The key role of peptidoglycan in the opsonization of Staphylococcus aureus.

In an effort to determine the staphylococcal cell surface component(s) of importance in opsonization, cell walls (peptidoglycan and teichoic acid) and peptidoglycan were isolated from Staphylococcus aureus strain H grown in [3H]glycine-containing broth. After incubation of the cell walls and peptidoglycan with various opsonic sources, uptake by human polymorphonuclear leukocytes was measured. The opsonic requirements for phagocytosis of cell walls and peptidoglycan were found to be similar to those of intact bacteria. Removal of teichoic acid from the cell wall did not affect opsonization. Likewise, a teichoic acid-deficient mutant strain of S. aureus H was opsonized in a manner similar to that of the parent strain. Immunoglobulin G functioned as the major heat-stable opsonic factor and both the classical and alternative pathways participated in opsonization. Kinetic studies revealed that opsonization of peptidoglycan, as well as C3-C9 consumption by peptidoglycan, proceeded at a slower rate via the alternative pathway (C2-deficient serum) than when the classical pathway was present (normal serum). The ability of peptidoglycan to activate C3-C9 was significantly reduced when normal and C2-deficient sera were preabsorbed with peptidoglycan at 2 degrees C suggesting that antibodies to peptidoglycan may be involved in activation of both the classical and alternative complement pathways. Thus, peptidoglycan appears to be the key cell wall component involved in staphylococcal opsonization, and it is suggested that host response to peptidoglycan, a major cell wall component of most gram-positive bacteria, may be related to the development of "natural immunity" to this group of microorganisms.

Solubilization and electrophoretic analysis of Staphylococcus aureus membrane proteins.

The protein composition of homogeneous Staphylococcus aureus 6538P cytoplasmic membranes was examined under denaturing electrophoretic conditions. A comparative analysis on the effectiveness of a variety of membrane solubilizing agents revealed the membrane protein extracts to be qualitatively similar as determined electrophoretically but different in the quantity of protein released; Zwittergent-314, sodium dodecyl sulfate, Triton X-100, Nonidet P-40, and sodium deoxycholate all proved to be effective solubilizing agents under the conditions examined. Fifty-five to sixty protein components were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis from homogeneous late-exponential phase membranes. The profile was unaffected when phenylmethylsulfonyl fluoride was included during membrane isolation and solubilization. Analysis of the solubilized membrane proteins by two-dimensional gel electrophoresis demonstrated in excess of 100 membrane protein components in a pH gradient between 3.5 to 7.7. The profile consisted of a heterogeneous mixture of mostly acidic components with isoelectric points between pH 4 and 5 and relative molecular weights between 158,000 and 35,000. Periodic acid-Schiff staining following sodium dodecyl sulfate gel electrophoresis revealed six to ten reactive bands with two of these bands also exhibiting a reaction with concanavalin A.

Improvement in the lipid extraction of staphylococcal cells by lysostaphin treatment.

A new method for extracting lipids from Staphylococcus aureus is described in this paper. The extracts of cells treated with lysostaphin are compared with those from untreated cells. The recovery of lipids improved with the enzymatic treatment. Tentative identification of the components of the lipid extracts has been carried out.

Crystallization and properties of staphylococcal leukocidin.

Two components (F and S) of leukocidin were isolated and purified from broth culture of V8 strain of Staphylococcus aureus. There was loss of leukocidin activity during the separation, which was regained by mixing the components. Each component was apparently homogeneous by several criteria. The F and S components were crystallized (by salting out with saturated (NH4)2SO4 at pH 7.0) in the form of square plates and very fine needles, respectively, and the molecular weights of the components were approx. 32 000 and 31 000, respectively. The isoelectric points of the F and S components were pH 9.08 and 9.39, respectively. No detectable half-cystine and free sulfhydryl groups were found in either component. Amino-terminal amino acid analysis of both components showed a single alanine residue. The F and S components of leukocidin acted synergistically on granulocytes from rabbit peripheral blood and a minimum lethal dose of 0.5 ng of each component destroyed all granulocytes at a concentration of 10(6) cells/20 microliter.

Alterations in the composition and bacteriophage-binding properties of walls of Staphylococcus aureus H grown in continuous culture in simplified defined media.

A nutritional mutant of Staphylococcus aureus H has been isolated and grown in media in which the only amino acids are arginine, cysteine, glutamic acid and proline. Walls of the bacteria grown in such media in continuous culture under potassium limitation differ in composition from walls of the bacteria grown in batch culture in rich nutrient broth in that they contain less glycine, the peptidoglycan component is less highly cross-linked and the teichoic acid component contains a reduced proportion of N-acetylglucosaminyl substituents. Walls of the potassium-limited bacteria retain the ability to bind bacteriophage 52a but are more susceptible to the action of lytic peptidases than are wall samples in which the peptidoglycan is more highly cross-linked. Teichoic acid was present in walls of the bacteria grown under phosphate limitation in the defined medium and these walls were also able to absorb bacteriophage 52a.

