Isolation of a serologically different compact-colony-forming active substance from strains of Staphylococcus aureus

To observe the possible serological heterogeneity of compact-colony-forming active substance (CCFAS), heat-killed vaccines were prepared by two strains of Staphylococcus aureus, strains SMU 1-46 and SMU 7931, cultured in 0.03 M trishydrochloride-buffered brain heart infusion, pH 8.4. After immunization with the vaccine in rabbits, antibody responses were observed during a period of six weeks after the immunization either by homologous and heterologous organisms using alkaline serum-soft agar technique. The results showed that remarkable antibody production was shown only against homologous strain, but not against heterologous strain. The antibodies were absorbed out only with highly purified preparation of CCFAS extracted from homologous strain and not with heterologous CCFAS. Differences of the major chemical composition of the substances showed that highly purified CCFAS extracted from strain SMU 7931 contained 2.84 and 2.04 times higher amounts of galactose and 2-amino-2-deoxy-D-galacturonic acid than those of CCFAS obtained from strain SMU 1-46.     

Localization of bacteriophage receptor, clumping factor, and protein A on the cell surface of Staphylococcus aureus.

The surface of several laboratory strains of Staphylococcus aureus were observed with a scanning electron microscope, and the presence of two morphologically characteristic structures--a ridge separating cell surface into old and new surfaces and a concentric circular structure--are described. These two structures seemed to be present universally on the surfaces of cells of the genus Staphylococcus. The removal of the circular structures by a mild treatment of the cell with trichloroacetic acid suggested that this structure seemed to represent circularly arranged teichoic acid. With experiments using morphologically recognizable markers among three of the cell wall components, clumping factor, phage receptor, and protein A, the clumping factor was proven to be specifically localized on the old surface; and more phage receptors were detected on the old surface than on the new surface, but protein A was present all over the cell surface. This indicated that the clumping factor and most of the phage receptors appeared on the cell wall surface in a late stage of the cell growth cycle, but protein A was present in an early stage of the growth. The idea of aging of the cell wall is discussed.     

Binding of staphylococcal cell surface polysaccharide to human fibrinogen

The interaction between the binding site of a polysaccharide (called compact colony forming active substance (CCFAS)), obtained from the cell surface of a strain of Staphylococcus, and human fibrinogen (HF) was investigated. The CCFAS was found to bind specifically to both the B beta and gamma chains of HF at pH 7.0 and 8.0, and the A alpha chain at pH 5.0. The binding of CCFAS with fibrinogen fragments obtained by digestion with plasmin were also investigated. Fragments with Mr of 55,000, 24,000, and 19,000 were the major bands precipitated by CCFAS at pH 7.0 and 8.0. Fragments with Mr of 85,000 and 75,000 bound to CCFAS at pH 5.0. Binding of CCFAS (7 micrograms) with fibrinogen could be inhibited by 1.2 micrograms of B beta chain and 1.5 micrograms gamma chain at alkaline pH or 6.2 micrograms of the A alpha chain at pH 5.0. CCFAS was, therefore, assumed to be specifically bonded with HF molecules, in the alkaline range at least, resulting in compact colony forming activity in serum soft agar and paracoagulation.     

Function of cell wall teichoic acid in thermally injured Staphylococcus aureus.

Thermally injured cells of Staphylococcus aureus lack the ability to grow on tryptic soy agar containing 7.5% NaCl. This injury phenomenon was examined in three strains of S. aureus: MF-31; H (Str); and, isolated from H (Str), 52A5, a mutant which lacks teichoic acid in the cell wall. Temperatures for sublethal heat treatment were selected to produce maximum injury with minimum death for each strain. Examination of isolated cell walls showed that magnesium was lost from the wall during heating, and that the degree of cell injury was accentuated when magnesium ions were either removed from or made unavailable to the cell. S. aureus 52A5 was more heat sensitive than its parent strain. Cells containing higher levels of wall teichoic acid generally showed less injury than normal cells. Cells with the weaker cation-binding polymer, teichuronic acid, in the cell wall generally showed greater injury. These data suggest that cell wall teichoic acid of S. aureus aids in the survival of the cell by the maintenance of an accessible surface pool of magnesium.     

