Degradation of methicillin-resistant Staphylococcus aureus biofilms using a chimeric lysin

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for a large number of chronic infections due to its ability to form robust biofilms. Herein, the authors evaluated the anti-biofilm activity of a Staphylococcus specific chimeric lysin ClyH on MRSA biofilms. ClyH is known to be active against planktonic MRSA cells in vitro and in vivo. The minimum concentrations for biofilm eradication (MCBE) of ClyH were 6.2–50?mg?l^?1, much lower than those of antibiotics. Scanning electron microscope (SEM) analysis revealed that ClyH eliminated MRSA biofilms through cell lytic activity in a time-dependent manner. Viable plate counts and kinetic analysis demonstrated that biofilms of different ages displayed varying susceptibility to ClyH. Together with previously demonstrated in vivo efficacy of ClyH against MRSA, the degradation efficacy against biofilms of different ages indicates that ClyH could be used to remove MRSA biofilms in vivo.     

The Capsular Polysaccharide of Staphylococcus aureus Is Attached to Peptidoglycan by the LytR-CpsA-Psr (LCP) Family of Enzymes

Envelope biogenesis in bacteria involves synthesis of intermediates that are tethered to the lipid carrier undecaprenol-phosphate. LytR-CpsA-Psr (LCP) enzymes have been proposed to catalyze the transfer of undecaprenol-linked intermediates onto the C6-hydroxyl of MurNAc in peptidoglycan, thereby promoting attachment of wall teichoic acid (WTA) in bacilli and staphylococci and capsular polysaccharides (CPS) in streptococci. S. aureus encodes three lcp enzymes, and a variant lacking all three genes (Δlcp) releases WTA from the bacterial envelope and displays a growth defect. Here, we report that the type 5 capsular polysaccharide (CP5) of Staphylococcus aureus Newman is covalently attached to the glycan strands of peptidoglycan. Cell wall attachment of CP5 is abrogated in the Δlcp variant, a defect that is best complemented via expression of lcpC in trans. CP5 synthesis and peptidoglycan attachment are not impaired in the tagO mutant, suggesting that CP5 synthesis does not involve the GlcNAc-ManNAc linkage unit of WTA and may instead utilize another Wzy-type ligase to assemble undecaprenyl-phosphate intermediates. Thus, LCP enzymes of S. aureus are promiscuous enzymes that attach secondary cell wall polymers with discrete linkage units to peptidoglycan.     

Rapid detection of Staphylococcus aureus strains having reduced susceptibility to vancomycin using a chemiluminescence-based drug-susceptibility test

Current drug-susceptibility tests used routinely in clinical laboratories sometimes fail to identify strains of Staphylococcus aureus with reduced susceptibility to vancomycin. To solve this problem, we have developed a more sensitive and rapid method that measures bacterial metabolic activity by a chemiluminescence-based technique. This method is able to discriminate such strains from vancomycin-susceptible S. aureus with a sensitivity and specificity of > 95%. This rapid and reliable method appears to be promising for detection of vancomycin-intermediate S. aureus strains in clinical laboratories, and may supersede classical susceptibility testing.     

The Beneficial Effect of Ginsenoside Rg1 on Schwann Cells Subjected to Hydrogen Peroxide Induced Oxidative Injury

Ginsenoside Rg1 (GRg1) has been considered to have therapeutic potential in promoting peripheral nerve regeneration and functional recovery after sciatic nerve injuries. However, the mechanism underlying the beneficial effect of GRg1 on peripheral nerve regeneration is currently unclear. The possible effect of GRg1 on Schwann cells (SCs), which were subjected to oxidative injury after nerve injury, might contribute to the beneficial effect of GRg1 on nerve regeneration. The present study was designed to investigate the potential beneficial effect of GRg1 on SCs exposed to oxidative injury. The oxidative injury to SCs was induced by hydrogen peroxide. The effect of GRg1 (50 μM) on SCs exposed to oxidative injury was measured by the levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH) and catalase (CAT) in SCs. The cell number and cell viability of SCs were evaluated through fluorescence observation and MTT assay. The apoptosis of SCs induced by oxidative injury was evaluated by an apoptosis assay. The expression and secretion of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were evaluated using RT-PCR, Western blotting, and an ELISA method. We found that GRg1 significantly up-regulated the level of SOD, GSH and CAT, and decreased the level of MDA in SCs treated with hydrogen peroxide. In addition, GRg1 has been shown to be able to inhibit the proapoptotic effect of hydrogen peroxide, as well as inhibit the detrimental effect of hydrogen peroxide on cell number and cell viability. Furthermore, GRg1 also increased the mRNA levels, protein levels and secretion of NGF and BDNF in SCs after incubation of hydrogen peroxide. Further study showed that preincubation with H89 (a PKA inhibitor) significantly inhibited the effects induced by hydrogen peroxide, indicating that the PKA pathway might be involved in the antioxidant effect and neurotrophic factors (NTFs) promoting effect of GRg1. In addition, a short-term in vivo study was performed to confirm and validate the antioxidant effect and nerve regeneration-promoting effect of GRg1 in a sciatic crush injury model in rats. We found that GRg1 significantly increased SOD, CAT and GSH, decreased MDA, as well as promoted nerve regeneration after crush injury. In conclusion, the present study showed that GRg1 is capable of helping SCs recover from the oxidative insult induced by hydrogen peroxide, which might account, at least in part, for the beneficial effect of GRg1 on nerve regeneration.     

