Purification and characterization of the Staphylococcus aureus bacillithiol transferase BstA

Background

Gram-positive bacteria in the phylum Firmicutes synthesize the low molecular weight thiol bacillithiol rather than glutathione or mycothiol. The bacillithiol transferase YfiT from Bacillus subtilis was identified as a new member of the recently discovered DinB/YfiT-like Superfamily. Based on structural similarity using the Superfamily program, we have determined 30 of 31 Staphylococcus aureus strains encode a single bacillithiol transferase from the DinB/YfiT-like Superfamily, while the remaining strain encodes two proteins.

Methods

We have cloned, purified, and confirmed the activity of a recombinant bacillithiol transferase (henceforth called BstA) encoded by the S. aureus Newman ORF NWMN_2591. Moreover, we have studied the saturation kinetics and substrate specificity of this enzyme using in vitro biochemical assays.

Results

BstA was found to be active with the co-substrate bacillithiol, but not with other low molecular weight thiols tested. BstA catalyzed bacillithiol conjugation to the model substrates monochlorobimane, 1-chloro-2,4-dinitrobenzene, and the antibiotic cerulenin. Several other molecules, including the antibiotic rifamycin S, were found to react directly with bacillithiol, but the addition of BstA did not enhance the rate of reaction. Furthermore, cells growing in nutrient rich medium exhibited low BstA activity.

Conclusions

BstA is a bacillithiol transferase from S. aureus that catalyzes the detoxification of cerulenin. Additionally, we have determined that bacillithiol itself might be capable of directly detoxifying electrophilic molecules.

General significance

BstA is an active bacillithiol transferase from S. aureus Newman and is the first DinB/YfiT-like Superfamily member identified from this organism. Interestingly, BstA is highly divergent from B. subtilis YfiT.     

The C‐type lectin receptor MGL senses N‐acetylgalactosamine on the unique Staphylococcus aureus ST395 wall teichoic acid

Staphylococcus aureus is a common skin commensal but is also associated with various skin and soft tissue pathologies. Upon invasion, S. aureus is detected by resident innate immune cells through pattern‐recognition receptors (PRRs), although a comprehensive understanding of the specific molecular interactions is lacking. Recently, we demonstrated that the PRR langerin (CD207) on epidermal Langerhans cells senses the conserved β‐1,4‐linked N‐acetylglucosamine (GlcNAc) modification on S. aureus wall teichoic acid (WTA), thereby increasing skin inflammation. Interestingly, the S. aureus ST395 lineage as well as certain species of coagulase‐negative staphylococci (CoNS) produce a structurally different WTA molecule, consisting of poly‐glycerolphosphate with α‐O‐N‐acetylgalactosamine (GalNAc) residues, which are attached by the glycosyltransferase TagN. Here, we demonstrate that S. aureus ST395 strains interact with the human Macrophage galactose‐type lectin (MGL; CD301) receptor, which is expressed by dendritic cells and macrophages in the dermis. MGL bound S. aureus ST395 in a tagN‐ and GalNAc‐dependent manner but did not interact with different tagN‐positive CoNS species. However, heterologous expression of Staphylococcus lugdunensis tagN in S. aureus conferred phage infection and MGL binding, confirming the role of this CoNS enzyme as GalNAc‐transferase. Functionally, the detection of GalNAc on S. aureus ST395 WTA by human monocyte‐derived dendritic cells significantly enhanced cytokine production. Together, our findings highlight differential recognition of S. aureus glycoprofiles by specific human innate receptors, which may affect downstream adaptive immune responses and pathogen clearance.     