Immunological characterization of an exopolysaccharide from the Staphylococcus aureus strain Smith diffuse

Exopolysaccharides (EXPs) of Staphylococcus aureus are associated with virulence in animal models. An EXP from the S. aureus strain Smith diffuse was previously detected in 64.3% of S. aureus clinical isolates. EXP was isolated from culture supernatants of this strain after DNAase, RNAase, phosphodiesterase I and lysostaphin treatment, and was further purified by cation-exchange and molecular-sieve chromatography. Isoelectric focusing revealed a pI of 3.6 for the EXP while the pI of teichoic acid was less than 2.7. Crossed immunoelectrophoresis with homologous Smith diffuse antisera indicated that the EXP contained two immunological components. A major precipitin line persisted after the antisera had been absorbed with the non-EXP-producing variant strain, Smith compact, while the second component was removed. Tandem immunoelectrophoresis also demonstrated that the EXP was distinct from teichoic acid. The EXP contained 2-amino-2-deoxyglucuronic acid, glucose, mannose and galactose. No fatty acids or nucleic acids were present and total protein content was less than 2%. Teichoic acid could not be demonstrated in the EXP, thus further substantiating the immunological studies. S. aureus EXP isolated by the present method can be used for further serological and virulence studies.     

Outermost-cell-surface changes in an encapsulated strain of Staphylococcus aureus after preservation by freeze-drying

The effects of drying time during freeze-drying on the outermost cell surface of an encapsulated strain of Staphylococcus aureus S-7 (Smith, diffuse) were investigated, with special attention paid to capsule and slime production. To quantify capsule and slime production, capsule antigen production and cellular characteristics such as growth type in serum-soft agar, cell volume index, and clumping factor reaction were examined. After freeze-drying the colonial morphology of strain S-7 was altered from a diffuse to a compact type in serum-soft agar. In accordance with these changes, the titer of the clumping factor reaction increased while the cell volume index, capsule and slime production, and capsule antigen production were markedly decreased in parallel with the period of freeze-drying. The ability of the strain to adhere to collagen, fibrinogen, and soybean lectin was also compared before and after freeze-drying. Fibrinogen levels slightly increased when 10% skim milk and 2% honey were used as cryoprotective agents and showed a remarkable increase when 0.05 M phosphate buffer was used as a control. Also, the ability of strain S-7 to adhere to soybean lectin declined, whereas no changes were observed for collagen under any conditions. Strain S-7 was phage nontypable before freeze-drying but the number of typable cells increased after freeze-drying; phage-typable cells reacted to phage 52 alone after 5 h of freeze-drying, but additional cells also proved to be phage typable to phage 42E after 10 h. Electron micrographs indicated that strain S-7, an encapsulated strain, was converted to an unencapsulated state after freeze-drying.(ABSTRACT TRUNCATED AT 250 WORDS)     

Outermost-cell-surface changes in an encapsulated strain of Staphylococcus aureus after preservation by freeze-drying.

The effects of drying time during freeze-drying on the outermost cell surface of an encapsulated strain of Staphylococcus aureus S-7 (Smith, diffuse) were investigated, with special attention paid to capsule and slime production. To quantify capsule and slime production, capsule antigen production and cellular characteristics such as growth type in serum-soft agar, cell volume index, and clumping factor reaction were examined. After freeze-drying the colonial morphology of strain S-7 was altered from a diffuse to a compact type in serum-soft agar. In accordance with these changes, the titer of the clumping factor reaction increased while the cell volume index, capsule and slime production, and capsule antigen production were markedly decreased in parallel with the period of freeze-drying. The ability of the strain to adhere to collagen, fibrinogen, and soybean lectin was also compared before and after freeze-drying. Fibrinogen levels slightly increased when 10% skim milk and 2% honey were used as cryoprotective agents and showed a remarkable increase when 0.05 M phosphate buffer was used as a control. Also, the ability of strain S-7 to adhere to soybean lectin declined, whereas no changes were observed for collagen under any conditions. Strain S-7 was phage nontypable before freeze-drying but the number of typable cells increased after freeze-drying; phage-typable cells reacted to phage 52 alone after 5 h of freeze-drying, but additional cells also proved to be phage typable to phage 42E after 10 h. Electron micrographs indicated that strain S-7, an encapsulated strain, was converted to an unencapsulated state after freeze-drying.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Properties of a Novel Pleiotropic Bacteriophage-Resistant Mutant of Staphylococcus aureus H

