Effect of Protein A on Adsorption of Bacteriophages to Staphylococcus aureus

Experiments were performed to determine if protein A influenced the association of bacteriophages with Staphylococcus aureus. Bacteriophage adsorption was compared in a S. aureus strain rich in protein A and mutants of this strain with very little protein A, in a strain with little protein A, and in mutants of this strain with increased protein A. In addition, the effect of growth in mannitol-salt broth and trypsin digestion (known to reduce protein A) on bacteriophage absorption was measured. There was an inverse relationship between protein A content of strains and the quantity of bacteriophage absorbed. However, no inhibition of staphylococcal phages was obtained with purified soluble protein A. Protein A as a surface component rendered the bacteria more resistant to adsorption of staphylococcal typing phages presumably by masking the phage receptor sites. When protein A-deficient mutants were incubated with bacteriophages, there was survival of staphylococci with increased protein A content probably due to a selective action.     

Escherichia coli strains of serogroup O139 isolated from oedema disease of swine bind fibronectin

Abstract 15 Escherichia coli strains of the serogroup O139, isolated from oedema disease of swine, were examined for their ability to interact with ¹²⁵I-labelled fibronectin. All strains were positive, and all except one showed higher fibronectin binding than Staphylococcus aureus strain Cowan 1 cells (to which fibronectin bound in the order of 15% of total protein added). 7 E. coli strains isolated from diarrhoea in young piglets were also tested, and 3 were positive. 2 of these strains showed higher binding than S. aureus Cowan 1 cells. E. coli strains expressing either K99 or K88 antigen were poor binders, comparable to cells of S. aureus strain Wood 46. There was no correlation between cell surface hydrophobicity, as determined by chromatography on Octyl-Sepharose, and the fibronectin-binding property.     

Surface properties of Staphylococcus aureus affecting chemiluminescence response of human phagocytes

To assess the surface properties of Staphylococcus aureus affecting the response of human phagocytes, the effects of the organisms with different surface properties on the chemiluminescence (CL) response of human phagocytes were examined. The magnitude of the phagocytic CL response to hydrophobic strains was significantly greater than that to hydrophilic strains, while no significant difference in the CL response was seen between protein A-deficient strains and their parent strains. The CL response to the hydrophilic organisms prepared from a hydrophobic strain by trypsin treatment decreased significantly. These results suggest that the phagocytic CL response to staphylococci depends on the hydrophobicity of the surface, but not on the presence of protein A. Two protein A-deficient strains which were isolated from protein A-positive strains showed identical hydrophobicity with their parent strains. All of the hydrophilic strains isolated from hydrophobic strains possessed protein A identical to that of their parent strains. Moreover, a hydrophilic strain could be isolated from a protein A-deficient, hydrophobic strain. These results strongly suggest that protein A is not solely responsible for the surface hydrophobicity of S. aureus.     

金黄色葡萄球菌凝集因子A的免疫原性

为研究金黄色葡萄球菌(Staphylococcus aureus)凝集因子A(ClfA)免疫原性及免疫保护作用,应用PCR方法扩增出金黄色葡萄球菌Newman、Wood46和HLJ23-1株的clfa基因并进行序列分析,再将Newman株的clfa基因插入到pQE-30载体上,导入宿主菌Escherichia coli M15(pREP4)并诱导表达和纯化ClfA重组蛋白。用纯化的ClfA免疫小鼠,检测血清中抗体和细胞因子水平,首次免疫后35 d时用金黄色葡萄球菌Wood46、HLJ23-1、Newman株对小鼠攻毒。结果发现:clfa基因序列高度保守;ClfA重组蛋白在E.coli M15中获得表达;在首次免疫后35 d时血清抗体效价和细胞因子浓度与对照组相比,均显著升高(P<0.05);攻毒结果为蛋白免疫组小鼠获得一定的免疫保护。由此表明,ClfA重组蛋白有较好的免疫原性和免疫保护力。     

Chemiluminescence response of human phagocytes against IgG-opsonized staphylococci with and without protein A activities

