Biomedical subjects

B J Wilkinson

Publications and source records attributed to B J Wilkinson.

At least 19 recordsLinked to original sources

PubMed1992

Autolysis of methicillin-resistant and -susceptible Staphylococcus aureus.

The autolytic activities, including unstimulated, Triton X-100-stimulated, and daptomycin-induced, of various sets of methicillin-resistant and related methicillin-susceptible strains were compared. Faster rates of autolysis were noted in two heterogeneous methicillin-resistant transductants than in their methicillin-susceptible parental recipients, in a heterogeneous resistant strain than in a susceptible derivative created by chemical mutagenesis, and in a homogeneous resistant strain than in a derivative that had decreased methicillin resistance and was created by transposon Tn551 mutagenesis. These results suggest that the presence of the methicillin resistance region, mec, either directly or indirectly through an interaction with other host genes, confers a faster rate of autolysis on strains. Various auxilliary genes are known to affect methicillin resistance expression, and one of these genes, femA, was necessary for the expression of this faster rate of autolysis. These differences in autolytic activities were not observed in isolated crude cell walls retaining autolytic activities, suggesting different modes of regulation of autolysins in intact cells and isolated walls. In contrast, one homogeneous, highly resistant strain, DU4916, had a lower autolytic activity than did derived heterogeneous resistant and susceptible strains created by chemical mutagenesis and a strain that had decreased resistance and was created by transposon mutagenesis. Our observations suggest that methicillin resistance expression is associated with an enhanced rate of autolysis, in heterogeneous resistant strains at least.

Autolysis↗

PubMed1992

Analysis of a peptidoglycan hydrolase gene from Staphylococcus aureus NCTC 8325.

We have investigated the expression of the peptidoglycan hydrolase gene (lytA) of Staphylococcus aureus NCTC 8325. Results from in vitro transcription-translation analysis, maxicell experiments, and Northern (RNA) blot analysis suggest that the lytA gene encodes a polypeptide of M(r) approximately 50,000. Physical mapping data indicate that the lytA gene originated from prophage 11 in the NCTC 8325 strain.

Chromosome Mapping↗

PubMed1992

Proline transport in Staphylococcus aureus: a high-affinity system and a low-affinity system involved in osmoregulation.

L-Proline enhanced the growth of Staphylococcus aureus in high-osmotic-strength medium, i.e., it acted as an osmoprotectant. Study of the kinetics of L-[14C]proline uptake by S. aureus NCTC 8325 revealed high-affinity (Km = 1.7 microM; maximum rate of transport [Vmax] = 1.1 nmol/min/mg [dry weight]) and low-affinity (Km = 132 microM; Vmax = 22 nmol/min/mg [dry weight]) transport systems. Both systems were present in a proline prototrophic variant grown in the absence of proline, although the Vmax of the high-affinity system was three to five times higher than that of the high-affinity system in strain 8325. Both systems were dependent on Na+ for activity, and the high-affinity system was stimulated by lower concentrations of Na+ more than the low-affinity system. The proline transport activity of the low-affinity system was stimulated by increased osmotic strength. The high-affinity system was highly specific for L-proline, whereas the low-affinity system showed a broader substrate specificity. Glycine betaine did not compete with proline for uptake through either system. Inhibitor studies confirmed that proline uptake occurred via Na(+)-dependent systems and suggested the involvement of the proton motive force in creating an Na+ gradient. Hyperosmotic stress (upshock) of growing cultures led to a rapid and large uptake of L-[14C]proline that was not dependent on new protein synthesis. It is suggested that the low-affinity system is involved in adjusting to increased environmental osmolarity and that the high-affinity system may be involved in scavenging low concentrations of proline.

Biological Transport↗

PubMed1992

Staphylococcus aureus osmoregulation: roles for choline, glycine betaine, proline, and taurine.

Choline, glycine betaine, and L-proline enhanced the growth of Staphylococcus aureus at high osmolarity (i.e., they acted as osmoprotectants) on various liquid and solid defined media, while an osmoprotective effect of taurine was shown only for cells growing on high-NaCl solid medium that lacked other osmoprotectants. Potassium pool levels were high, and there was little difference in levels in cells grown at different osmolarities. Glycine betaine accumulated to high levels in osmotically stressed cells, and choline was converted to glycine betaine. Proline and taurine also accumulated in response to osmotic stress but to lower levels than glycine betaine.

Amino Acids↗

PubMed1991

Sequence analysis of a Staphylococcus aureus gene encoding a peptidoglycan hydrolase activity.

