基于重组溶葡球菌酶和ATP生物发光技术特异定量检测金黄色葡萄球菌

基于重组溶葡球菌酶和ATP生物发光法建立特异定量检测金黄色葡萄球菌的方法。优化设计合成溶葡球菌酶序列,构建重组表达载体pQE30-Lys,转化至大肠杆菌M15并诱导表达,镍柱纯化得到目的蛋白。利用重组溶葡球菌酶和ATP生物发光法特异定量检测金黄色葡萄球菌并与平板计数对比。成功表达了重组溶葡球菌酶,并建立了特异定量检测金黄色葡萄球菌的方法,与平板计数具有显著线性关系。本研究建立的将重组溶葡球菌酶和ATP生物发光法相结合的检测方法操作快捷简单,具有良好的应用前景。     

Comparative toxicity studies on bromochloroacetate,dibromoacetate, and bromodichloroacetate in J774A.1 macrophages: Roles of superoxide anion and protein carbonyl compounds

The brominated and mixed bromo‐chloro‐haloacetates, such as dibromoacetate (DBA), bromochloroacetate (BCA), and bromodichloroacetate (BDCA), are by‐products of water chlorination and are found at lower levels than the fully chlorinated acetates in the drinking water. The toxicities of the compounds were assessed in J774A.1 cells and were found to induce concentration‐dependent increases in cell death and superoxide anion and protein carbonyl compounds production. Compared to the previously tested concentrations of dichoroacetate (DCA) and trichloroacetate (TCA) in the same cell line, the tested haloacetates induced similar effects on cellular viability and superoxide anion production but at DBA and BCA concentrations that were approximately 40–160 times lower than those of DCA and TCA, and at BDCA concentrations that were 4–16 times lower than those of DCA and TCA. Also, production of super oxide anion, protein carbonyl compounds, and induction of phagocytic activation are suggested to play a role in their toxicity.     

Effect of SXWS/WSXWS peptides on chemotaxis and adhesion of the macrophage‐like cell line J774

WSXWS motif is a conserved amino acid sequence that is present in type I cytokine receptors. This motif that can be found both in the ligand binding chains and signal transducer molecule of the receptors with different amino acids at the position “X” plays a role in the receptor folding, ligand binding and signal transduction as well. Structural analysis proved that WSEWS motif of IL‐6R is located in a highly accessible location in the protein. Structural properties and chemotaxis of a tetrapeptide library with SXWS sequence, where X was the 19 proteinogenic amino acids except cystein were systematically studied earlier. It has been proved that C‐terminal amidation and the identity of amino acid X had a pronounced influence on the chemotactic properties but less of the structure of the peptides. Here, we present our findings on the effect of a tetrapeptide and a pentapeptide library with the sequence of SXWS and WSXWS on the chemotaxis and adhesion of J774 murine macrophage cell line. We studied the effect of the presence/absence of N‐terminal tryptophan and the different amino acids at the X position on these physiological responses. Results indicated that amino acid X had a marked influence on chemotaxis, adhesion as well as on proliferation induced by (W)SXWS peptides. Elongation of SXWS sequence with a tryptophan at the N terminus also altered pronouncedly all the physiological responses of the cells studied. A good correlation could be observed between the chemotaxis and the proliferation and physicochemical parameters of the amino acid X. Copyright © 2015 John Wiley & Sons, Ltd.     

Cloning and sequencing of the gene, fmtC, which affects oxacillin resistance in methicillin-resistant Staphylococcus aureus

Two Tn551 insertional mutants with reduced methicillin resistance were isolated from methicillin-resistant Staphylococcus aureus KSA8. These two mutants showed increased susceptibility to beta-lactam antibiotics and bacitracin, but not to fosfomycin and vancomycin. Tn551 in these mutants was inserted into the same gene, termed fmtC. The fmtC gene has an open reading frame of 840 amino acid residues with an estimated molecular mass of 96.9 kDa. The N-terminal half of the deduced FmtC protein is very hydrophobic, implying that this protein is a membrane-associated protein.     