The effects of immunological IgG binding to Staphylococcus aureus and IgG binding via protein A on the chemiluminescence (CL) response of human phagocytes were examined. The results obtained by enzyme immunoassay showed a clear correlation between the magnitude of the CL response and amount of IgG on protein A-deficient HL-87 strain. Despite no difference in protein A activity between 209P and Cowan I strains, the CL response to IgG-opsonized 209P cells was lower than that to Cowan I cells similarly opsonized. Moreover, the CL response to opsonized HL-87 cells was identical with that of opsonized Cowan I cells, which was a protein A-rich parent strain of the HL-87. The protein A activity of Cowan I cells was significantly decreased when the cells were treated with the Fc fraction of IgG before opsonization, but such a treatment did not change the phagocytic CL response. These results strongly suggest that IgG bound to protein A via its Fc portion has no effect on the phagocytic CL response and that IgG immunologically bound to S. aureus is responsible for the opsonization of the bacteria.     

Chemical characterization of a new surface antigenic polysaccharide from a mutant of Staphylococcus aureus

A mutant of Staphylococcus aureus H was isolated by virtue of its inability to agglutinate with antibodies against teichoic acid of S. aureus. Immunological studies revealed that the mutant, S. aureus T, possessed a new surface antigen in addition to having the antigenic determinant of the wild-type strain, the ribitol teichoic acid. The presence of this additional surface component rendered strain T resistant to staphylococcal typing phages, presumably by masking the phage-receptor sites. The polymer was separated from teichoic acid by chromatography on diethylaminoethyl cellulose and was shown to be composed of two amino sugars, N-acetyl-d-fucosamine and N-acetyl-d-mannosamin uronic acid.     

Bovine whey proteins inhibit the interaction of Staphylococcus aureus and bacteriophage K

AIMS: To understand the potential use of bacteriophage K to treat bovine Staphylococcus aureus mastitis, we studied the role of whey proteins in the inhibition of the phage-pathogen interaction in vitro. METHODS AND RESULTS: The interaction of bacteriophage K and S. aureus strain Newbould 305 was studied in raw bovine whey and serum. Incubation of S. aureus with phage in whey showed that the bacteria are more resistant to phage lysis when grown in whey and also bovine serum. Whey collected from 23 animals showed a wide variation in the level of phage-binding inhibition. The role of the protein component of milk whey in this inhibition was established; treatment of the whey by heat, proteases and ultrafiltration removed the inhibitory activity. Brief exposure of S. aureus cells to whey, followed by resuspension in broth, also reduced phage binding. Microscopy showed the adhesion of extracellular material to the S. aureus cell surface following exposure to whey. Chromatographic fractionation of the whey demonstrated that the inhibitory proteins were present in the high molecular weight fraction. CONCLUSIONS: The adsorption of whey proteins to the S. aureus cell surface appeared to inhibit phage attachment and thereby hindered lysis. The inhibitory whey proteins are of high molecular weight in their native form and may sterically block phage attachment sites on the cell surface. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings have implications for any future use of phage therapy in the treatment of mastitis, and other diseases, caused by S. aureus. This pathogen is predicted to be much more resistant to phage treatment in vivo than would be expected from in vitro broth culture experiments.     

Electrophoretic and immunochemical characteristics of proteins of Rickettsia prowazekii strains of various virulence

PAAG-electrophoresis of the isogenic pair of Rickettsia prowazekii strains E and Evir lysates demonstrate the similarity in polypeptide tracks. The different electrophoretic mobility of the Mr 30 Kd protein from these strains as compared with the mobility of analogous protein from the standard virulent Breinl strain is registered. In immunoblot experiments the specific rabbit antiserums obtained on the 30th day of infection with the Breinl, E or Evir strains demonstrate the presence of the different main antigens 60 Kd or 70 Kd. The difference evidently reflects the specificity of development of two forms of infection by the strains having different virulence. The surface tris-soluble antigens of Rickettsia prowazekii have the similar polypeptide contents and immunochemical properties. The main component of tris-soluble antigens Mr 130 Kd protein is not strain specific having the common thermolabile epitope.     

