive skin and soft tissue infections (SSTIs) in both the hospital and community settings, and it is also becoming increasingly antibiotic resistant. Quorum sensing (QS) mediated by the accessory gene regulator (agr) controls virulence factor production essential for causing SSTIs. We recently identified ω-hydroxyemodin (OHM), a polyhydroxyanthraquinone isolated from solid-phase cultures of Penicillium restrictum, as a suppressor of QS and a compound sought for the further characterization of the mechanism of action. At concentrations that are nontoxic to eukaryotic cells and subinhibitory to bacterial growth, OHM prevented agr signaling by all four S. aureus agr alleles. OHM inhibited QS by direct binding to AgrA, the response regulator encoded by the agr operon, preventing the interaction of AgrA with the agr P2 promoter. Importantly, OHM was efficacious in a mouse model of S. aureus SSTI. Decreased dermonecrosis with OHM treatment was associated with enhanced bacterial clearance and reductions in inflammatory cytokine transcription and expression at the site of infection. Furthermore, OHM treatment enhanced the immune cell killing of S. aureus in vitro in an agr-dependent manner. These data suggest that bacterial disarmament through the suppression of S. aureus QS may bolster the host innate immune response and limit inflammation.
Subject(s)
Anti-Bacterial Agents/pharmacology , Emodin/analogs & derivatives , Inflammation/prevention & control , Methicillin-Resistant Staphylococcus aureus/drug effects , Quorum Sensing/drug effects , Staphylococcal Infections/drug therapy , Staphylococcal Infections/microbiology , Animals , Bacterial Proteins/genetics , Cytokines/biosynthesis , Emodin/pharmacology , Humans , In Vitro Techniques , Inflammation/etiology , Inflammation/pathology , Leukocytes/microbiology , Mice , Models, Molecular , Rabbits , Staphylococcal Infections/pathology , Trans-Activators/genetics , Virulence Factors/metabolismABSTRACT
Bacterial signaling systems are prime drug targets for combating the global health threat of antibiotic resistant bacterial infections including those caused by Staphylococcus aureus. S. aureus is the primary cause of acute bacterial skin and soft tissue infections (SSTIs) and the quorum sensing operon agr is causally associated with these. Whether efficacious chemical inhibitors of agr signaling can be developed that promote host defense against SSTIs while sparing the normal microbiota of the skin is unknown. In a high throughput screen, we identified a small molecule inhibitor (SMI), savirin (S. aureus virulence inhibitor) that disrupted agr-mediated quorum sensing in this pathogen but not in the important skin commensal Staphylococcus epidermidis. Mechanistic studies employing electrophoretic mobility shift assays and a novel AgrA activation reporter strain revealed the transcriptional regulator AgrA as the target of inhibition within the pathogen, preventing virulence gene upregulation. Consistent with its minimal impact on exponential phase growth, including skin microbiota members, savirin did not provoke stress responses or membrane dysfunction induced by conventional antibiotics as determined by transcriptional profiling and membrane potential and integrity studies. Importantly, savirin was efficacious in two murine skin infection models, abating tissue injury and selectively promoting clearance of agr+ but not Δagr bacteria when administered at the time of infection or delayed until maximal abscess development. The mechanism of enhanced host defense involved in part enhanced intracellular killing of agr+ but not Δagr in macrophages and by low pH. Notably, resistance or tolerance to savirin inhibition of agr was not observed after multiple passages either in vivo or in vitro where under the same conditions resistance to growth inhibition was induced after passage with conventional antibiotics. Therefore, chemical inhibitors can selectively target AgrA in S. aureus to promote host defense while sparing agr signaling in S. epidermidis and limiting resistance development.