Immunostimulatory effect of spinach aqueous extract on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages

We herein report the immunostimulatory effect of spinach aqueous extract (SAE) on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages. SAE significantly enhanced the production of interleukin (IL)-6 and tumor necrosis factor-α by both J774.1 cells and peritoneal macrophages by enhancing the expression levels of these cytokine genes. In addition, the phagocytosis activity of J774.1 cells was facilitated by SAE. Immunoblot analysis revealed that SAE activates mitogen-activated protein kinase and nuclear factor-κB cascades. It was found that SAE activates macrophages through not only TLR4, but also other receptors. The production of IL-6 was significantly enhanced by peritoneal macrophages from SAE-administered BALB/c mice, suggesting that SAE has a potential to stimulate macrophage activity in vivo. Taken together, these data indicate that SAE would be a beneficial functional food with immunostimulatory effects on macrophages.     

The induction of oxidative stress and cellular death by the drinking water disinfection by-products,dichloroacetate and trichloroacetate in J774.A1 cells

The in vitro toxicity of the drinking water disinfection by products dichloroacetate (DCA) and trichloroacetate (TCA) were studied using the J774A.1 macrophage cell line. DCA and TCA were added to cell cultures at concentrations ranging between 8-32 mM and incubated for 24, 36 and 60 h. DCA and TCA effects on cellular viability, lactate dehydrogenase (LDH) release and superoxide anion (SA) production by the cells, as well as superoxide dismutase (SOD) activities of the cells were determined. DCA and TCA caused time- and concentration-dependent increases in cellular death, in LDH release and production of SA by the cells. The compounds also caused modulations in SOD activities of the cells, with increases observed at the lower concentrations and/or shorter periods of incubations and suppression with the higher concentrations and/or longer periods of incubation. The results of the study indicate that DCA and TCA induce macrophage activation and that the activation is associated with cellular toxicity. Also, DCA and TCA are found to be equitoxic to J774.A1 cells.     

Induction of apoptosis in macrophage cell line,J774, by the cell-free supernatant from Pseudomonas aeruginosa

Pseudomonas aeruginosa is able to secrete many virulence factors that are cytotoxic towards eukaryotic cells. To investigate the effect of the bacterium on macrophages, we obtained cell-free supernatants from P. aeruginosa (Pa) IID1117 (elastase-positive and protease-positive) and Pa IID1130 (elastase-positive and protease-negative). After 6 hr of incubation with the cell-free supernatant from the Pa IID1117 strain, the viability of J774 macrophages was shown to be significantly reduced (47.5+/-11%), but not Pa IID1130 (96.4+/-1.6%) at a concentration of 10% (v/v) compared to control J774 macrophages without any supernatant (97.2+/-1.7%) by the detection of trypan blue dye exclusion. The death of cells was further demonstrated to be due to apoptosis characterized by chromatin condensation and apoptotic bodies by Hoechst 33258 staining, DNA fragmentation by agarose gel electrophoresis and terminal deoxynucleotidyl transferase-mediated d-UTP nick end labeling (TUNEL). An activated subunit was found to be released from procaspase-3 in cell lysate. But in the presence of protease inhibitor, the apoptosis was completely blocked. The findings indicate that the Pa IID1117 strain is capable of inducing apoptosis in J774 macrophages. The apoptosis induced by the cell-free supernatant from Pa IID1117 strain is suggested to be dependent on protease, but not elastase.     

Possible participation of the Rho/Rho-associated coiled-coil-forming kinase pathway in the cell death of Cryptococcus neoformans caused by Staphylococcus aureus adherence

We here report the apoptotic death of a fungus, Cryptococcus neoformans (C. neoformans), in response to adherence of the pathogenic bacterium Staphylococcus aureus (S. aureus). In co-culture, cryptococcal actin was visibly aggregated. To investigate the mechanism of death, the participation of small GTP(guanosine triphosphate)-binding proteins belonging to the Rho subfamily, which regulate the actin cytoskeleton, was explored. C. neoformans was cultured with S. aureus in the presence of N-(4-pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide (Y-27632), an inhibitor of Rho-associated coiled-coil forming kinase (ROCK), a downstream effector of Rho. Death of C. neoformans was significantly reduced by the inhibitor. Concomitantly, Y-27632 prevented the aggregation of actin. Therefore, it was concluded that the Rho/ROCK pathway is involved in cell death induced by adherence stress. Increased expression of the voltage-dependent anion channel (VDAC), located in the mitochondrial outer membrane, has previously been observed in the apoptosis-like death of C. neoformans in the presence of hydrogen peroxide. Ruthenium red (RuR), which binds to VDAC and inhibits cytochrome c release, was used to determine the involvement of VDAC following adherence stress caused by S. aureus. RuR treatment increased the viability of C. neoformans co-cultured with S. aureus in a dose dependent manner. These findings suggest that Rho-ROCK signaling could be involved, via a mitochondrial pathway, in the apoptosis-like death of C. neoformans induced by the adherence of S. aureus.     