SbnG,a Citrate Synthase in Staphylococcus aureus: A NEW FOLD ON AN OLD ENZYME*

In response to iron deprivation, Staphylococcus aureus produces staphyloferrin B, a citrate-containing siderophore that delivers iron back to the cell. This bacterium also possesses a second citrate synthase, SbnG, that is necessary for supplying citrate to the staphyloferrin B biosynthetic pathway. We present the structure of SbnG bound to the inhibitor calcium and an active site variant in complex with oxaloacetate. The overall fold of SbnG is structurally distinct from TCA cycle citrate synthases yet similar to metal-dependent class II aldolases. Phylogenetic analyses revealed that SbnG forms a separate clade with homologs from other siderophore biosynthetic gene clusters and is representative of a metal-independent subgroup in the phosphoenolpyruvate/pyruvate domain superfamily. A structural superposition of the SbnG active site to TCA cycle citrate synthases and site-directed mutagenesis suggests a case for convergent evolution toward a conserved catalytic mechanism for citrate production.     

Complex Structure and Biochemical Characterization of the Staphylococcus aureus Cyclic Diadenylate Monophosphate (c-di-AMP)-binding Protein PstA,the Founding Member of a New Signal Transduction Protein Family

Signaling nucleotides are integral parts of signal transduction systems allowing bacteria to cope with and rapidly respond to changes in the environment. The Staphylococcus aureus PII-like signal transduction protein PstA was recently identified as a cyclic diadenylate monophosphate (c-di-AMP)-binding protein. Here, we present the crystal structures of the apo- and c-di-AMP-bound PstA protein, which is trimeric in solution as well as in the crystals. The structures combined with detailed bioinformatics analysis revealed that the protein belongs to a new family of proteins with a similar core fold but with distinct features to classical PII proteins, which usually function in nitrogen metabolism pathways in bacteria. The complex structure revealed three identical c-di-AMP-binding sites per trimer with each binding site at a monomer-monomer interface. Although distinctly different from other cyclic-di-nucleotide-binding sites, as the half-binding sites are not symmetrical, the complex structure also highlighted common features for c-di-AMP-binding sites. A comparison between the apo and complex structures revealed a series of conformational changes that result in the ordering of two anti-parallel β-strands that protrude from each monomer and allowed us to propose a mechanism on how the PstA protein functions as a signaling transduction protein.     

Methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase as a target for bis-indole alkaloids with antibacterial activities