Crystallization of the delta toxin of Staphylococcus aureus.

Delta toxin is a small cytolytic polypeptide produced and secreted by the organism Staphylococcus aureus and belongs to a family of surface-active toxins that exhibit pronounced effects on a wide variety of cellular membranes. Although this class of proteins has been much studied by a wide variety of physical techniques, no consensus has been reached on their mode of action. Therefore, in order to investigate their role in causing membrane damage, a structural analysis of the delta toxin has been initiated. Crystals of this protein have been grown by dialysis against mixtures of 2-methylpentan-2,4-diol and water. These crystals are relatively insensitive to radiation damage and diffract to high resolution. The results of this study should provide a valuable insight into the cytolytic properties of these molecules.

Preliminary crystallographic data for exfoliative toxin B from Staphylococcus aureus.

The serotype B of exfoliative toxin, isolated from Staphylococcus aureus, strain TC 142, has been crystallized. The monoclinic crystals belong to space group P21, with a = 55.9 A, b = 107.9 A, c = 42.8 A, and beta = 90.9 degrees. The asymmetric unit contains two molecules of molecular weight 30,000.

Staphylococcus aureus tetrapeptide with high chemotactic potency and efficacy for human leukocytes.

A chemotactic tetrapeptide from culture fluids of Staphylococcus aureus was purified to homogeneity by reverse-phase high-pressure liquid chromatography. The peptide comprises equimolar methionine, leucine, isoleucine, and phenylalanine. It inhibited binding of fluoresceinated fMet-Leu-Phe-Lys to human monocytes, which showed that it interacted with the formyl-methionyl peptide receptor and suggested that it was a formyl-methionyl peptide. Based on a comparison of dose-response curves for inhibition of fluoresceinated fMet-Leu-Phe-Lys binding, the relative affinity of the peptide for the receptor was comparable to that of fMet-Leu-Phe-Lys. At optimal concentrations, chemotactic efficacy (percentage of monocytes migrating to the attractant) was 53 +/- 4%, in contrast to 36 +/- 3% for the reference attractant fMet-Leu-Phe. Since approximately 60% of human monocytes have formyl-peptide receptors, the bacterial peptide is capable of attracting all receptor-bearing monocytes.

Interactions between protein-gold complexes and cell surfaces: a method for precise quantitation.

We have developed a rapid and precise electron microscope technique for the quantitation of gold particles in suspension using latex microspheres as a reference (EM latex technique). This technique allowed us to determine the specific absorption of colloidal gold at its absorption maximum (520 nm) and the average number of ligands ([125I]IgG) bound to one gold particle. On the basis of these values important binding characteristics of protein-gold complexes to cell surfaces were analyzed in a model system consisting of Staphylococcus aureus with protein A on the cell wall as a specific binding site for IgG-Au. Our observations showed that the number of binding sites represented by one IgG-gold complex depended primarily on the particle size, with one 20-nm IgG-Au corresponding to 15 and one 6-nm IgG-Au to 2.5 binding sites. Hence, the efficiency of binding of IgG-Au complexes increased with decreasing gold particle size. Saturation of binding sites, however, was not achieved. The technique also made possible the determination of the affinity between IgG-Au complexes and the cell surface; this affinity can either be regarded as a characteristic of the ligand IgG or of the gold particle. We observed that the affinity of IgG decreased with the size of the gold particles to which IgG was bound, whereas the affinity of the entire gold particle increased with particle size. The EM latex technique for quantitation of gold particles extends the general use of protein-gold complexes to the quantitative characterization of their interaction with cell surface constituents.

The epidermolytic toxin of Staphylococcus aureus: its failure to bind to cells and its detection in blister fluids of patients with bullous impetigo.

Radioiodinated staphylococcal epidermolytic toxin was found not to bind to erythrocytes, blood leukocytes, trypsin-dispersed keratinocytes, epidermis or whole skin. Moreover the toxin could not be found to bind to murine epithelia by indirect immunofluorescence methods. However, the toxin, measured by radioimmunoassay, could be eluted from the skin of mice undergoing epidermolysis following intraperitoneal injection of toxinogenic Staphylococcus aureus. Furthermore, epidemolysin was measured in the blister fluid of 3 of 5 children with bullous impetigo but not in blister fluid from control patients with other blistering eruptions. Thus epidermolysin has been demonstrated to be present in lesions of the staphylococcal epidermolytic toxin syndrome but its mechanism of action does not involve binding to cells.