猪源致病性金黄色葡萄球菌的分离鉴定及其耐药性分析

目的鉴定引起猪渗出性皮炎的病原,并分析猪源致病性金黄色葡萄球菌的耐药性,为临床用药提供依据。方法采集患渗出性皮炎的仔猪标本进行细菌分离培养,联合应用形态学检查、生理生化试验和PCR方法鉴定分离菌株,并进行致病性和药物敏感性试验。结果先后从病猪标本中分离鉴定获得PSA1、PSA2、PSA3和PSA4四株金黄色葡萄球菌,其中PSA1和PSA3分离株的致病性较强。药敏试验结果显示PSA1、PSA2和PSA3分离株为MRSA菌株,PSA4分离株为MSSA菌株。MRSA菌株对14种抗菌药物均呈现不同程度的耐药,尤其是对青霉素、链霉素、四环素、强力霉素、环丙沙星和氧氟沙星等6种抗菌药物的耐药率达100%。所有分离株对万古霉素与替考拉宁均敏感。结论合肥地区猪渗出性表皮炎的病原为金黄色葡萄球菌。猪源致病性金黄色葡萄球菌合肥分离株具有多重耐药性,治疗猪渗出性皮炎应建立在体外药敏试验的基础上,有针对性选择抗菌药物。     

Translation of RNAIII, the Staphylococcus aureus agr regulatory RNA molecule, can be activated by a 3'-end deletion

Abstract RNAIII, an RNA molecule shown to encode δ-hemolysin and independently to regulate toxin synthesis in Staphylococcus aureus , is transcribed at the mid-exponential phase of growth, while its target genes are activated 2 h later, at the post-exponential phase of growth. We show here that the translation of RNAIII to the 26-amino acid peptide δ-hemolysin is delayed by 1 h, and that this delay is abolished when the 3'-end of this molecule is deleted. We suggest that structural changes of RNAIII to a translatable form of the molecule precede its regulation of target gene expression.     

Mechanisms of internalization of apoptotic and necrotic L929 cells by a macrophage cell line studied by electron microscopy

Rapid and efficient phagocytic removal of dying cells is a key feature of apoptosis. In necrotic caspase-independent modes of death, the role and extent of phagocytosis is not well documented. To address this issue, we studied at the ultrastructural level the phagocytic response to dying cells in an in vitro phagocytosis assay with a mouse macrophage cell line (Mf4/4). As target cells, murine L929sAhFas cells were induced to die by TNFR1-mediated necrosis or by Fas-mediated apoptosis. Apoptotic L929sAhFas cells are taken up by complete engulfment of apoptotic bodies as single entities forming a tight-fitting phagosome, thus resembling the "zipper"-like mechanism of internalization. In contrast, primary and secondary necrotic cells were internalized by a macropinocytotic mechanism with formation of multiple ruffles by the ingesting macrophage. Ingestion of necrotic cellular material was invariably taking place after the integrity of the cell membrane was lost and did not occur as discrete particles, in contrast to apoptotic material that is surrounded by an intact membrane. Although nuclei of necrotic cells have been observed in the vicinity of macrophages, no uptake of necrotic nuclei was observed. The present report provides a basis for future studies aimed at discovering molecular pathways that precede these diverse mechanisms of uptake.     

Structure-Function Analysis of Heterodimer Formation,Oligomerization, and Receptor Binding of the Staphylococcus aureus Bi-component Toxin LukGH

The bi-component leukocidins of Staphylococcus aureus are important virulence factors that lyse human phagocytic cells and contribute to immune evasion. The γ-hemolysins (HlgAB and HlgCB) and Panton-Valentine leukocidin (PVL or LukSF) were shown to assemble from soluble subunits into membrane-bound oligomers on the surface of target cells, creating barrel-like pore structures that lead to cell lysis. LukGH is the most distantly related member of this toxin family, sharing only 30–40% amino acid sequence identity with the others. We observed that, unlike other leukocidin subunits, recombinant LukH and LukG had low solubility and were unable to bind to target cells, unless both components were present. Using biolayer interferometry and intrinsic tryptophan fluorescence we detected binding of LukH to LukG in solution with an affinity in the low nanomolar range and dynamic light scattering measurements confirmed formation of a heterodimer. We elucidated the structure of LukGH by x-ray crystallography at 2.8-Å resolution. This revealed an octameric structure that strongly resembles that reported for HlgAB, but with important structural differences. Structure guided mutagenesis studies demonstrated that three salt bridges, not found in other bi-component leukocidins, are essential for dimer formation in solution and receptor binding. We detected weak binding of LukH, but not LukG, to the cellular receptor CD11b by biolayer interferometry, suggesting that in common with other members of this toxin family, the S-component has the primary contact role with the receptor. These new insights provide the basis for novel strategies to counteract this powerful toxin and Staphylococcus aureus pathogenesis.     