Involvement of caspase‐1 in inflammasomes activation and bacterial clearance in S. aureus‐infected osteoblast‐like MG‐63 cells

Staphylococcus aureus, a versatile Gram‐positive bacterium, is the main cause of bone and joint infections (BJI), which are prone to recurrence. The inflammasome is an immune signaling platform that assembles after pathogen recognition. It activates proteases, most notably caspase‐1 that proteolytically matures and promotes the secretion of mature IL‐1β and IL‐18. The role of inflammasomes and caspase‐1 in the secretion of mature IL‐1β and in the defence of S. aureus‐infected osteoblasts has not yet been fully investigated. We show here that S. aureus‐infected osteoblast‐like MG‐63 but not caspase‐1 knock‐out CASP1 ^?/?MG‐63 cells, which were generated using CRISPR‐Cas9 technology, activate the inflammasome as monitored by the release of mature IL‐1β. The effect was strain‐dependent. The use of S. aureus deletion and complemented phenole soluble modulins (PSMs) mutants demonstrated a key role of PSMs in inflammasomes‐related IL‐1β production. Furthermore, we found that the lack of caspase‐1 in CASP1 ^?/?MG‐63 cells impairs their defense functions, as bacterial clearance was drastically decreased in CASP1 ^?/? MG‐63 compared to wild‐type cells. Our results demonstrate that osteoblast‐like MG‐63 cells play an important role in the immune response against S. aureus infection through inflammasomes activation and establish a crucial role of caspase‐1 in bacterial clearance.     

A unique Ni2+ ‐dependent and methylglyoxal‐inducible rice glyoxalase I possesses a single active site and functions in abiotic stress response

The glyoxalase system constitutes the major pathway for the detoxification of metabolically produced cytotoxin methylglyoxal (MG) into a non‐toxic metabolite d ‐lactate. Glyoxalase I (GLY I) is an evolutionarily conserved metalloenzyme requiring divalent metal ions for its activity: Zn²⁺ in the case of eukaryotes or Ni²⁺ for enzymes of prokaryotic origin. Plant GLY I proteins are part of a multimember family; however, not much is known about their physiological function, structure and metal dependency. In this study, we report a unique GLY I (OsGLYI‐11.2) from Oryza sativa (rice) that requires Ni²⁺ for its activity. Its biochemical, structural and functional characterization revealed it to be a monomeric enzyme, possessing a single Ni²⁺ coordination site despite containing two GLY I domains. The requirement of Ni²⁺ as a cofactor by an enzyme involved in cellular detoxification suggests an essential role for this otherwise toxic heavy metal in the stress response. Intriguingly, the expression of OsGLYI‐11.2 was found to be highly substrate inducible, suggesting an important mode of regulation for its cellular levels. Heterologous expression of OsGLYI‐11.2 in Escherichia coli and model plant Nicotiana tabacum (tobacco) resulted in improved adaptation to various abiotic stresses caused by increased scavenging of MG, lower Na⁺/K⁺ ratio and maintenance of reduced glutathione levels. Together, our results suggest interesting links between MG cellular levels, its detoxification by GLY I, and Ni²⁺ – the heavy metal cofactor of OsGLYI‐11.2, in relation to stress response and adaptation in plants.     

Drosophila proteins interacting with metallothioneins: A metal‐dependent recognition

Metallothioneins (MTs) are ubiquitous, low‐molecular weight, cysteine‐rich proteins. Despite a well‐established protective role in metal excess detoxification, there is little data about their putative physiological functions, commonly assumed to be metal homeostasis and redox equilibrium. Protein–protein interactions should have provided useful information to unveil unsuspected functions, but reports on MT interactions are scarce. This is probably due to the MT metal‐dependent 3D structure, a fact that has been seldom taken into account when performing proteomic interaction assays. In the present work, we have detected that the two major D. melanogaster isoforms (MtnA and MtnB) interact with the peroxiredoxin (Prx) encoded by the gene Jafrac1, both in a clear metal‐dependent pattern. The MT–Prx interaction is further confirmed in Saccharomyces cerevisiae by assaying both yeast MTs (Crs5p and Cup1p) versus Tsa1p and Tsa2p, the Jafrac1 homologous Prxs in this organism. Thus, a new methodological approach to detect MT‐interacting proteins in different proteomes is established on the basis of assaying MTs in the form of different metal complexes. Furthermore, new perspectives to investigate the often hypothesized contribution of MTs to the redox physiological networks are open.     