A phage-resistant mutant of Staphylococcus aureus H (Sm(R)), S. aureus 52A5, was previously shown to lack polymeric teichoic acid. This paper characterizes other phenotypic differences between the strains. In broth cultures the mutant cells grew more slowly, were larger, and formed much larger clumps than the parent strain. The clumps of cells appeared to be covalently linked and could only be separated by mild sonic energy-a process which yielded viable cells. Mutant and parent cells autolyzed at equal rates, whereas isolated cell walls of the mutant strain autolyzed faster than the wild type. Nevertheless, the specific activity of the autolytic enzyme in the wild type soluble fraction was much higher than in the mutant. In contrast to the parent, strain 52A5 failed to accumulate nucleotide-bound murein precursors when treated with penicillin. Mutant strains with these characteristics were repeatedly isolated both spontaneously and by chemical mutagenesis. Strain 52A5 was shown to be fully revertible. Thus, it appears to be a pleiotropic mutation, and the possible nature of the defect which causes these varied effects is discussed.     

Nature and Origins of Phosphorus Compounds in Isolated Cell Walls of Staphylococcus aureus

Preparations of purified cell walls from Staphylococcus aureus were shown to contain small amounts of phospholipid and glycerol teichoic acid. Since these are components of the cell membrane, it is probable that the wall itself contains no lipid, but does retain fragments of membrane because of physical connections between wall and membrane. In walls of S. aureus strain 52A5, which completely lacks ribitol teichoic acid, the only phosphorylated compound identified as a genuine wall component was a phosphorylated derivative of murein that gave rise to muramic acid phosphate on acid hydrolysis. Muramic acid phosphate was also identified in hydrolysates of walls from S. aureus H and strain 52A2.     

Staphylococcus aureus adherence to influenza A virus-infected and control cell cultures: evidence for multiple adhesins

During major epidemics with influenza, there is an increased number of pneumonias due to Staphylococcus aureus with a subsequent high mortality rate. We have postulated that influenza A virus infection of host cells promotes the adherence of S. aureus ultimately resulting in bacterial superinfection. In the present study we compared the adherence of seven strains of 3H-labeled S. aureus to Madin-Darby canine kidney (MDCK) cell monolayers, uninfected and infected with influenza A/FM/1/47 virus. Test strains included: Cowan I; a Cowan I protein A-deficient mutant (PA-); EMS, a protein A and clumping factor-deficient mutant; HSmR; 52A5, a teichoic acid-deficient mutant of HSmR; M, an encapsulated strain; and, No. 1071, a clinical isolate. By radioassay, six of the seven strains demonstrated significantly enhanced adherence to virus-infected cell monolayers compared to uninfected controls; only the M strain was adherence negative. Surface hydrophobicity of the staphylococci did not correlate with their ability to adhere. Four strains of labeled staphylococci (Cowan I, PA-, EMS, and No. 1071), untreated or treated with 2.5% trypsin, 1.25% protease, or by autoclaving, were tested in the radioassay. Protease treatment, which was more effective than trypsin treatment, reduced adherence of all four test strains by 74-96%. Results of heat treatment suggested the presence of both thermolabile and thermostable adhesins. Staphylococcal thermal extracts, profiled by anion-exchange HPLC, were used to pretreat monolayers in a blocking radioassay. Adherence was decreased to control cells (9-78%) and to virus-infected cells (56-90%). The data suggest that multiple distinct surface proteins mediate the binding of S. aureus to uninfected and influenza A virus-infected cells.     

Defect in biosynthesis of the linkage unit between peptidoglycan and teichoic acid in a bacteriophage-resistant mutant of Staphylococcus aureus.

The biosynthesis of the linkage region between peptidoglycan and the ribitol teichoic acid was investigated in the bacteriophage-resistant, teichoic acid-less mutant Staphylococcus aureus 52A5 (Chatterjee et al., J. Bacteriol. 100:846--853, 1969). Membrane preparations of this strain were found to be incapable of forming the first intermediate of the biosynthetic pathway, namely, the transfer of N-acetyl-D-glucosamine (GlcNAc) from UDP-GlcNAc to the acceptor molecule, which presumbably is undecaprenol phosphate (R. Bracha and L. Glaser, Biochem. Biophys. Res. Commun. 72:1091--1098, 1976). The addition of heat-inactivated membrane preparations of S. aureus 52A2 (which normally has ribitol teichoic acid) that had been preincubated with UDP-GlcNAc to membranes of strain 52A5 enabled the synthesis of teichoic acid. These data suggest that the mutational defect in the teichoic acid-less organism is in the synthesis of the first compound of the linkage unit, and this is apparently the reason for its absence in the cell walls.     