Rapid detection of enterotoxigenic Staphylococcus aureus harbouring genes for four classical enterotoxins, SEA, SEB, SEC and SED, by loop-mediated isothermal amplification assay

AIM: The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) assay targeting the genes for the four classical enterotoxins, SEA, SEB, SEC and SED, in Staphylococcus aureus. METHODS AND RESULTS: Specific primers were designed which target each specific sequence of the enterotoxin genes. With 30 strains of Staph. aureus, the results of the LAMP assay to each enterotoxin, SEA, SEB, SEC and SED, completely accorded with the results of polymerase chain reaction (PCR) assay. Enterotoxin production, determined by a reverse passive latex agglutination assay, strongly correlated with the presence of the corresponding genes. Amplification was not observed when 14 strains of nonenterotoxigenic Staph. aureus and 20 strains consisting of 19 bacterial species other than Staph. aureus were tested. In addition, the sensitivity of the LAMP assay was generally higher than that of conventional PCR assay and it rapidly detected enterotoxigenic Staph. aureus strains within 60 min. CONCLUSIONS: The LAMP assay developed in this study is rapid, specific and sensitive for the detection of enterotoxigenic Staph. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: The method is suitable for clinical diagnosis and food safety applications.     

Evaluation of antibacterial activity induced by Staphylococcus aureus and Ent A in the hemolymph of Spodoptera littoralis

The problem of antibiotic resistance considers one of the most dangerous challenges facing the medical field. So, it is necessary to find substitutions to conventional antibiotics. Antimicrobial peptides (AMPs) are a bio-functional derivative that have been observed as one of the important solutions to such upcoming crisis. Owing to their role as the first line of defense against bacteria, fungi, and viruses. This study was conducted to induce the immune response of Spodoptera littoralis larvae by inoculation of sub lethal doses of Staphylococcus aureus and its enterotoxin. Since Staphylococcal enterotoxin A (SEA) considers the major causative agents of Staphylococcal food poisoning, our study oriented to purify and characterize this toxin to provoke its role in yielding AMPs with broad spectrum antimicrobial activity. A great fluctuation was recorded in the biochemical properties of immunized hemolymph not only in the total protein content but also protein banding pattern. Protein bands of ∼22 kDa (attacin-like) and ∼15 kDa (lysozyme-like) were found to be common between the AMPs induced as a result of both treatments. While protein bands of molecular weight ∼70 kDa (phenoloxidase-like) and ∼14 kDa (gloverin-like) were found specific for SEA treatment. Chromatographic analysis using HPLC for the induced AMPs showed different types of amino acids appeared with differences in their quantities and velocities. These peptides exhibited noticeable antimicrobial activity against certain Gram-positive and Gram-negative bacteria. In conclusion, the antimicrobial potential of the antimicrobial peptides (AMP) induced in the larval hemolymph of S. littoralis will be a promising molecule for the development of new therapeutic alternatives.     

The binding mechanism,multiple binding modes,and allosteric regulation of Staphylococcus aureus Sortase A probed by molecular dynamics simulations

Sortase enzymes are vitally important for the virulence of gram‐positive bacteria as they play a key role in the attachment of surface proteins to the cell wall. These enzymes recognize a specific sorting sequence in proteins destined to be displayed on the surface of the bacteria and catalyze the transpeptidation reaction that links it to a cell wall precursor molecule. Because of their role in establishing pathogenicity, and in light of the recent rise of antibiotic‐resistant bacterial strains, sortase enzymes are novel drug targets. Here, we present a study of the prototypical sortase protein Staphylococcus aureus Sortase A (SrtA). Both conventional and accelerated molecular dynamics simulations of S. aureus SrtA in its apo state and when bound to an LPATG sorting signal (SS) were performed. Results support a binding mechanism that may be characterized as conformational selection followed by induced fit. Additionally, the SS was found to adopt multiple metastable states, thus resolving discrepancies between binding conformations in previously reported experimental structures. Finally, correlation analysis reveals that the SS actively affects allosteric pathways throughout the protein that connect the first and the second substrate binding sites, which are proposed to be located on opposing faces of the protein. Overall, these calculations shed new light on the role of dynamics in the binding mechanism and function of sortase enzymes.     