Antibacterial and membrane‐damaging activities of β‐bungarotoxin B chain

This study investigates whether the B chain of β‐bungarotoxin exerted antibacterial activity against Escherichia coli (Gram‐negative bacteria) and Staphylococcus aureus (Gram‐positive bacteria) via its membrane‐damaging activity. The B chain exhibited a growth inhibition effect on E. coli but did not show a bactericidal effect on S. aureus. The B‐chain bactericidal action on E. coli positively correlated with an increase in membrane permeability in the bacterial cells. Lipopolysaccharide (LPS) layer destabilization and lipoteichoic acid (LTA) biosynthesis inhibition in the cell wall increased the B‐chain bactericidal effect on E. coli and S. aureus. The B chain induced leakage and fusion in E. coli and S. aureus membrane‐mimicking liposomes. Compared with LPS, LTA notably suppressed the membrane‐damaging activity and fusogenicity of the B chain. The B chain showed similar binding affinity with LPS and LTA, whereas LPS and LTA binding differently induced B‐chain conformational change as evidenced by the circular dichroism spectra. Taken together, our data indicate that the antibacterial action of the B chain is related to its ability to induce membrane permeability and suggest that the LPS‐induced and LTA‐induced B‐chain conformational change differently affects the bactericidal action of the B chain. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

X‐ray crystallographic structure of BshB,the zinc‐dependent deacetylase involved in bacillithiol biosynthesis

Many gram‐positive bacteria produce bacillithiol to aid in the maintenance of redox homeostasis and degradation of toxic compounds, including the antibiotic fosfomycin. Bacillithiol is produced via a three‐enzyme pathway that includes the action of the zinc‐dependent deacetylase BshB. Previous studies identified conserved aspartate and histidine residues within the active site that are involved in metal binding and catalysis, but the enzymatic mechanism is not fully understood. Here we report two X‐ray crystallographic structures of BshB from Bacillus subtilis that provide insight into the BshB catalytic mechanism.     

Staphylococcus aureus lactate‐ and malate‐quinone oxidoreductases contribute to nitric oxide resistance and virulence

Staphylococcus aureus is a Gram‐positive pathogen that resists many facets of innate immunity including nitric oxide (NO·). Staphylococcus aureus NO‐resistance stems from its ability to evoke a metabolic state that circumvents the negative effects of reactive nitrogen species. The combination of l ‐lactate and peptides promotes S. aureus growth at moderate NO‐levels, however, neither nutrient alone suffices. Here, we investigate the staphylococcal malate‐quinone and l ‐lactate‐quinone oxidoreductases (Mqo and Lqo), both of which are critical during NO‐stress for the combined utilization of peptides and l ‐lactate. We address the specific contributions of Lqo‐mediated l ‐lactate utilization and Mqo‐dependent amino acid consumption during NO‐stress. We show that Lqo conversion of l ‐lactate to pyruvate is required for the formation of ATP, an essential energy source for peptide utilization. Thus, both Lqo and Mqo are essential for growth under these conditions making them attractive candidates for targeted therapeutics. Accordingly, we exploited a modelled Mqo/Lqo structure to define the catalytic and substrate‐binding residues.We also compare the S. aureus Mqo/Lqo enzymes to their close relatives throughout the staphylococci and explore the substrate specificities of each enzyme. This study provides the initial characterization of the mechanism of action and the immunometabolic roles for a newly defined staphylococcal enzyme family.     

Regulation of basal and oxidative stress‐triggered jasmonic acid‐related gene expression by glutathione

Glutathione is a determinant of cellular redox state with roles in defence and detoxification. Emerging concepts suggest that this compound also has functions in cellular signalling. Here, we report evidence that glutathione plays potentially important roles in setting signalling strength through the jasmonic acid (JA) pathway. Firstly, we show that basal expression of JA‐related genes is correlated with leaf glutathione content when the latter is manipulated either genetically or pharmacologically. Secondly, analyses of an oxidative stress signalling mutant, cat2, reveal that up‐regulation of the JA pathway triggered by intracellular oxidation requires accompanying glutathione accumulation. Genetically blocking this accumulation in a cat2 cad2 line largely annuls H₂O₂‐induced expression of JA‐linked genes, and this effect can be rescued by exogenously supplying glutathione. While most attention on glutathione functions in biotic stress responses has been focused on the thiol‐regulated protein NPR1, a comparison of JA‐linked gene expression in cat2 cad2 and cat2 npr1 double mutants provides evidence that glutathione acts through other components to regulate the response of this pathway to oxidative stress. Our study provides new information implicating glutathione as a factor determining basal JA gene expression and suggests novel glutathione‐dependent control points that regulate JA signalling in response to intracellular oxidation.     