A rapid method to quantitate non-labeled RNA species in bacterial cells.

We have developed a rapid method to quantitate specific bacterial RNA species. The method measures the steady-state level of RNA, produces a linear response over more than a 16-fold range of RNA concentration, and can be used for Staphylococcus aureus, Escherichia coli and Bacillus subtilis. In this method, a sheared whole-cell lysate of approx. 7 x 10(8) organisms, prepared as for plasmid screening, is separated on agarose, blotted to a nitrocellulose filter, hybridized with a radiolabeled DNA probe, and autoradiographed. The RNA species are quantitated by counting the radioactive bands on the filter. We have applied the method to the measurement of mRNA induction of the genes encoding beta-lactamase, ermC rRNA methylase, and the alpha-complementing fragment of beta-galactosidase. Upon induction, a ten-fold increase in the mRNA for each gene was observed. The peak mRNA level occurred after 30 min for beta-lactamase, 20 min for beta-galactosidase, and 5 min for the ermC rRNA methylase.

Measurement of staphylococcal protein A and detection of protein A-carrying staphylococcus strains by a competitive ELISA method.

A competitive ELISA for the estimation of staphylococcal protein A is described. Tetanus toxoid is insolubilized on polystyrene and incubated with human antitoxin, which renders the Fc-piece of this antibody freely accessible to protein A. The binding of the latter is demonstrated by its competition with protein A-phosphatase conjugate. The method has been shown to be sensitive and reproducible. It has been used for the detection of protein A in culture supernatants and on living pathogenic staphylococci. The test could therefore be of diagnostic value. Protein A is also present in extracts of food contaminated with enterotoxic staphylococci. It can be eliminated by a simple absorption with insoluble porcine IgG.

Purification of staphylococcal enterotoxins A and E by immunoaffinity chromatography using a murine monoclonal antibody with dual specificity for both of these toxins.

An immunosorbent column was prepared by coupling a murine monoclonal antibody (MAb) with dual specificity for staphylococcal enterotoxins A (SEA) and E (SEE) to Affi-Gel 10. Purification of both SEA and SEE from culture supernatants was carried out with the immunosorbent column using 0.2 M acetic acid containing 0.15 M NaCl as eluant. The yields obtained were approximately 76% for SEA and 70% for SEE. Purified SEA and SEE were found to be immunologically and electrophoretically homogeneous. Immunoaffinity chromatography using a MAb with dual specificity proved to be valuable in the purification of SEA and SEE, not only from the standpoint of percentage recovery, but also because of the degree of purity and the ease of purification.

Improved purification of leukocidin from Staphylococcus aureus and toxin distribution among hospital strains.

For purification of F and S components of the Panton-Valentine leukocidin, an easy three-step method using fast protein liquid chromatography was developed to replace the time-consuming purification procedures previously published. This technique enabled the recovery of 13 and 17 mg of purified F and S, respectively, per litre of culture supernatant. Affinity-purified neutralizing polyclonal antibodies were obtained against each individual component. One hundred and thirty-nine Staphylococcus aureus strains isolated from various clinical samples of hospitalized patients were screened by immunoprecipitation for Panton-Valentine leukocidin (PVL) production. Only 8 strains produced PVL; all originated from injured superficial soft tissues. Contrary to widespread opinion, the 8 PVL-producing strains were never associated with severe infection.

A rapid assay for protein-A in Staph. aureus strains, using immunomagnetic monosized polymer particles.

Monosized magnetic polystyrene beads, Dynabeads M-450, coated with sheep IgG were used in an agglutination test for the detection of cell-surface protein-A, based on Fc-protein-A binding. Ninety-three per cent of the bovine Staphylococcus aureus strains isolated from clinical mastitis expressed surface protein-A. The agglutination test was rapid compared to FITC-labelled IgG binding or dog-serum agar precipitation test for protein-A, and was significantly correlated with these methods. Scanning electron micrographs demonstrated binding of protein-A positive bacteria to the particle surface, thus bridging the beads and forming the agglutination lattice. The microsphere agglutination assay described is a quick, simple and reproducible method for detecting surface protein-A in staphylococci.

A new method for purification of staphylocoagulase by a bovine prothrombin-Sepharose column.

Staphylocoagulase was isolated from a culture filtrate of Staphylococcus aureus, strain st-213, by a two step purification procedure of chromatography on a bovine prothrombin-Sepharose 4B affinity column and gel filtration on Sephadex G-25. The yield of the coagulase activity ranged from 75--83% and the purified preparation gave a single precipitin line in immunodiffusion tests against anti-crude and anti-purified staphylocoagulase sera. However, the final product was shown to contain one major and two minor components by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Chemical analysis of the material indicated that it does not contain any cystine residues and that its NH2-terminal residue is a single isoleucine.