Immunochemical studies of Staphylococcus aureus Oeding-Haukenes antigen a5: a phosphorus-containing polysaccharide

Antigen a5 was isolated from strain 830 of Staphylococcus aureus by autolysis in phosphate buffer followed by alcohol precipitation. Purification was principally achieved by affinity chromatography on wheat germ agglutinin ultrogel and on anti-S. aureus teichoic acid immunosorbent. The a5 antigen was weakly immunogenic in rabbits. Chemical analysis showed that a5 is a teichoic acid composed of ribitol phosphate, N-acetylglucosamine and alanine. It has similar physico-chemical properties to the wall beta-N-acetylglucosamine ribitol teichoic acid of S. aureus but is serologically distinct.     

Isolation and Characterization of Teichoic Acid-Like Substance as an Adhesin of Staphylococcus aureus to HeLa Cells

A cell wall component that bound to HeLa cells (HeLa cell-binding CWC) was isolated from a clinical isolate of Staphylococcus aureus. The HeLa cell-binding CWC was resistant to heat (100 C, 1 hr) and proteases, did not stain with Coomassie Brilliant Blue R-250 on SDS-PAGE but stained as a broad band with antiserum against the strain on Western blots. These data suggest that the HeLa cell-binding CWC is not a protein, and may be teichoic acid. Purified teichoic acid bound to HeLa cells, whereas fractions without teichoic acid did not. In Western blots, HeLa cell-binding CWC appeared as a broad band of less than 35 kDa, similar to that of purified teichoic acid. These data suggest that the HeLa cell-binding CWC obtained in this study is teichoic acid. Teichoic acid inhibited S. aureus adherence to HeLa cells and bound to the cells time and dose dependently, in a saturable and reversible manner, and therefore appears to be an adhesin of S. aureus to HeLa cells.     

Effect of teichoic acid on resistance to the membrane-lytic agent of Streptococcus zymogenes

Davie, Joseph M. (Indiana University, Bloomington), and Thomas D. Brock. Effect of teichoic acid on resistance to the membrane-lytic agent of Streptococcus zymogenes. J. Bacteriol. 92:1623-1631. 1966.-The resistance of Streptococcus zymogenes to its own lytic agent has been shown to be due to the production of a specific, inhibitory teichoic acid. A survey of streptococcal strains showed that only strains resistant to the lytic agent produced the specific inhibitor. In addition, the inhibitor can be removed from spheroplasts of resistant strains, thereby making them sensitive to the lysin. Throughout the early part of the growth cycle, the inhibitor is associated with the cell and cannot be found in the medium. During late logarithmic phase, however, the inhibitor is released into the medium by the cells, and therefore is a contributing factor to the apparent lability of the lytic agent. The purified, inhibitory teichoic acid contains ribitol, phosphate, glucose, and d-alanine. The alkaline lability of the biological activity of the teichoic acid was correlated with the hydrolysis of the d-alanine. A streptococcal strain which is sensitive to the membrane-lytic agent produced an inactive ribitol teichoic acid which lacks the ester-linked d-alanine, whereas a lysin-resistant mutant of this strain produces a teichoic acid which contains d-alanine and which has inhibitory activity.     

红色诺卡氏菌Nr-8206株培养形态与其有效物质及杂质关系分析

筛选红色诺卡氏菌（Nocardia rubra）Nr-8206株适宜投产的最佳培养形态。将红色诺卡氏菌Nr-8206株复壮,通过涂布、形态学考察筛选典型菌落形态。通过发酵技术获得各种菌落形态菌株的生物量,进一步通过细胞破碎、化学提纯等方法获得细胞壁多糖产物,紫外可见光分光光度法进行有效物质含量测定及杂质的检测。结果表明,红色诺卡氏菌Nr-8206株的最佳菌落形态为菌落直径1.68 mm、橘色、有突起、有褶皱、菌落边缘丝状,编号RY2。进行菌株RY2发酵,其菌体量最多,经破碎、提纯后,其有效物质糖含量及胞壁酸含量均高于其他形态的菌落,并高于出发菌株Nr-8206,且其杂质蛋白质残余量更低,杂质更容易去除。该研究可供生产企业以该菌株作为工作菌株时提供形态选择参考。     