Subject(s)
Anti-Bacterial Agents/therapeutic use , Bacterial Proteins/antagonists & inhibitors , Immunity, Innate/drug effects , Quinazolinones/therapeutic use , Quorum Sensing/drug effects , Staphylococcal Skin Infections/drug therapy , Staphylococcus aureus/drug effects , Trans-Activators/antagonists & inhibitors , Triazoles/therapeutic use , Animals , Anti-Bacterial Agents/adverse effects , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Line, Transformed , Drug Discovery , Genes, Reporter/drug effects , High-Throughput Screening Assays , Macrophages/drug effects , Macrophages/immunology , Macrophages/metabolism , Macrophages/microbiology , Male , Mice, Hairless , Mice, Knockout , Molecular Conformation , Molecular Docking Simulation , Molecular Targeted Therapy/adverse effects , Mutation , Phagocytosis/drug effects , Promoter Regions, Genetic/drug effects , Quinazolinones/adverse effects , Quinazolinones/chemistry , Quinazolinones/pharmacology , Skin/drug effects , Skin/microbiology , Staphylococcal Skin Infections/immunology , Staphylococcal Skin Infections/microbiology , Staphylococcus aureus/growth & development , Staphylococcus aureus/immunology , Staphylococcus aureus/physiology , Staphylococcus epidermidis/drug effects , Staphylococcus epidermidis/growth & development , Staphylococcus epidermidis/immunology , Staphylococcus epidermidis/physiology , Trans-Activators/chemistry , Trans-Activators/genetics , Trans-Activators/metabolism , Triazoles/adverse effects , Triazoles/chemistry , Triazoles/pharmacologyABSTRACT
Nitric oxide synthase (NOS), a flavo-hemoprotein, is responsible for biosynthesis of nitric oxide (NO) in mammals. Three NOS isoforms, iNOS, eNOS and nNOS (inducible, endothelial, and neuronal NOS), achieve their biological functions by tight control of interdomain electron transfer (IET) process through interdomain interactions. In particular, the FMN-heme IET is essential in coupling electron transfer in the reductase domain with NO synthesis in the heme domain by delivery of electrons required for O2 activation at the catalytic heme site. Emerging evidence indicates that calmodulin (CaM) activates NO synthesis in eNOS and nNOS by a conformational change of the FMN domain from its shielded electron-accepting (input) state to a new electron-donating (output) state, and that CaM is also required for proper alignment of the FMN and heme domains in the three NOS isoforms. In the absence of a structure of full-length NOS, an integrated approach of spectroscopic, rapid kinetic and mutagenesis methods is required to unravel regulation mechanism of the FMN-heme IET process. This is to investigate the roles of the FMN domain motions and the docking between the primary functional FMN and heme domains in regulating NOS activity. The recent developments in this area that are driven by the combined approach are the focuses of this review. A better understanding of the roles of interdomain FMN/heme interactions and CaM binding may serve as a basis for the rational design of new selective modulators of the NOS enzymes.
Subject(s)
Flavin Mononucleotide/metabolism , Heme/chemistry , Nitric Oxide Synthase/chemistry , Nitric Oxide Synthase/metabolism , Calmodulin/chemistry , Calmodulin/metabolism , Electron Transport , Flavin Mononucleotide/chemistry , Heme/metabolism , Kinetics , Nitric Oxide Synthase Type II/chemistry , Nitric Oxide Synthase Type II/metabolism , Nitric Oxide Synthase Type III/chemistry , Nitric Oxide Synthase Type III/metabolismABSTRACT
Nitric oxide (NO) production by mammalian NO synthase (NOS) is believed to be regulated by the docking of the flavin mononucleotide (FMN) domain in one subunit of the dimer onto the heme domain of the adjacent subunit. Glu546, a conserved charged surface residue of the FMN domain in human inducible NOS (iNOS), is proposed to participate in the interdomain FMN/heme interactions [Sempombe et al. Inorg. Chem.2011, 50, 6869-6861]. In the present work, we further investigated the role of the E546 residue in the FMN-heme interdomain electron transfer (IET), a catalytically essential step in the NOS enzymes. Laser flash photolysis was employed to directly measure the FMN-heme IET kinetics for the E546N mutant of human iNOS oxygenase/FMN (oxyFMN) construct. The temperature dependence of the IET kinetics was also measured over the temperature range of 283-304 K to determine changes in the IET activation parameters. The E546N mutation was found to retard the IET by significantly raising the activation entropic barrier. Moreover, pulsed electron paramagnetic resonance data showed that the geometry of the docked FMN/heme complex in the mutant is basically the same as in the wild type construct, whereas the probability of formation of such a complex is about twice lower. These results indicate that the retarded IET in the E546N mutant is not caused by an altered conformation of the docked FMN/heme complex, but by a lower population of the IET-active conformation. In addition, the negative activation entropy of the mutant is still substantially lower than that of the holoenzyme. This supports a mechanism by which the FMN domain can modify the IET through altering probability of the docked state formation.
