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M Vaara

Publications and source records attributed to M Vaara.

At least 91 records · Page 5Linked to original sources
PubMed1989

rfaP mutants of Salmonella typhimurium.

Salmonella typhimurium rfaP mutants were isolated and characterised with respect to their sensitivity towards hydrophobic antibiotics and detergents, and their lipopolysaccharides were chemically analysed. The rfaP mutants were selected after diethylsulfate mutagenesis or as spontaneous mutants. The mutation in two independent mutants SH7770 (line LT2) and SH8551 (line TML) was mapped by cotransduction with cysE to the rfa locus. The mutants were sensitive to hydrophobic antibiotics (clindamycin, erythromycin and novobiocin) and detergents (benzalkoniumchloride and sodium dodecyl sulfate). Analysis of their lipopolysaccharides by chemical methods and by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed that their saccharide portion was, to a large extent, of chemotype Rc with small proportions of material containing a more complete core oligosaccharide and O-specific chains. Only 2.5 mol phosphate/mol lipopolysaccharide was found whereas the phosphate content of the lipopolysaccharide of a galE mutant strain was 4.8 mol. Thus the rfaP mutant lipopolysaccharides lacked more than two phosphate residues. Assessment of the location of phosphate groups in rfaP lipopolysaccharides revealed the presence of at least 2 mol phosphate in lipid A, indicating that the core oligosaccharide was almost devoid of phosphate. The chemical, physiological and genetic data obtained for these mutants are in full agreement with those reported earlier for rfaP mutants of Salmonella minnesota.

Anti-Bacterial Agents↗
PubMed1989

Isolation, cloning, and primary structure of a cationic 16-kDa outer membrane protein of Salmonella typhimurium.

By using acid-urea polyacrylamide gel electrophoresis, two cationic proteins were found in the isolated outer membranes of Salmonella typhimurium SH5014. Also, all the other enterobacterial strains studied (five additional strains of S. typhimurium, one strain of Salmonella minnesota, and three strains of Escherichia coli K12) had those proteins. The most abundant (OMB2) was purified in preparative acid-urea polyacrylamide gel electrophoresis and reversed-phase high pressure liquid chromatography (HPLC). It had a molecular mass of 16 kDa, a pI above 10.0, and was rich in arginine and lysine. 72% of the total amino acid sequence was determined by sequencing several HPLC-purified proteolytic fragments and 55 amino acids from the NH2 terminus. Furthermore, we isolated by molecular cloning the corresponding gene, named it ompH, and determined its nucleotide sequence. By combining protein and nucleotide sequence data, we determined the primary structure of the entire OmpH protein. It consists of 141 amino acids, possesses regions very rich in basic amino acids, and has a molecular mass of 15,862 kDa.

Amino Acid Sequence↗
PubMed1989

Sodium hexametaphosphate sensitizes Pseudomonas aeruginosa, several other species of Pseudomonas, and Escherichia coli to hydrophobic drugs.

Many gram-negative bacteria are known to be remarkably resistant to hydrophobic noxious agents by virtue of their outer membranes (OM). We investigated, by using four different assay methods, the ability of sodium hexametaphosphate (HMP) to disrupt this OM barrier. (i) In the growth inhibition assay, HMP was found to sensitize strains of Pseudomonas aeruginosa to all the hydrophobic probes tested (rifampin, fusidic acid, dactinomycin, sodium dodecyl sulfate, and Triton X-100). A concentration of 0.3% HMP decreased the MICs of the probes by a factor of approximately 10, and maximally even a 30-fold sensitization was found with 1% HMP. (ii) In the bactericidal assay, 0.3% HMP decreased the MBC of the hydrophobic probe rifampin by a factor of approximately 30. (iii) In the bacteriolytic assay, 0.1% HMP sensitized the target bacteria to lysis by sodium dodecyl sulfate and Triton X-100. (iv) In the fluorescent-probe binding assay, HMP drastically enhanced the binding of fluorescent N-phenyl naphthylamine to the membranes of the target cells. In addition to P. aeruginosa, P. fluorescens, P. putida, P. fragi, and Escherichia coli were susceptible to the OM permeability-increasing action of HMP, while P. cepacia was resistant.

Anti-Bacterial Agents↗
PubMed1988

Effective inhibition of cardiolipin-binding antibodies in gram-negative infections by bacterial lipopolysaccharide.