Eosinophil cationic protein high-affinity binding to bacteria-wall lipopolysaccharides and peptidoglycans

The eosinophil cationic protein (ECP) is an eosinophil-secreted RNase involved in the immune host defense, with a cytotoxic activity against a wide range of pathogens. The protein displays antimicrobial activity against both Gram-negative and Gram-positive strains. The protein can destabilize lipid bilayers, although the action at the membrane level can only partially account for its bactericidal activity. We have now shown that ECP can bind with high affinity to the bacteria-wall components. We have analyzed its specific association to lipopolysaccharides (LPSs), its lipid A component, and peptidoglycans (PGNs). ECP high-affinity binding capacity to LPSs and lipid A has been analyzed by a fluorescent displacement assay, and the corresponding dissociation constants were calculated using the protein labeled with a fluorophor. The protein also binds in vivo to bacteria cells. Ultrastructural analysis of cell bacteria wall and morphology have been visualized by scanning and transmission electron microscopy in both Escherichia coli and Staphylococcus aureus strains. The protein damages the bacteria surface and induces the cell population aggregation on E. coli cultures. Although both bacteria strain cells retain their shape and no cell lysis is patent, the protein can induce in E. coli the outer membrane detachment. ECP also activates the cytoplasmic membrane depolarization in both strains. Moreover, the depolarization activity on E. coli does not require any pretreatment to overcome the outer membrane barrier. The protein binding to the bacteria-wall surface would represent a first encounter step key in its antimicrobial mechanism of action.     

Identification of a carotenoid-binding protein in the cytoplasmic membrane from the heterotrophic cyanobacterium Synechocystis sp. strain PCC6714.

We isolated a carotenoid-binding protein from the cytoplasmic membrane of the cyanobacterium Synechocystis sp. strain PCC6714. The polypeptide demonstrated a characteristic mobility shift when electrophoresed in lithium dodecyl sulfate-polyacrylamide gels. The protein migrated with an apparent molecular mass of 35 kilodaltons when solubilized at 0 degrees C, but after solubilization at 70 degrees C, the protein migrated as a 45-kilodalton species. The carotenoid-binding protein accumulated only in autotrophically grown cells; cytoplasmic membranes prepared from photoheterotrophically grown cells lacked this component.     

Mycobacterium bovis BCG induces high mobility group box 1 protein release from monocytic cells

High mobility group box 1 protein (HMGB-1), a nuclear protein is a critical cytokine that mediates the response to infection, injury and inflammation. The aim of our study was to elaborate a reliable in vitro model to investigate whether Mycobacterium bovis BCG is able to induce HMGB-1 secretion from the monocytic U-937 cells. Western blot technique was applied for the detection of HMGB-1 from supernatants of cells, following induction with LPS, Staphylococcus aureus, and Mycobacterium bovis BCG. HMGB-1 was subjected to MALDI-TOF mass and PSD analysis. Quantitation of the secreted HMGB-1 was performed by ELISA. The BCG strain induced higher amounts of secreted HMGB-1 than LPS or Staphylococcus aureus. The translocation of the HMGB-1 to the cytoplasm following infection of cells with BCG was demonstrated by immunofluorescence examinations. CONCLUSION: Our pilot experiments draw attention the to HMGB-1-inducing ability of Mycobacterium bovis. Assessment of the pathophysiological role of this late cytokine in mycobacterial infections demands further in vitro and in vivo examinations.     