The nucleotide (nt) sequence of a 2.0-kb NheI-XbaI DNA fragment containing a peptidoglycan hydrolase-encoding gene, lytA, tentatively identified as encoding an N-acetylmuramyl-L-alanine amidase, from Staphylococcus aureus, was determined. The nt sequencing revealed an open reading frame (ORF) of 1443 bp with a consensus ribosome-binding site located 7 nt upstream from the ATG start codon. The primary amino acid (aa) sequence deduced from the nt sequence revealed a putative protein of 481 aa residues with an Mr of 53815. Comparison of the aa sequence of the ORF with aa sequences in the GenBank data base (version 63, March 1990) revealed that the C-terminal sequence showed significant homology to the C-terminal sequence of lysostaphin from Staphylococcus simulans biovar staphylolyticus.

Amino Acid Sequence↗

PubMed1991

Antibacterial activity and penicillin-binding protein affinity of new cephalosporin derivatives in Staphylococcus aureus and Escherichia coli.

A series of C3-substituted cephalosporins with different levels of oxidation was synthesized and the activities of eight compounds, including affinity for penicillin-binding proteins (PBPs), in Staphylococcus aureus and Escherichia coli were studied. The more hydrophobic cephalosporins, with a thiophene moiety at the C7 position, were more active than C7-methoxyiminoaminothiazole derivatives against S. aureus H (beta-lactam-susceptible), and compounds with a thioether function at C3 were more active than those with a sulphonyl function at this position. In contrast, the most active compounds against E. coli were hydrophilic, zwitterionic C7-methoxyimino-aminothiazole C3-thioether cephalosporins. The physicochemical properties of the most active compounds were in accord with those predicted for rapid outer membrane penetration. However, among the thiophene cephalosporins the dianionic sulphonyl compounds were more active than the monoanionic thioether derivatives. There was a reasonably good correlation between MICs and PBP affinities for S. aureus H. The compounds had most affinity for PBP 1 and least for PBP 4. The two most potent compounds had high affinities for PBPs 1, 2 and 3. The compounds had low affinities for the beta-lactam-insensitive PBP 2a of methicillin-resistant S. aureus strain DU4916-K7. The novel cephalosporins had the highest affinities for PBPs 1a/1b and 3 of E. coli DCO. The major morphological response of E. coli to the compounds was cell elongation.

Bacterial Proteins↗

PubMed1990

Basic features of the staphylococcal heat shock response.

The major heat shock proteins of Staphylococcus aureus had apparent Mrs of 84,000, 76,000, and 60,000, and other prominent proteins of Mrs 66,000, 51,000, 43,000 and 24,000 were also induced. Staphylococcus epidermidis showed a similar response. These proteins were also induced by CdCl2, ethanol and apparently osmotic stress (1.71 M NaCl or 2.25 M sucrose). Most of the proteins sedimented with the membrane fraction, but the Mr 60,000 protein remained in the cytoplasm.

Cesium↗

PubMed1990

Cloning and expression of a Staphylococcus aureus gene encoding a peptidoglycan hydrolase activity.

A gene of Staphylococcus aureus PS47 encoding lytic activity was cloned and expressed in Escherichia coli. Deletion analysis of a recombinant plasmid carrying a 7.4-kilobase-pair fragment (kbp) of S. aureus DNA suggested that the gene was located within a 2.5-kbp EcoRI-XbaI fragment. Analysis of extracts of E. coli harboring recombinant plasmids on denaturing polyacrylamide gels containing purified cell walls of S. aureus showed a clearing zone by a polypeptide of apparent Mr 23,000. The release of dinitrophenylalanine but not reducing groups from purified cell walls by a cell extract of recombinant E. coli suggested that we had cloned an N-acetylmuramyl-L-alanine amidase.

Cell Wall↗

PubMed1990

Neutrophil chemotactic activity of peptidoglycan. A comparison between Staphylococcus aureus and Staphylococcus epidermidis.

The ability of peptidoglycan from Staphylococcus epidermidis and Staphylococcus aureus to generate in human serum chemoattractant for peripheral blood neutrophils was studied. It was shown that PG from the two bacteria was able to induce chemotactic activity in normal human serum. Sonication of PG was required to generate this activity. Very little or no activity was generated in heat-treated or C5-deficient human serum by PG, indicating that PG treatment of serum resulted in generation of chemoattractants by activation of complement. Kinetics studies employing C2-deficient or MgEGTA-chelated serum revealed that S. epidermidis induced chemotactic activity by activating the alternative complement pathway. The alternative complement activation induced by S. epidermidis occurred rapidly and was completed after 15 min, whereas S. aureus activated the alternative pathway much more slowly, with activation reaching a maximum at 60 min. The rapid activation of the alternative complement pathway by S. epidermidis PG may partly explain why this bacterium does not normally cause infections in healthy individuals.

Antigens, Bacterial↗

PubMed1979

Inhibition of alternative complement pathway opsonization by group A streptococcal M protein.