Novel classes of antimicrobials are needed to address the emergence of multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). We have recently identified pyruvate kinase (PK) as a potential novel drug target based upon it being an essential hub in the MRSA interactome (Cherkasov, A., Hsing, M., Zoraghi, R., Foster, L. J., See, R. H., Stoynov, N., Jiang, J., Kaur, S., Lian, T., Jackson, L., Gong, H., Swayze, R., Amandoron, E., Hormozdiari, F., Dao, P., Sahinalp, C., Santos-Filho, O., Axerio-Cilies, P., Byler, K., McMaster, W. R., Brunham, R. C., Finlay, B. B., and Reiner, N. E. (2011) J. Proteome Res. 10, 1139-1150; Zoraghi, R., See, R. H., Axerio-Cilies, P., Kumar, N. S., Gong, H., Moreau, A., Hsing, M., Kaur, S., Swayze, R. D., Worrall, L., Amandoron, E., Lian, T., Jackson, L., Jiang, J., Thorson, L., Labriere, C., Foster, L., Brunham, R. C., McMaster, W. R., Finlay, B. B., Strynadka, N. C., Cherkasov, A., Young, R. N., and Reiner, N. E. (2011) Antimicrob. Agents Chemother. 55, 2042-2053). Screening of an extract library of marine invertebrates against MRSA PK resulted in the identification of bis-indole alkaloids of the spongotine (A), topsentin (B, D), and hamacanthin (C) classes isolated from the Topsentia pachastrelloides as novel bacterial PK inhibitors. These compounds potently and selectively inhibited both MRSA PK enzymatic activity and S. aureus growth in vitro. The most active compounds, cis-3,4-dihyrohyrohamacanthin B (C) and bromodeoxytopsentin (D), were identified as highly potent MRSA PK inhibitors (IC(50) values of 16-60 nM) with at least 166-fold selectivity over human PK isoforms. These novel anti-PK natural compounds exhibited significant antibacterial activities against S. aureus, including MRSA (minimal inhibitory concentrations (MIC) of 12.5 and 6.25 μg/ml, respectively) with selectivity indices (CC(50)/MIC) >4. We also report the discrete structural features of the MRSA PK tetramer as determined by x-ray crystallography, which is suitable for selective targeting of the bacterial enzyme. The co-crystal structure of compound C with MRSA PK confirms that the latter is a target for bis-indole alkaloids. It elucidates the essential structural requirements for PK inhibitors in "small" interfaces that provide for tetramer rigidity and efficient catalytic activity. Our results identified a series of natural products as novel MRSA PK inhibitors, providing the basis for further development of potential novel antimicrobials.     

Extracellular Fibrinogen-binding Protein (Efb) from Staphylococcus aureus Inhibits the Formation of Platelet-Leukocyte Complexes

Extracellular fibrinogen-binding protein (Efb) from Staphylococcus aureus inhibits platelet activation, although its mechanism of action has not been established. In this study, we discovered that the N-terminal region of Efb (Efb-N) promotes platelet binding of fibrinogen and that Efb-N binding to platelets proceeds via two independent mechanisms: fibrinogen-mediated and fibrinogen-independent. By proteomic analysis of Efb-interacting proteins within platelets and confirmation by pulldown assays followed by immunoblotting, we identified P-selectin and multimerin-1 as novel Efb interaction partners. The interaction of both P-selectin and multimerin-1 with Efb is independent of fibrinogen. We focused on Efb interaction with P-selectin. Excess of P-selectin extracellular domain significantly impaired Efb binding by activated platelets, suggesting that P-selectin is the main receptor for Efb on the surface of activated platelets. Efb-N interaction with P-selectin inhibited P-selectin binding to its physiological ligand, P-selectin glycoprotein ligand-1 (PSGL-1), both in cell lysates and in cell-free assays. Because of the importance of P-selectin-PSGL-1 binding in the interaction between platelets and leukocytes, we tested human whole blood and found that Efb abolishes the formation of platelet-monocyte and platelet-granulocyte complexes. In summary, we present evidence that in addition to its documented antithrombotic activity, Efb can play an immunoregulatory role via inhibition of P-selectin-PSGL-1-dependent formation of platelet-leukocyte complexes.     

Identification of a domain critical for Staphylococcus aureus LukED receptor targeting and lysis of erythrocytes

Leukocidin ED (LukED) is a pore-forming toxin produced by Staphylococcus aureus, which lyses host cells and promotes virulence of the bacteria. LukED enables S. aureus to acquire iron by lysing erythrocytes, which depends on targeting the host receptor Duffy antigen receptor for chemokines (DARC). The toxin also targets DARC on the endothelium, contributing to the lethality observed during bloodstream infection in mice. LukED is comprised of two monomers: LukE and LukD. LukE binds to DARC and facilitates hemolysis, but the closely related Panton–Valentine leukocidin S (LukS-PV) does not bind to DARC and is not hemolytic. The interaction of LukE with DARC and the role this plays in hemolysis are incompletely characterized. To determine the domain(s) of LukE that are critical for DARC binding, we studied the hemolytic function of LukE–LukS-PV chimeras, in which areas of sequence divergence (divergence regions, or DRs) were swapped between the toxins. We found that two regions of LukE''s rim domain contribute to hemolysis, namely residues 57–75 (DR1) and residues 182–196 (DR4). Interestingly, LukE DR1 is sufficient to render LukS-PV capable of DARC binding and hemolysis. Further, LukE, by binding DARC through DR1, promotes the recruitment of LukD to erythrocytes, likely by facilitating LukED oligomer formation. Finally, we show that LukE targets murine Darc through DR1 in vivo to cause host lethality. These findings expand our biochemical understanding of the LukE–DARC interaction and the role that this toxin-receptor pair plays in S. aureus pathophysiology.     