Subject(s)
Flavin Mononucleotide/metabolism , Heme/metabolism , Nitric Oxide Synthase Type II/metabolism , Electron Spin Resonance Spectroscopy , Electron Transport , Humans , Kinetics , Molecular Docking Simulation , Nitric Oxide Synthase Type II/chemistry , Nitric Oxide Synthase Type II/genetics , Oxidation-Reduction , Point Mutation , Protein Conformation , Protein Structure, Tertiary , Spectrometry, Fluorescence , Spectrum Analysis, RamanABSTRACT
Staphylococcus aureus contains an autoinducing quorum-sensing system encoded within the agr operon that coordinates expression of virulence genes required for invasive infection. Allelic variation within agr has generated four agr specific groups, agr I-IV, each of which secretes a distinct autoinducing peptide pheromone (AIP1-4) that drives agr signaling. Because agr signaling mediates a phenotypic change in this pathogen from an adherent colonizing phenotype to one associated with considerable tissue injury and invasiveness, we postulated that a significant contribution to host defense against tissue damaging and invasive infections could be provided by innate immune mechanisms that antagonize agr signaling. We determined whether two host defense factors that inhibit AIP1-induced agrI signaling, Nox2 and apolipoprotein B (apoB), also contribute to innate control of AIP3-induced agrIII signaling. We hypothesized that apoB and Nox2 would function differently against AIP3, which differs from AIP1 in amino acid sequence and length. Here we show that unlike AIP1, AIP3 is resistant to direct oxidant inactivation by Nox2 characteristic ROS. Rather, the contribution of Nox2 to defense against agrIII signaling is through oxidation of LDL. ApoB in the context of oxLDL, and not LDL, provides optimal host defense against S. aureus agrIII infection by binding the secreted signaling peptide, AIP3, and preventing expression of the agr-driven virulence factors which mediate invasive infection. ApoB within the context of oxLDL also binds AIP 1-4 and oxLDL antagonizes agr signaling by all four agr alleles. Our results suggest that Nox2-mediated oxidation of LDL facilitates a conformational change in apoB to one sufficient for binding and sequestration of all four AIPs, demonstrating the interdependence of apoB and Nox2 in host defense against agr signaling. These data reveal a novel role for oxLDL in host defense against S. aureus quorum-sensing signaling.
Subject(s)
Apolipoproteins B/metabolism , Bacterial Proteins/metabolism , Membrane Glycoproteins/physiology , NADPH Oxidases/physiology , Quorum Sensing/physiology , Receptors, LDL/metabolism , Staphylococcal Infections/microbiology , Staphylococcus aureus/physiology , Trans-Activators/metabolism , Animals , Blotting, Western , Disease Models, Animal , Female , Gene Expression Regulation, Bacterial , Immunity, Innate , Immunoassay , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , NADPH Oxidase 2 , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Staphylococcal Infections/metabolism , Staphylococcal Infections/pathology , Surface Plasmon ResonanceABSTRACT
Mammalian nitric oxide synthases (NOSs) are enzymes responsible for oxidation of L-arginine (L-Arg) to nitric oxide (NO). Mechanisms of reactions at the catalytic heme site are not well understood, and it is of current interest to study structures of the heme species that activates O(2) and transforms the substrate. The NOS ferrous-NO complex is a close mimic of the obligatory ferric (hydro)peroxo intermediate in NOS catalysis. In this work, pulsed electron-nuclear double resonance (ENDOR) was used to probe the position of the l-Arg substrate at the NO(â¢)-coordinated ferrous heme center(s) in the oxygenase domain of rat neuronal NOS (nNOS). The analysis of (2)H and (15)N ENDOR spectra of samples containing d(7)- or guanidino-(15)N(2) labeled L-Arg has resulted in distance estimates for the nearby guanidino nitrogen and the nearby proton (deuteron) at C(δ). The L-Arg position was found to be noticeably different from that in the X-ray crystal structure of nNOS ferrous-NO complex [Li et al. J. Biol. Inorg. Chem.2006, 11, 753-768], with the nearby guanidino nitrogen being ~0.5 Å closer to, and the nearby H(δ) about 1 Å further from, the NO ligand than in the X-ray structure. The difference might be related to the structural constraints imposed on the protein by the crystal. Importantly, in spite of its closer position, the guanidino nitrogen does not form a hydrogen bond with the NO ligand, as evidenced by the absence of significant isotropic hfi constant for N(g1). This is consistent with the previous reports that it is not the L-Arg substrate itself that would most likely serve as a direct proton donor to the diatomic ligands (NO and O(2)) bound to the heme.