Synthesis and Evaluation of a Conjugate Vaccine Composed of Staphylococcus aureus Poly-N-Acetyl-Glucosamine and Clumping Factor A

The increasing frequency, severity and antimicrobial resistance of Staphylococcus aureus infections has made the development of immunotherapies against this pathogen more urgent than ever. Previous immunization attempts using monovalent antigens resulted in at best partial levels of protection against S. aureus infection. We therefore reasoned that synthesizing a bivalent conjugate vaccine composed of two widely expressed antigens of S. aureus would result in additive/synergetic activities by antibodies to each vaccine component and/or in increased strain coverage. For this we used reductive amination, to covalently link the S. aureus antigens clumping factor A (ClfA) and deacetylated poly-N-β-(1-6)-acetyl-glucosamine (dPNAG). Mice immunized with 1, 5 or 10 μg of the dPNAG-ClfA conjugate responded in a dose-dependent manner with IgG to dPNAG and ClfA, whereas mice immunized with a mixture of ClfA and dPNAG developed significantly lower antibody titers to ClfA and no antibodies to PNAG. The dPNAG-ClfA vaccine was also highly immunogenic in rabbits, rhesus monkeys and a goat. Moreover, affinity-purified, antibodies to ClfA from dPNAG-ClfA immune serum blocked the binding of three S. aureus strains to immobilized fibrinogen. In an opsonophagocytic assay (OPKA) goat antibodies to dPNAG-ClfA vaccine, in the presence of complement and polymorphonuclear cells, killed S. aureus Newman and, to a lower extent, S. aureus Newman ΔclfA. A PNAG-negative isogenic mutant was not killed. Moreover, PNAG antigen fully inhibited the killing of S. aureus Newman by antisera to dPNAG-ClfA vaccine. Finally, mice passively vaccinated with goat antisera to dPNAG-ClfA or dPNAG-diphtheria toxoid conjugate had comparable levels of reductions of bacteria in the blood 2 h after infection with three different S. aureus strains as compared to mice given normal goat serum. In conclusion, ClfA is an immunogenic carrier protein that elicited anti-adhesive antibodies that fail to augment the OPK and protective activities of antibodies to the PNAG cell surface polysaccharide.     

Invasion of bone cells by Staphylococcus epidermidis

Bone implants infected with Staphylococcus epidermidis often require surgical intervention because of the failure of antibiotic treatment. The reasons why such infections are resistant to therapy are poorly understood. We have previously reported that another bacterium, Staphylococcus aureus, can invade bone cells and thereby evade antimicrobial therapy. In this study we have investigated the hypothesis that S. epidermidis can also invade bone cells and may therefore explain the difficulties of treating infections with this organism. We found that S. epidermidis was capable of invading bone cells but that there were significant strain dependent differences in this capacity. A recombinant protein encompassing the D1-D4 repeat region of S. aureus fibronectin-binding protein B completely inhibited internalization of S. aureus but failed to block internalization of S. epidermidis. Similarly a blocking antibody to alpha5beta1 integrin inhibited internalization of S. aureus by bone cells but had no effect on the uptake of S. epidermidis. Therefore unlike S. aureus, S. epidermidis does not gain entrance into bone cells through a fibronectin bridge between the alpha5beta1 integrin and a bacterial adhesin.     

Inhibitory effects of D-amino acids on Staphylococcus aureus biofilm development

Biofilms are communities of cells held together by a self-produced extracellular matrix typically consisting of protein, exopolysaccharide, and often DNA. A natural signal for biofilm disassembly in Bacillus subtilis is certain D-amino acids, which are incorporated into the peptidoglycan and trigger the release of the protein component of the matrix. D-amino acids also prevent biofilm formation by the related Gram-positive bacterium Staphylococcus aureus. Here we employed fluorescence microscopy and confocal laser scanning microscopy to investigate how D-amino acids prevent biofilm formation by S. aureus. We report that biofilm formation takes place in two stages, initial attachment to surfaces, resulting in small foci, and the subsequent growth of the foci into large aggregates. D-amino acids did not prevent the initial surface attachment of cells but blocked the subsequent growth of the foci into larger assemblies of cells. Using protein- and polysaccharide-specific stains, we have shown that D-amino acids inhibited the accumulation of the protein component of the matrix but had little effect on exopolysaccharide production and localization within the biofilm. We conclude that D-amino acids act in an analogous manner to prevent biofilm development in B. subtilis and S. aureus. Finally, to investigate the potential utility of D-amino acids in preventing device-related infections, we have shown that surfaces impregnated with D-amino acids were effective in preventing biofilm growth.     