Quaternary variations in the structural assembly of N‐acetylglucosamine‐6‐phosphate deacetylase from Pasteurella multocida

N‐acetylglucosamine 6‐phosphate deacetylase (NagA) catalyzes the conversion of N‐acetylglucosamine‐6‐phosphate to glucosamine‐6‐phosphate in amino sugar catabolism. This conversion is an essential step in the catabolism of sialic acid in several pathogenic bacteria, including Pasteurella multocida, and thus NagA is identified as a potential drug target. Here, we report the unique structural features of NagA from P. multocida (PmNagA) resolved to 1.95 Å. PmNagA displays an altered quaternary architecture with unique interface interactions compared to its close homolog, the Escherichia coli NagA (EcNagA). We confirmed that the altered quaternary structure is not a crystallographic artifact using single particle electron cryo‐microscopy. Analysis of the determined crystal structure reveals a set of hot‐spot residues involved in novel interactions at the dimer‐dimer interface. PmNagA binds to one Zn²⁺ ion in the active site and demonstrates kinetic parameters comparable to other bacterial homologs. Kinetic studies reveal that at high substrate concentrations (~10‐fold the K_M), the tetrameric PmNagA displays hysteresis similar to its distant neighbor, the dimeric Staphylococcus aureus NagA (SaNagA). Our findings provide key information on structural and functional properties of NagA in P. multocida that could be utilized to design novel antibacterials.     

Methylglyoxal resistance in Bacillus subtilis: contributions of bacillithiol‐dependent and independent pathways

Methylglyoxal (MG) is a toxic by‐product of glycolysis that damages DNA and proteins ultimately leading to cell death. Protection from MG is often conferred by a glutathione‐dependent glyoxalase pathway. However, glutathione is absent from the low‐GC Gram‐positive Firmicutes, such as Bacillus subtilis. The identification of bacillithiol (BSH) as the major low‐molecular‐weight thiol in the Firmicutes raises the possibility that BSH is involved in MG detoxification. Here, we demonstrate that MG can rapidly and specifically deplete BSH in cells, and we identify both BSH‐dependent and BSH‐independent MG resistance pathways. The BSH‐dependent pathway utilizes glyoxalase I (GlxA, formerly YwbC) and glyoxalase II (GlxB, formerly YurT) to convert MG to d ‐lactate. The critical step in this pathway is the activation of the KhtSTU K⁺ efflux pump by the S‐lactoyl‐BSH intermediate, which leads to cytoplasmic acidification. We show that cytoplasmic acidification is both necessary and sufficient for maximal protection from MG. Two additional MG detoxification pathways operate independent of BSH. The first involves three enzymes (YdeA, YraA and YfkM) which are predicted to be homologues of glyoxalase III that converts MG to d ‐lactate, and the second involves YhdN, previously shown to be a broad specificity aldo‐keto reductase that converts MG to acetol.     

Rice Exo70 interacts with a fungal effector,AVR‐Pii,and is required for AVR‐Pii‐triggered immunity

Vesicle trafficking including the exocytosis pathway is intimately associated with host immunity against pathogens. However, we still have insufficient knowledge about how it contributes to immunity, and how pathogen factors affect it. In this study, we explore host factors that interact with the Magnaporthe oryzae effector AVR‐Pii. Gel filtration chromatography and co‐immunoprecipitation assays identified a 150 kDa complex of proteins in the soluble fraction comprising AVR‐Pii and OsExo70‐F2 and OsExo70‐F3, two rice Exo70 proteins presumably involved in exocytosis. Simultaneous knockdown of OsExo70‐F2 and F3 totally abrogated Pii immune receptor‐dependent resistance, but had no effect on Pia‐ and Pik‐dependent resistance. Knockdown levels of OsExo70‐F3 but not OsExo70‐F2 correlated with reduction of Pii function, suggesting that OsExo70‐F3 is specifically involved in Pii‐dependent resistance. Under our current experimental conditions, over‐expression of AVR‐Pii or knockdown of OsExo70‐F2 and ‐F3 genes in rice did not affect the virulence of compatible isolates of M. oryzae. AVR‐Pii interaction with OsExo70‐F3 appears to play a crucial role in immunity triggered by Pii, suggesting a role for OsExo70 as a decoy or helper in Pii/AVR‐Pii interactions.     