An 100-kDa arachidonate-mobilizing phospholipase A2 in mouse spleen and the macrophage cell line J774. Purification, substrate interaction and phosphorylation by protein kinase C.

A phospholipase A2 hydrolyzing arachidonic-acid-containing phospholipids has been purified 5600-fold from mouse spleen and to near homogeneity from the macrophage cell line J774. A molecular mass of 100 kDa for the enzyme was estimated by SDS/PAGE, while it migrated as a 70-kDa protein upon gel chromatography. The enzyme from both sources showed the same characteristics as that previously identified in murine peritoneal macrophages [Wijkander, J. & Sundler, R. (1989), FEBS Lett. 244, 51-56], i.e. it was totally dependent on Ca2+ with half-maximal activity at approximately 0.7 microM and hydrolyzed arachidonoyl phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol equally well. Also, the platelet-activating-factor precursor, 1-O-alkyl-2-arachidonoylglycerophosphocholine, was hydrolyzed to a similar extent. A preference for arachidonoylphosphatidylcholine over oleoylphosphatidylcholine was seen both with sonicated vesicles and labeled macrophage membranes as substrate. Ca(2+)-dependent interaction of the enzyme with sonicated vesicles composed of neutral phospholipids led to rapid initial hydrolysis, followed by loss of catalytic activity. Such inactivation did not occur with vesicles of pure anionic phospholipids, or with membranes prepared from macrophages. Phospholipase A2, purified from J774 cells, was rapidly phosphorylated by protein kinase C type-II, leading to incorporation of approximately 0.5 mol phosphate/mol enzyme.     

Comparative studies of the immunogenicity and protective potential of biofilm vs planktonic Staphylococcus aureus vaccine against bovine mastitis using non-invasive mouse mastitis as a model system

This study was undertaken to compare the immunogenicity and protective potential of biofilm vs planktonic Staphylococcus aureus vaccine for the prevention of mastitis using the mouse as a model system. Mice immunized with formalin-killed whole cell vaccine of S. aureus residing in a biofilm when delivered via an intramammary route produced a cell mediated immune response. Mice immunized with this biofilm vaccine showed significant reductions in colonization by S. aureus in mammary glands, severity of clinical symptoms and tissue damage in mammary glands in comparison with the mice immunized with formalin-killed whole cells of planktonic S. aureus. The planktonic vaccine administered by a subcutaneous route produced a significantly higher humoral immune response (IgG₁ and IgG) than the biofilm vaccine. However, considering the host response, tissue damage, the clinical severity and colonization of S. aureus in mammary glands, the biofilm vaccine performed better in immunogenicity and protective potential when administered by the intramammary route.     

Effects of superoxide dismutase and polyclonal tumor necrosis factor-alpha antibodies on chloroacetate-induced cellular death and superoxide anion production by J774.A1 macrophages

Dichloroacetate (DCA) and trichloroacetate (TCA) are by-products that are formed during the process of water chlorination and have been previously shown to induce superoxide anion (SA) production and cellular death when added to J774.A1 macrophage cultures. In this study, the effects of superoxide dismutase (SOD) and polyclonal tumor necrosis factor-alpha (TNF-alpha) antibodies on DCA- and TCA-induced SA production and cellular death have been tested on the J774.A1 macrophage cultures. TCA and DCA were added to different cultures either alone, each at a concentration of 16 mM, or in combination with SOD (2-12 units/ml), or with TNF-alpha antibodies (10 and 25 units/ml). Cells were incubated for 48 h, after which cellular death/viability, lactate dehydrognase (LDH) leakage by the cells, and SA production by the cells were determined. While TCA and DCA caused significant cellular toxicity, indicated by reduction in cellular viability and increases in LDH leakage and SA production, SOD addition resulted in significant reduction of the effects induced by the compounds. On the other hand, addition of TNF-alpha antibodies to the DCA- and TCA-treated cultures resulted in significant reduction of DCA- but not TCA-induced cellular death and SA production by the cells. Although these results suggest a significant role for SA in DCA- and TCA-induced cellular death, they may also suggest two different mechanisms for the chloroacetate-induced SA production by the cells.