Hydrogen Peroxide in Plants： a Versatile Molecule of the Reactive Oxygen Species Network

Plants often face the challenge of severe environmental conditions, which include various biotic and abiotic stresses that exert adverse effects on plant growth and development. During evolution, plants have evolved complex regulatory mechanisms to adapt to various environmental stressors. One of the consequences of stress is an increase in the cellular concentration of reactive oxygen species （ROS）, which are subsequently converted to hydrogen peroxide （H2O2）. Even under normal conditions, higher plants produce ROS during metabolic processes. Excess concentrations of ROS result in oxidative damage to or the apoptotic death of cells. Development of an antioxidant defense system in plants protects them against oxidative stress damage. These ROS and, more particularly, H2O2, play versatile roles in normal plant physiological processes and in resistance to stresses. Recently, H2O2 has been regarded as a signaling molecule and regulator of the expression of some genes in cells. This review describes various aspects of H2O2 function, generation and scavenging, gene regulation and cross-links with other physiological molecules during plant growth, development and resistance responses.     

Redox Control of Protein Arginine Methyltransferase 1 (PRMT1) Activity

Elevated levels of asymmetric dimethylarginine (ADMA) correlate with risk factors for cardiovascular disease. ADMA is generated by the catabolism of proteins methylated on arginine residues by protein arginine methyltransferases (PRMTs) and is degraded by dimethylarginine dimethylaminohydrolase. Reports have shown that dimethylarginine dimethylaminohydrolase activity is down-regulated and PRMT1 protein expression is up-regulated under oxidative stress conditions, leading many to conclude that ADMA accumulation occurs via increased synthesis by PRMTs and decreased degradation. However, we now report that the methyltransferase activity of PRMT1, the major PRMT isoform in humans, is impaired under oxidative conditions. Oxidized PRMT1 displays decreased activity, which can be rescued by reduction. This oxidation event involves one or more cysteine residues that become oxidized to sulfenic acid (-SOH). We demonstrate a hydrogen peroxide concentration-dependent inhibition of PRMT1 activity that is readily reversed under physiological H₂O₂ concentrations. Our results challenge the unilateral view that increased PRMT1 expression necessarily results in increased ADMA synthesis and demonstrate that enzymatic activity can be regulated in a redox-sensitive manner.     

Glutamine-Fructose-6-Phosphate Transaminase 2 (GFPT2) Is Upregulated in Breast Epithelial–Mesenchymal Transition and Responds to Oxidative Stress

Breast cancer cells that have undergone partial epithelial–mesenchymal transition (EMT) are believed to be more invasive than cells that have completed EMT. To study metabolic reprogramming in different mesenchymal states, we analyzed protein expression following EMT in the breast epithelial cell model D492 with single-shot LFQ supported by a SILAC proteomics approach. The D492 EMT cell model contains three cell lines: the epithelial D492 cells, the mesenchymal D492M cells, and a partial mesenchymal, tumorigenic variant of D492 that overexpresses the oncogene HER2. The analysis classified the D492 and D492M cells as basal-like and D492HER2 as claudin-low. Comparative analysis of D492 and D492M to tumorigenic D492HER2 differentiated metabolic markers of migration from those of invasion. Glutamine-fructose-6-phosphate transaminase 2 (GFPT2) was one of the top dysregulated enzymes in D492HER2. Gene expression analysis of the cancer genome atlas showed that GFPT2 expression was a characteristic of claudin-low breast cancer. siRNA-mediated knockdown of GFPT2 influenced the EMT marker vimentin and both cell growth and invasion in vitro and was accompanied by lowered metabolic flux through the hexosamine biosynthesis pathway (HBP). Knockdown of GFPT2 decreased cystathionine and sulfide:quinone oxidoreductase (SQOR) in the transsulfuration pathway that regulates H₂S production and mitochondrial homeostasis. Moreover, GFPT2 was within the regulation network of insulin and EGF, and its expression was regulated by reduced glutathione (GSH) and suppressed by the oxidative stress regulator GSK3-β. Our results demonstrate that GFPT2 controls growth and invasion in the D492 EMT model, is a marker for oxidative stress, and associated with poor prognosis in claudin-low breast cancer.