Influence of Sae‐regulated and Agr‐regulated factors on the escape of Staphylococcus aureus from human macrophages

Although Staphylococcus aureus is not a classical intracellular pathogen, it can survive within phagocytes and many other cell types. However, the pathogen is also able to escape from cells by mechanisms that are only partially understood. We analysed a series of isogenic S. aureus mutants of the USA300 derivative JE2 for their capacity to destroy human macrophages from within. Intracellular S. aureus JE2 caused severe cell damage in human macrophages and could efficiently escape from within the cells. To obtain this full escape phenotype including an intermittent residency in the cytoplasm, the combined action of the regulatory systems Sae and Agr is required. Mutants in Sae or mutants deficient in the Sae target genes lukAB and pvl remained in high numbers within the macrophages causing reduced cell damage. Mutants in the regulatory system Agr or in the Agr target gene psmα were largely similar to wild‐type bacteria concerning cell damage and escape efficiency. However, these strains were rarely detectable in the cytoplasm, emphasizing the role of phenol‐soluble modulins (PSMs) for phagosomal escape. Thus, Sae‐regulated toxins largely determine damage and escape from within macrophages, whereas PSMs are mainly responsible for the escape from the phagosome into the cytoplasm. Damage of macrophages induced by intracellular bacteria was linked neither to activation of apoptosis‐related caspase 3, 7 or 8 nor to NLRP3‐dependent inflammasome activation.     

Tyrosinase‐catalyzed oxidation of resveratrol produces a highly reactive ortho‐quinone: Implications for melanocyte toxicity

trans‐Resveratrol (3,5,4′‐trihydroxy‐trans‐stilbene, RES), a naturally occurring polyphenol, has recently attracted increased interest as a health‐beneficial agent. However, based on its p‐substituted phenol structure, RES is expected to be a substrate for tyrosinase and to produce a toxic o‐quinone metabolite. The results of this study demonstrate that the oxidation of RES by tyrosinase produces 4‐(3′,5′‐dihydroxy‐trans‐styrenyl)‐1,2‐benzoquinone (RES‐quinone), which decays rapidly to an oligomeric product (RES‐oligomer). RES‐quinone was identified after reduction to its corresponding catechol, known as piceatannol. RES‐quinone reacts with N‐acetylcysteine, a small thiol, to form a diadduct and a triadduct, which were identified by NMR and MS analyses. The production of a triadduct is not common for o‐quinones, suggesting a high reactivity of RES‐quinone. RES‐quinone also binds to bovine serum albumin through its cysteine residue. RES‐oligomer can oxidize GSH to GSSG, indicating its pro‐oxidant activity. These results suggest that RES could be cytotoxic to melanocytes due to the binding of RES‐quinone to thiol proteins.     

Reduced‐oxidized difference spectral analysis and chemiluminescence‐based Scatchard analysis demonstrate selective binding of myeloperoxidase to microbes