Structural elucidation of the extracellular and cell-wall teichoic acids of Staphylococcus aureus MN8m, a biofilm forming strain

Extracellular teichoic acid, an essential constituent of the biofilm produced by Staphylococcus epidermidis strain RP62A, is also an important constituent of the extracellular matrix of another biofilm producing strain, Staphylococcus aureus MN8m. The structure of the extracellular and cell wall teichoic acids of the latter strain was studied by NMR spectroscopy and capillary electrophoresis-mass spectrometry. Both teichoic acids were found to be a mixture of two polymers, a (1-->5)-linked poly(ribitol phosphate), substituted at the 4-position of ribitol residues with beta-GlcNAc, and a (1-->3)-linked poly(glycerol phosphate), partially substituted with the D-Ala at 2-position of glycerol residue. Such mixture is unusual for S. aureus.     

Cross-linked peptidoglycan mediates lysostaphin binding to the cell wall envelope of Staphylococcus aureus

Staphylococcus simulans bv. staphylolyticus secretes lysostaphin, a bacteriocin that cleaves pentaglycine cross bridges in the cell wall of Staphylococcus aureus. The C-terminal cell wall-targeting domain (CWT) of lysostaphin is required for selective binding of this bacteriocin to S. aureus cells; however, the molecular target for this was unknown. We used purified green fluorescent protein fused to CWT (GFP-CWT) to reveal species-specific association of the reporter with staphylococci. GFP-CWT bound S. aureus cells as well as purified peptidoglycan sacculi. The addition of cross-linked murein, disaccharides linked to interconnected wall peptides, blocked GFP-CWT binding to staphylococci, whereas murein monomers or lysostaphin-solubilized cell wall fragments did not. S. aureus strain Newman variants lacking the capacity for synthesizing polysaccharide capsule (capFO), poly-N-acetylglucosamine (icaAC), lipoprotein (lgt), cell wall-anchored proteins (srtA), or the glycolipid anchor of lipoteichoic acid (ypfP) bound GFP-CWT similar to wild-type staphylococci. A tagO mutant strain, defective in the synthesis of polyribitol wall teichoic acid attached to the cell wall envelope, displayed increased GFP-CWT binding. In contrast, a femAB mutation, reducing both the amount and the length of peptidoglycan cross-linking (monoglycine cross bridges), showed a dramatic reduction in GFP-CWT binding. Thus, the CWT domain of lysostaphin directs the bacteriocin to cross-linked peptidoglycan, which also serves as the substrate for its glycyl-glycine endopeptidase domain.     

Effects of fibronectin on the compact colony formation in staphylococci

Fifty-two unencapsulated strains of Staphylococcus aureus, including strains of Wood 46 and Cowan I, formed compact colonies in fibronectin -soft agar. However, 20 encapsulated strains of Staphylococcus aureus and 50 strains of Staphylococcus epidermidis showed diffuse growth in the medium. The results suggest that another possible cellular factor, other than protein A, is involved in the binding of the cell surface with fibronectin and that it would be one of factors in forming compact colonies in serum-soft agar.     

Inhibition of Fibrinogen Reaction by Polysaccharide of Encapsulated Staphylococcus aureus

Encapsulated and nonencapsulated strains of Staphylococcus aureus which lack coagulase or clumping factor (bound coagulase), or both, were examined for the antigen associated with the fibrinogen-cell clumping reaction. Extracts of the cells were tested for the ability to react with fibrinogen or to inhibit fibrinogen precipitation. Antisera prepared against encapsulated (coagulase-positive, clumping factor-negative) variants, as well as against nonencapsulated wild-type (coagulase-positive, clumping factor-positive) S. aureus strains, contained high titers of clumping-inhibiting antibody. When coagulase-negative, clumping factor-negative mutants were the immunizing agents, antisera contained no demonstrable clumping-inhibiting antibody. Phenol extracts of all coagulase-positive strains tested precipitated fibrinogen, regardless of the ability of cells to clump in the presence of fibrinogen. Polysaccharide extracts of encapsulated, clumping factor-negative strains inhibited this fibrinogen-precipitating activity, whereas similar extracts of nonencapsulated staphylococci did not inhibit the fibrinogen reaction. From these results, it appeared that the coagulase-positive, encapsulated staphylococci which do not clump in fibrinogen solution possess clumping factor, but that their capsular polysaccharide inhibits clumping activity. These findings suggested a closer association of clumping factor and coagulase than is now recognized.