Occurrence of ribitol-containing lipoteichoic acid in Staphylococcus aureus H and its glycosylation.

A ribitol-containing lipoteichoic acid was obtained from the 20,000 x g supernatant fraction of Staphylococcus aureus H by extraction with Triton X-100 followed by fractionation on Sepharose 6B and DEAE-cellulose columns. The purified lipoteichoic acid was composed of phosphate, glycerol, glucose, glucosamine, ribitol, and fatty acids in a molar ratio of 1 : 0.9 : 0.06 : 0.03 : 0.09 : 0.07. Based on the structural analysis of fragments from alkali and HF hydrolysis, the lipoteichoic acid appears to consist of three moieties, namely a ribitol phosphate oligomer, poly(glycerol phosphate) which has about 30 glycerol phosphate units, and beta-glucosyl-beta-glucosyl(1 leads to 1)diacylglycerol. N-Acetylglucosamine was linked to the ribitol residues. The lipoteichoic acid serves as an acceptor of glycosyl moieties from UDP-glucose and UDP-N-acetylglucosamine in the enzyme reaction catalyzed by the membrane preparation. The rate of enzymatic glycosylation was increased by prior treatment of the lipoteichoic acid with N-acetyl-beta-D-glucosaminidase. The glycosylation seems to occur at the ribitol residues of the lipoteichoic acid.

Enzymatic properties of staphylothrombin, an active molecular complex formed between staphylocoagulase and human prothrombin.

Staphylocoagulase with a molecular weight of 64,000 and subspecies ranging in molecular weight from 36,000 to 64,000 were purified by affinity column chromatography on bovine prothrombin-Sepharose 4B from the culture filtrates of the Staphylococcus aureus strains, st-213 and 104. The samples containing all molecular species from both strains had the same NH2-terminal sequence, Ile-Val-Thr-Lys-Asp-Tyr-Ser-Lys-Glu-, implying that the molecular heterogeneity was due to proteolytic degradation to some extent of the COOH-terminal portion during cultivation or purification. Staphylocoagulase (Mr = 64,000) from strain st-213 formed an active complex, "staphylothrombin," with human prothrombin in a molar ratio of 1 to 1.1. Staphylothrombin was unstable at 37 degrees C and some portions of staphylocoagulase in the complex were rapidly degraded into small fragments, together with the fragmentation of prothrombin into prethrombin 1 and prothrombin fragment 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent fluorography for the products of prothrombin activation by staphylocoagulase in the presence of [3H]diisopropylphosphofluoridate (DFP) demonstrated the formation of a DFP-sensitive active site in the prothrombin molecule, and no cleavage of the Arg-Ile bond linking the A and B chains of alpha-thrombin was found. The enzymatic properties including the pH-dependency of the activity, substrate specificity and behavior towards thrombin inhibitors of staphylothrombin differed from those of alpha-thrombin, although the active site titration of staphylothrombin with p-nitrophenyl-p'-guanidinobenzoate showed 0.95 +/- 0.2 mol of active site/mol of enzyme.

Staphylococcal protein A; its preparation and an application to rubella serology.

Good yields of staphylococcal protein A are obtained by growing the staphylococcus Cowan type 1 on cellophane agar. The activity of these preparations in removing immunoglobulin G (IgG) from human serum can be readily measured by the Mancini radial-diffusion technique and the correct in-use dilution determined. Treatment with protein A of sera from women with a history of rubella may help in the identification of those having specific antibody in the IgM and IgA fractions. This relatively simple procedure may have worthwhile application in the diagnosis of rubella.

Coagulase production by strains of Staphylococcus aureus of differing resistance characters: a comparison of two traditional methods with a latex agglutination system detecting both clumping factor and protein A.

Five groups of strains of Staphylococcus aureus (54 in total) were tested by slide and tube coagulase methods with rabbit and human plasma, and the results were compared with a latex test for both clumping factor and Protein A (Staphaurex, Wellcome Foundation). The five groups comprised: epidemic methicillin resistant S aureus (group 1); other methicillin resistant S aureus (group 2); other resistant S aureus (group 3); other S aureus (group 4); and a group of reference strains, not all true S aureus (group 5). Groups 1, 3, and 4 gave consistently strong positive results with the tube test and the latex test and less strong positive results with the slide test. Group 2 strains sometimes gave weak or negative results in slide and latex tests, but tube tests with both types of plasma were strongly positive. Only within group 5 strains were negative results in the tube test found. Group 1 strains showed no diminution in expression of free coagulase or of clumping factor. The latex test was more sensitive than the slide test but less sensitive than the tube test. Doubtful or negative slide test or latex test results, particularly with strains resistant to methicillin, should be checked by a tube coagulase test.