Group A streptococcal M protein is known to be antiphagocytic; however, the exact basis for this property has not been established. In this study the hypothesis was tested that cell wall--associated M protein inhibits phagocytosis by interfering with bacterial opsonization. Two strains of group A Streptococcus pyogenes, CS44 (M+) and CS64 (an M- variant of CS44), were radiolabeled, and after incubation in serum these organisms were exposed to human polymorphonuclear leukocytes. Phagocytosis was quantitated by measurement of leukocyte-associated radioactivity. The contributions of complement and of immunoglobulin to streptococcal opsonization were evaluated by use of serum from a variety of sources. The results revealed that the M- strain was efficiently opsonized via the alternative complement pathway in a relative absence of immunoglobulins. In contrast, the M+ strain was poorly opsonized by all sera tested. These findings suggest that streptococcal M protein in some way prevents bacterial opsonization via the alternative complement pathway and that this property of M protein may partly explain its antiphagocytic characteristic.

Antigens, Bacterial↗

PubMed1979

Cryptic peptidoglycan and the antiphagocytic effect of the Staphylococcus aureus capsule: model for the antiphagocytic effect of bacterial cell surface polymers.

The antiphagocytic effect of the Staphylococcus aureus capsule is known to be related to its ability to interfere with opsonization by normal human serum. In this study, evidence is presented with isolated cell surface components which indicates that the capsule hinders opsonization by masking cell wall peptidoglycan. In contrast to intact, encapsulated S. aureus M cells, peptidoglycan particles isolated from the organism were efficiently opsonized by normal human serum and phagocytized by human polymorphonuclear leukocytes. Cell wall particles retaining capsular material were opsonized less efficiently than peptidoglycan. Studies comparing the opsonic capacities of normal, C2-deficient, and heat-inactivated sera led to the conclusion that both the classical and the alternative complement pathways contribute to the opsonization of peptidoglycan in normal human serum. It appears that the capsule interferes with opsonization via both of these complement pathways. Serum from rabbits immunized with S. aureus M had significant heat-stable opsonic activity for the intact organism and cell walls retaining capsular material, but not for peptidoglycan. A general model is proposed to explain how antiphagocytic cell surface polymers may inhibit bacterial opsonization and thereby impede natural immunity.

Cell Wall↗

PubMed1979

Staphylococcus aureus cell surface: capsule as a barrier to bacteriophage adsorption.

Encapsulated Staphylococcus aureus strains M and Smith diffuse bound phage 84 and 52A much less efficiently than their unencapsulated counterparts, M variant and Smith compact. It is proposed that the capsule acts as a barrier excluding phage from interaction with their receptor (cell wall peptidoglycan and teichoic acid). Inefficient phage adsorption by encapsulated staphylococci may explain, in part, the poor phage typing of such strains.

Adsorption↗

PubMed1979

Chemotaxigenesis by cell surface components of Staphylococcus aureus.

In an attempt to delineate the staphylococcal cell surface components of importance in chemotaxigenesis, we incubated intact Staphylococcus aureus H, crude cell walls, purified cell walls, peptidoglycan, teichoic acid, and cell membranes with human sera. The results reported indicate that both crude cell walls and purified cell walls, as well as peptidoglycan, were potent chemotaxigens. These particles led to the generation in normal human serum of a factor that was chemotactic for human polymorphonuclear leukocytes. Cell wall peptidoglycan and teichoic acid both appeared to play a role in chemotaxigenesis. Kinetic studies employing C2-deficient serum and immunoglobulin-deficient serum revealed that optimal chemotaxigenesis required the presence of an intact classical complement pathway, as well as antibody. Granulocyte aggregometry studies showed that significant levels of C5a were generated in normal serum and that this activated complement component appears to be a major chemotactic factor produced in serum upon interaction with staphylococcal cell wall components.

Cell Membrane↗

PubMed1979

Localization of the third component of complement on the cell wall of encapsulated Staphylococcus aureus M: implications for the mechanism of resistance to phagocytosis.

Encapsulated Staphylococcus aureus strains are more virulent than unencapsulated staphylococci, and this phenomenon has been associated with decreased opsonization of encapsulated bacteria by normal human serum. Peptidoglycan, a major cell wall component of S. aureus, has been shown to promote opsonization of this bacterial species by certain components of the serum complement system. However, when the processes of complement activation and opsonization of encapsulated staphylococci have been studied, it has been found that encapsulated bacteria activate complement efficiently and C3 is bacteria associated, yet these organisms are not efficiently phagocytized by human polymorphonuclear leukocytes. In this study, the hypothesis was tested that opsonically active molecules are not on the true external surface of encapsulated organisms but rather are cell wall associated and thus "hidden" from human polymorphonuclear leukocytes. By using immunoelectronmicroscopy, C3 was found to be localized on the cell wall of an encapsulated S. aureus strain after incubation of the organism in normal human serum. When shrinkage of the capsule was prevented by treatment with anticapsular antibody, the C3 was again shown to be cell wall associated and located beneath the bacterial capsule. These results suggest that encapsulated S. aureus may resist phagocytosis because opsonically active C3 molecules are not exposed at the true external surface of the bacterium.