Multiple mechanisms for the activation of human platelet aggregation by Staphylococcus aureus: roles for the clumping factors ClfA and ClfB,the serine-aspartate repeat protein SdrE and protein A

The ability of Staphylococcus aureus cells to induce platelet aggregation has long been recognized. However, despite several attempts to identify the mechanisms involved in this interaction, the nature of the bacterial receptors required remains poorly understood. Using genetic manipulation, this study for the first time provides clear evidence that several S. aureus surface proteins participate in the inter-action with platelets. Mutants of S. aureus strain Newman lacking one or more surface proteins were tested for their ability to stimulate platelet aggregation. This approach was complemented by the expression of a number of candidate proteins in the non-aggregating Gram-positive bacterium Lacto-coccus lactis. S. aureus-induced aggregation was monophasic and was dependent on the platelet receptor GPIIb/IIIa. The fibrinogen-binding proteins, clumping factors A and B and the serine-aspartate repeat protein SdrE could each induce aggregation when expressed in L. lactis. Although protein A expressed in L. lactis was not capable of inducing aggregation independently, it enhanced the aggregation response when expressed on the surface of S. aureus. Thus, S. aureus has multiple mechanisms for stimulating platelet aggregation. Such functional redundancy suggests that this phenomenon may be important in the pathogenesis of invasive diseases such as infective endocarditis.     

Identification of the autoagglutination factor of Hms- cells of the plague agent

A search for cellular components responsible for autoagglutination (AA) in broth and salt solutions of Hms- cells of the plague agent Yersinia pestis was performed. The AA- mutants were obtained using vaccine strain Y. pestis EV76 derivative containing one species-specific plasmid pYP. The mutants were shown to differ from the parent strain by the decreased surface hydrophobicity, insensitivity to plague diagnostic L-413c bacteriophage and negative haemagglutination reaction with antibodies to F1 capsular substance of the plague agent. The mutants did not differ from the parent strain by electrophoretic mobility and immunochemical activity of LPS but were characterized by the absence of a 17 kDa protein on the cell surface. The AA+ cells that lost this protein after weak alkali extraction were less hydrophobic and failed to express AA in 0.5 M ammonium sulfate. After the extraction, the cells lost the ability to neutralize L-413c and to react with the anti-F1 antibodies, while both activities as well as 17 kDa protein were detected in the extracts. Thus, the 17 kDa protein is suggested to be a hydrophobic surface antigen which acts as a receptor of the L-413c bacteriophage and represents an AA factor of Hms- cells of Y. pestis.     

Identification and molecular characterization of a putative regulatory locus that affects autolysis in Staphylococcus aureus.

Previously in our laboratory, a PCR-based strategy was used to isolate potential sensor gene fragments from the Staphyloccus aureus genome. One DNA fragment was isolated that shared strong sequence similarity to genes encoding bacterial sensor proteins, indicating that it originated from within a potential staphylococcal sensor protein gene. In this study, the DNA surrounding the PCR product origin was cloned and sequenced. This analysis revealed the presence of two genes, termed lytS and lytR, whose deduced amino acid sequences were similar to those of members of the two-component regulatory system family of proteins. S. aureus cells containing an insertional disruption of lytS exhibited a marked propensity to form aggregates in liquid culture, suggesting that alterations in cell surface components exist in this strain. Transmission electron microscopic examination of these cells revealed that the cell surface was rough and diffuse and that a large proportion of the cell population had lysed. The lytS mutant also exhibited increased autolysis and an altered level of murein hydrolase activity produced compared with the parental strain, NCTC 8325-4. These data suggest that the lytS and lytR gene products control the rate of autolysis in S. aureus by affecting the intrinsic murein hydrolase activity associated with the cell.     