Subject(s)
Arginine/analysis , Ferrous Compounds/metabolism , Nitric Oxide Synthase Type I/metabolism , Nitric Oxide/metabolism , Animals , Arginine/metabolism , Biocatalysis , Electron Spin Resonance Spectroscopy , Ferrous Compounds/chemistry , Models, Molecular , Nitric Oxide/chemistry , Nitric Oxide Synthase Type I/chemistry , Oxidation-Reduction , RatsABSTRACT
In the crystal structure of a calmodulin (CaM)-bound FMN domain of human inducible nitric oxide synthase (NOS), the CaM-binding region together with CaM forms a hinge, and pivots on an R536(NOS)/E47(CaM) pair (Xia et al. J Biol Chem 284:30708-30717, 2009). Notably, isoform-specific human inducible NOS S562 and C563 residues form hydrogen bonds with the R536 residue through their backbone oxygens. In this study, we investigated the roles of the S562 and C563 residues in the NOS FMN-heme interdomain electron transfer (IET), the rates of which can be used to probe the interdomain FMN/heme alignment. Human inducible NOS S562K and C563R mutants of an oxygenase/FMN (oxyFMN) construct were made by introducing charged residues at these sites as found in human neuronal NOS and endothelial NOS isoforms, respectively. The IET rate constant of the S562K mutant is notably decreased by one third, and its flavin fluorescence intensity per micromole per liter is diminished by approximately 24 %. These results suggest that a positive charge at position 562 destabilizes the hydrogen-bond-mediated NOS/CaM alignment, resulting in slower FMN-heme IET in the mutant. On the other hand, the IET rate constant of the C563R mutant is similar to that of the wild-type, indicating that the mutational effect is site-specific. Moreover, the human inducible NOS oxyFMN R536E mutant was constructed to disrupt the bridging CaM/NOS interaction, and its FMN-heme IET rate was decreased by 96 %. These results demonstrated a new role of the isoform-specific serine residue of the key CaM/FMN(NOS) bridging site in regulating the FMN-heme IET (possibly by tuning the alignment of the FMN and heme domains).
Subject(s)
Heme/metabolism , Nitric Oxide Synthase Type II/chemistry , Nitric Oxide Synthase Type II/metabolism , Protein Isoforms/metabolism , Amino Acid Sequence , Calmodulin/chemistry , Calmodulin/metabolism , Electron Transport , Flavin Mononucleotide/chemistry , Flavin Mononucleotide/metabolism , Heme/chemistry , Humans , Hydrogen Bonding , Models, Molecular , Molecular Sequence Data , Mutation , Nitric Oxide Synthase Type II/genetics , Protein Isoforms/chemistry , Sequence Alignment , Spectrometry, FluorescenceABSTRACT
Mammalian nitric oxide synthase (NOS) is a flavo-hemoprotein that catalyzes the oxidation of L-arginine to nitric oxide. Information about the relative alignment of the heme and FMN domains of NOS is important for understanding the electron transfer between the heme and FMN centers, but no crystal structure data for NOS holoenzyme are available. In our previous work [Astashkin, A. V.; Elmore, B. O.; Fan, W.; Guillemette, J. G.; Feng, C. J. Am. Chem. Soc. 2010, 132, 12059-12067], the distance between the imidazole-coordinated low-spin Fe(III) heme and FMN semiquinone in a human inducible NOS (iNOS) oxygenase/FMN construct has been determined by pulsed electron paramagnetic resonance (EPR). The orientation of the Fe-FMN radius vector, R(Fe-FMN), with respect to the heme g-frame was also determined. In the present study, pulsed electron-nuclear double resonance (ENDOR) investigation of the deuterons at carbons C2 and C5 in the deuterated coordinated imidazole was used to determine the relative orientation of the heme g-frame and molecular frame, from which R(Fe-FMN) can be referenced to the heme molecular frame. Numerical simulations of the ENDOR spectra showed that the g-factor axis corresponding to the low-field EPR turning point is perpendicular to the heme plane, whereas the axis corresponding to the high-field turning point is in the heme plane and makes an angle of about 80° with the coordinated imidazole plane. The FMN-heme domain docking model obtained in the previous work was found to be in qualitative agreement with the combined experimental results of the two pulsed EPR works.