Myeloperoxidase (MPO), a microbicidal haloperoxidase of neutrophil leukocytes, was observed to selectively bind to bacteria. Binding was quantified by dithionite‐reduced minus oxidized (R? O) difference spectral analysis. Escherichia coli and Pseudomonas aeruginosa showed large MPO binding by R? O difference spectral analysis, whereas Streptococcus sanguinis did not. For increased sensitivity, free and microbe‐bound MPO and chloroperoxidase (CPO) activities were quantified by acid‐optimum haloperoxidase‐dependent chemiluminescence (CL) measurements, and these data were used for Scatchard analysis. The MPO bound/free (B/F) CL ratio was 49.5 for P. aeruginosa, 14.6 for Staphylococcus aureus, 2.8 for E. coli, 0.7 for Candida albicans and 0.4 for S. sanguinis. By comparison, the CPO B/F CL ratio was 0.03 for P. aeruginosa, 0.09 for S. aureus, 0.31 for E. coli, 0.18 for C. albicans and 0.16 for S. sanguinis. As a member of the lactic acid family of bacteria and a viridans streptococcus, S. sanguinis does not synthesize cytochromes and is catalase‐negative. The metabolic products of S. sanguinis, i.e. lactic acid and hydrogen peroxide, provide optimal acidity and substrate for MPO oxidation of chloride to hypochlorite. Hypochlorite can react with organic substrates to yield dehydrogenated or chlorinated products, but when peroxide is not limiting, hypochlorite reacts with peroxide yielding singlet oxygen. The reactivity of hypochlorite is dependent on substrate availability. The microsecond half‐life of electronically excited singlet oxygen restricts reactivity to within a radius of <0.25 µm; i.e. the reactivity of singlet oxygen is both substrate and half‐life dependent. Poor MPO binding provides protection and possibly competitive advantage to viridans streptococci. Copyright © 2010 John Wiley & Sons, Ltd.     

Expression of Caenorhabditis elegans PCS in the AtPCS1‐deficient Arabidopsis thaliana cad1‐3 mutant separates the metal tolerance and non‐host resistance functions of phytochelatin synthases

Phytochelatin synthases (PCS) play key roles in plant metal tolerance. They synthesize small metal‐binding peptides, phytochelatins, under conditions of metal excess. Respective mutants are strongly cadmium and arsenic hypersensitive. However, their ubiquitous presence and constitutive expression had long suggested a more general function of PCS besides metal detoxification. Indeed, phytochelatin synthase1 from Arabidopsis thaliana (AtPCS1) was later implicated in non‐host resistance. The two different physiological functions may be attributable to the two distinct catalytic activities demonstrated for AtPCS1, that is the dipeptidyl transfer onto an acceptor molecule in phytochelatin synthesis, and the proteolytic deglycylation of glutathione conjugates. In order to test this hypothesis and to possibly separate the two biological roles, we expressed a phylogenetically distant PCS from Caenorhabditis elegans in an AtPCS1 mutant. We confirmed the involvement of AtPCS1 in non‐host resistance by showing that plants lacking the functional gene develop a strong cell death phenotype when inoculated with the potato pathogen Phytophthora infestans. Furthermore, we found that the C. elegans gene rescues phytochelatin synthesis and cadmium tolerance, but not the defect in non‐host resistance. This strongly suggests that the second enzymatic function of AtPCS1, which remains to be defined in detail, is underlying the plant immunity function.     

Extremely low‐frequency electromagnetic fields affect the immune response of monocyte‐derived macrophages to pathogens

This study aimed to determine the effect of extremely low‐frequency electromagnetic fields (ELF‐EMF) on the physiological response of phagocytes to an infectious agent. THP‐1 cells (human monocytic leukemia cell line) were cultured and 50 Hz, 1 mT EMF was applied for 4–6 h to cells induced with Staphylococcus aureus or interferon gamma/lipopolysaccharide (IFγ/LPS). Alterations in nitric oxide (NO), inducible nitric oxide synthase (iNOS) levels, heat shock protein 70 levels (hsp70), cGMP levels, caspase‐9 activation, and the growth rate of S. aureus were determined. The growth curve of exposed bacteria was lower than the control. Field application increased NO levels. The increase was more prominent for S. aureus‐induced cells and appeared earlier than the increase in cells without field application. However, a slight decrease was observed in iNOS levels. Increased cGMP levels in response to field application were closely correlated with increased NO levels. ELF‐EMF alone caused increased hsp70 levels in a time‐dependent manner. When cells were induced with S. aureus or IFγ/LPS, field application produced higher levels of hsp70. ELF‐EMF suppressed caspase‐9 activation by a small extent. These data confirm that ELF‐EMF affects bacterial growth and the response of the immune system to bacterial challenges, suggesting that ELF‐EMF could be exploited for beneficial uses. Bioelectromagnetics 31:603–612, 2010. © 2010 Wiley‐Liss, Inc.