金黄色葡萄球菌AgrA/C双组分信号转导模型的构建与功能评价

【背景】抗生素的无序使用加剧了耐药性金黄色葡萄球菌超级菌株的出现,由其引发的感染已成为最难解决的感染性疾患。在生物体系外构建AgrA/C双组分系统的跨膜信号转导过程,对解决金黄色葡萄球菌的耐药性问题和发现新型抗菌药物具有重要的研究意义。【目的】人工模拟构建金黄色葡萄球菌AgrA/C双组分信号转导模型,为生物体外研究金黄色葡萄球菌双组分信号转导的机制及以其为靶点的药物筛选提供新途径。【方法】在大肠杆菌宿主细胞中大量表达AgrA和Agr C蛋白,利用亲和层析和分子筛凝胶层析对其进行分离纯化,利用非放射性凝胶阻滞实验(EMSA)检测AgrA蛋白活性,并检测Agr C激酶活性;进而利用脂质体介导法在体外组装AgrA/C双组分信号转导模型,应用EMSA方法进行评价。【结果】分离纯化得到AgrA和Agr C蛋白,二者纯度均达到90%以上,均具有活性。在生物体系外构建了金黄色葡萄球菌AgrA/C双组分信号转导模型,该系统可增强AgrA对DNA的延滞作用,具有信号传递功能。【结论】初步构建AgrA/C双组分信号转导模型,该模型具有信号传递能力,有望作为针对金黄色葡萄球菌开发新型抗菌药物的筛选平台。     

Identification and partial characterization of two major proteins of Mr 47,000 synthesized by bovine retinal endothelial cells in culture.

Biosynthetic experiments with cultured bovine retinal endothelial cells have identified a glycoprotein of Mr 47,000 (Gp47) as a major component secreted into the medium. Gp47 is a non-collagenous glycoprotein with a pI of 4.6-5.5, which does not bind to either gelatin-Sepharose or heparin-Sepharose but is retained by concanavalin A-Sepharose. The Mr of this species decreases to approx. 42,000 in the presence of tunicamycin, indicating that it contains asparagine-linked oligosaccharides. A second protein of Mr 47,000 (P47) is present in the cell layer/matrix of these cultured cells. The electrophoretic mobility of P47 remains unaltered when synthesized in the presence of tunicamycin. Peptide-mapping experiments using N-chlorosuccinimide and Staphylococcus aureus V8 proteinase demonstrate that Gp47 and P47 are distinct proteins, and are not related to colligin, a membrane-bound collagen-receptor protein of similar size, or to SPARC, a major secreted product of parietal endodermal cells and sparse cultures of aortic endothelial cells.     

Effect of milk proteins on adhesion of bacteria to stainless steel surfaces.

Stainless steel coupons were treated with skim milk and subsequently challenged with individual bacterial suspensions of Staphylococcus aureus, Pseudomonas fragi, Escherichia coli, Listeria monocytogenes, and Serratia marcescens. The numbers of attached bacteria were determined by direct epifluorescence microscopy and compared with the attachment levels on clean stainless steel with two different surface finishes. Skim milk was found to reduce adhesion of S. aureus, L. monocytogenes, and S. marcescens. P. fragi and E. coli attached in very small numbers to the clear surfaces, making the effect of any adsorbed protein layer difficult to assess. Individual milk proteins alpha-casein, beta-casein, kappa-casein, and alpha-lactalbumin were also found to reduce the adhesion of S. aureus and L. monocytogenes. The adhesion of bacteria to samples treated with milk dilutions up to 0.001% was investigated. X-ray photoelectron spectroscopy was used to determine the proportion of nitrogen in the adsorbed films. Attached bacterial numbers were inversely related to the relative atomic percentage of nitrogen on the surface. A comparison of two types of stainless steel surface, a 2B and a no. 8 mirror finish, indicated that the difference in these levels of surface roughness did not greatly affect bacterial attachment, and reduction in adhesion to a milk-treated surface was still observed. Cross-linking of adsorbed proteins partially reversed the inhibition of bacterial attachment, indicating that protein chain mobility and steric exclusion may be important in this phenomenon.