Subject(s)
Heme/chemistry , Imidazoles/chemistry , Nitric Oxide Synthase Type II/chemistry , Electron Spin Resonance Spectroscopy , Humans , Models, MolecularABSTRACT
The FMN-heme intraprotein electron transfer (IET) kinetics in a human inducible NOS (iNOS) oxygenase/FMN construct were determined by laser flash photolysis as a function of solution viscosity (1.0-3.0 cP). In the presence of ethylene glycol or sucrose, an appreciable decrease in the IET rate constant value was observed with an increase in the solution viscosity. The IET rate constant is inversely proportional to the viscosity for both viscosogens. This demonstrates that viscosity, and not other properties of the added viscosogens, causes the dependence of IET rates on the solvent concentration. The IET kinetics results indicate that the FMN-heme IET in iNOS is gated by a large conformational change of the FMN domain. The kinetics and NOS flavin fluorescence results together indicate that the docked FMN/heme state is populated transiently.
Subject(s)
Flavin Mononucleotide/metabolism , Heme/metabolism , Nitric Oxide Synthase Type II/chemistry , Nitric Oxide Synthase Type II/metabolism , Buffers , Electron Transport , Ethylene Glycol/chemistry , Humans , Kinetics , Protein Conformation , Protein Structure, Tertiary , Solutions , Spectrometry, Fluorescence , Sucrose/chemistry , ViscosityABSTRACT
We have obtained low-temperature magnetic circular dichroism (MCD) spectra for ferric cyano complexes of the wild type and E546N mutant of a human inducible nitric oxide synthase (iNOS) oxygenase/flavin mononucleotide (oxyFMN) construct. The mutation at the FMN domain has previously been shown to modulate the MCD spectra of the l-arginine-bound ferric iNOS heme (Sempombe, J.; et al. J. Am. Chem. Soc. 2009, 131, 6940-6941). The addition of l-arginine to the wild-type protein causes notable changes in the CN(-)-adduct MCD spectrum, while the E546N mutant spectrum is not perturbed. Moreover, the MCD spectral perturbation observed with l-arginine is absent in the CN(-) complexes incubated with N-hydroxy-L-arginine, which is the substrate for the second step of NOS catalysis. These results indicate that interdomain FMN-heme interactions exert a long-range effect on key heme axial ligand-substrate interactions that determine substrate oxidation pathways of NOS.
Subject(s)
Circular Dichroism , Ferric Compounds/metabolism , Flavin Mononucleotide , Magnetics , Mutant Proteins/chemistry , Mutation , Nitric Oxide Synthase Type II/chemistry , Humans , Models, Molecular , Mutant Proteins/genetics , Mutant Proteins/metabolism , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Protein Structure, Tertiary , Substrate SpecificityABSTRACT
Mammalian nitric oxide synthase (NOS) is a homodimeric flavo-hemoprotein that catalyzes the oxidation of L-arginine to nitric oxide (NO). Regulation of NO biosynthesis by NOS is primarily through control of interdomain electron transfer (IET) processes in NOS catalysis. The IET from the flavin mononucleotide (FMN) to heme domains is essential in the delivery of electrons required for O(2) activation in the heme domain and the subsequent NO synthesis by NOS. The NOS output state for NO production is an IET-competent complex of the FMN-binding domain and heme domain, and thereby it facilitates the IET from the FMN to the catalytic heme site. The structure of the functional output state has not yet been determined. In the absence of crystal structure data for NOS holoenzyme, it is important to experimentally determine the Fe...FMN distance to provide a key calibration for computational docking studies and for the IET kinetics studies. Here we used the relaxation-induced dipolar modulation enhancement (RIDME) technique to measure the electron spin echo envelope modulation caused by the dipole interactions between paramagnetic FMN and heme iron centers in the [Fe(III)][FMNH(*)] (FMNH(*): FMN semiquinone) form of a human inducible NOS (iNOS) bidomain oxygenase/FMN construct. The FMNH(*)...Fe distance has been directly determined from the RIDME spectrum. This distance (18.8 +/- 0.1 A) is in excellent agreement with the IET rate constant measured by laser flash photolysis [Feng, C. J.; Dupont, A.; Nahm, N.; Spratt, D.; Hazzard, J. T.; Weinberg, J.; Guillemette, J.; Tollin, G.; Ghosh, D. K. J. Biol. Inorg. Chem. 2009, 14, 133-142].