Animals↗

PubMed1978

Characterization and identification of coagulase-negative, heat-stable deoxyribonuclease-positive staphylococci.

Various characteristics of 13 coagulase-negative, weakly heat-stable deoxyribonuclease-positive staphylococci from human, veterinary and food sources were determined in an effort to identify them. Nine of the isolates were identified as coagulase-negative Staphylococcus aureus (2), Staphylococcus xylosus (2), Staphylococcus simulans (3), Staphylococcus capitis (1) and Staphylococcus sciuri subsp. lentus (1); the other four isolates, from food and veterinary sources, could not be identified as currently accepted or proposed species. Teichoic acid and peptidoglycan compositions were used as key taxonomic characteristics. The determination of heat-stable deoxyribonuclease activity can be useful to detect coagulase-negative S. aureus strains. However, this activity also appears to be present in strains of other staphylococcal species.

Coagulase↗

PubMed1978

Influence of encapsulation on staphylococcal opsonization and phagocytosis by human polymorphonuclear leukocytes.

In previous studies, encapsulated Staphylococcus aureus strains have been shown to resist phagocytosis. In this investigation, the nature of the interference with phagocytosis by human polymorphonuclear leukocytes was examined by studying the opsonization of two pairs of unencapsulated (Smith compact and M variant) and encapsulated (Smith diffuse and M) S. aureus strains. The uptake of [3H]glycine-labeled bacteria by normal leukocytes was quantitatively measured after incubation of bacteria in pooled serum, C2-deficient serum, immunoglobulin-deficient serum, and serum from a rabbit immunized with S. aureus M. The presence of a capsule was found to interfere with opsonization by both the classical and alternative pathways of complement as well as by heat-stable opsonic factors in nonimmune human serum. This interference was significantly greater in the case of the S. aureus M strain than in the case of the Smith diffuse strain. The only effective opsonic source for S. aureus M was immune rabbit serum. It is proposed that encapsulation of S. aureus strains interferes with phagocytosis by preventing effective bacterial opsonization.

Antibodies, Bacterial↗

PubMed1978

Activation of complement by cell surface components of Staphylococcus aureus.

The abilities of intact Staphylococcus aureus H, crude cell walls (CCW), purified cell walls (PCW, peptidoglycan [PG] and covalently linked teichoic acid), peptidoglycan, and cell membranes (CM) to activate the complement system in normal human serum, C2-deficient serum, and immunoglobulin-deficient serum were compared. On a weight basis, PCW was the most active fraction; intact organisms and CCW were about equally effective; and PG was least active in causing complement consumption in normal serum. CM also activated complement but did not give a clear dose-response relationship in the concentrations used. Kinetic studies revealed that C3-C9 consumption occurred at a significantly slower rate in C2-deficient serum, indicating that intact organisms, PCW, and PG may activate the complement system via the classical and alternative pathways in normal serum. C3-C9 consumption was also slower in immunoglobulin-deficient serum than in normal serum, implying that immunoglobulins play a role in attaining maximum rates of complement activation. In all sera studied, PG was less active in complement activation than PCW. These results indicate that a number of cell surface components of S. aureus can play a role in complement activation by this organism and that the presence of teichoic acid has a significant enhancing effect in this regard.

Cell Membrane↗

PubMed1978

Dichotomy between opsonization and serum complement activation by encapsulated staphylococci.

Previous studies have demonstrated that encapsulated Staphylococcus aureus strains are not effectively opsonized by the serum complement system. Encapsulated staphylococci thereby "resist phagocytosis." To test whether this phenomenon might be explained by an inability of encapsulated strains to activate complement, the relationship between staphylococcal opsonization and serum complement activation was studied. Although encapsulation was found to interfere with opsonization by pooled human serum (human polymorphonuclear leukocytes phagocytized significantly fewer encapsulated bacteria than unencapsulated bacteria after incubation in this opsonic source), encapsulated (S. aureus M and Smith diffuse) and unencapsulated (S. aureus M variant and Smith compact) strains had similar capacities for complement activation as measured by C3-C9 consumption. When C2-deficient and immunoglobulin-deficient sera were studied, again C3-C9 consumption was not influenced by the presence or absence of a capsule. In addition, C3 was detected on the surface of both S. aureus M and M variant strains after incubation in pooled serum and staining with fluorescein-conjugated anti-C3 antibody. Thus, encapsulated staphylococci are not effectively opsonized even though complement is activated and C3 is present on the bacterial surface. The exact mechanism by which the capsule interferes with opsonization is still not known; however, inhibition of complement activation appears not to be the explanation of this phenomenon.

Antibodies↗