Anticardiolipin antibodies (ACA) were detected by solid-phase enzyme immunoassay in the majority of sera from patients with Gram-positive and Gram-negative bacterial infections. The response involved all the major immunoglobulin classes IgG, IgM, and IgA. The specificity of the ACA was studied in competitive inhibition experiments with three putative antigens: cardiolipin, lipopolysaccharide (LPS) isolated from Salmonella minnesota, strain Re 595, and synthetic Escherichia coli lipid A. The binding of IgG class ACA from the sera of five patients with Gram-negative infections was effectively inhibited by LPS, whereas 100-fold more cardiolipin was required for comparable inhibition. Pure lipid A was a less effective inhibitor of anticardiolipin activity than LPS. This pattern of reactivity was not seen in sera from patients with Gram-positive infections, syphilis, or systemic lupus erythematosus. Our findings suggest that cardiolipin may not be the inducing antigen for the cardiolipin-binding antibodies that develop in Gram-negative infections.

Antibody Specificity↗
PubMed1988

Effect of small cationic leukocyte peptides (defensins) on the permeability barrier of the outer membrane.

Defensins are small cationic antibacterial peptides that are abundant in polymorphonuclear leukocytes from human and other sources (T. Ganz, M. Selsted, D. Szklarek, S. Harwig, K. Daher, D. F. Bainton, and R. J. Lehrer, J. Clin. Invest. 76:1427-1435, 1985). We studied whether subinhibitory concentrations of defensins increase the outer membrane (OM) permeability of Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa to hydrophobic probes, as do many other polycations that have been studied previously. Throughout the study, we used polymyxin B nonapeptide (PMBN) as a reference peptide. PMBN has a known potent OM permeability-increasing action. As a sharp contrast to PMBN, subinhibitory concentrations of defensins did not permeabilize (or, under some test conditions, permeabilized very slightly) the OM to the probes that were used (rifampin and Triton X-100). At bacteriostatic or bactericidal defensin concentrations, some degree of synergism with rifampin was seen.

Blood Bactericidal Activity↗
PubMed1987

Reversible binding of Salmonella typhimurium lipopolysaccharides by immobilized protamine.

The ability of agarose-linked protamine to bind Salmonella typhimurium lipopolysaccharides was investigated. Radioactively labelled lipopolysaccharides were isolated both from a smooth strain (SH6749, labelled with [14C]galactose) and from a rough strain (SH5014, lipopolysaccharide chemotype Rb2, labelled with [3H]acetate). From 50-micrograms samples of the lipopolysaccharides, protamine-agarose columns bound 99.5-99.9% of the input radioactivity. The binding efficacy was not affected by pH in the range from 3.7 to 10.5. Maximal binding capacity of protamine-agarose for highly soluble (triethylamine form) lipopolysaccharide of SH5014 was estimated to be 13.5 mg/ml packed adsorbent. The bound lipopolysaccharides could be totally released from the columns and recovered by elution with the anionic detergent sodium deoxycholate, or with 0.5 M NaCl in the presence of the uncharged detergent Triton X-100. By analysis in sodium dodecyl sulfate/polyacrylamide gels, the macromolecular quality of the recovered lipopolysaccharide was shown to be identical to that of the original. Protamine-agarose chromatography can thus be applied to purify lipopolysaccharide preparations, and to separate as well as concentrate lipopolysaccharides from dilute solutions without altering their composition. This application was challenged with water as well as insulin solution experimentally contaminated with radiolabelled lipopolysaccharide. While the insulin protein did not bind to the protamine-agarose, the contaminating lipopolysaccharide was effectively trapped.

Binding Sites↗
PubMed1987

Analysis of polyamines as their dabsyl derivatives by reversed-phase high-performance liquid chromatography.

The polyamines putrescine, cadaverine, spermidine, and spermine and the corresponding mono-N-acetylpolyamines can be separated as their dimethylaminoazobenzenesulfonyl derivatives in a single analysis in less than 22 min. The method employs reversed-phase high-performance liquid chromatography (Spherisorb S5 ODS2 column) with an acetonitrile/acetate buffer gradient elution system and detection in the visible (436 nm) region. The detection limit for a single dimethylaminoazobenzenesulfonylpolyamine is less than 2 pmol.

Cadaverine↗
PubMed1986

Chain length heterogeneity of lipopolysaccharide released from Salmonella typhimurium by ethylenediaminetetraacetic acid or polycations.

Cells of two smooth Salmonella typhimurium strains (SL696 and SH4247) were treated with ethylenediaminetetraacetic acid (EDTA) and the polycations poly(L-lysine) and protamine to monitor both quantitatively and qualitatively the release of [14C] galactose-labelled lipopolysaccharide into the medium to find out whether these effector substances caused selective release of certain fractions from the initially heterogenous lipopolysaccharide population. Each one of the substances released considerable amounts of lipopolysaccharide into the medium. Analysis by sodium dodecyl sulphate/polyacrylamide gel electrophoresis followed by autoradiography showed that the total lipopolysaccharide (from isolated membranes) and the released materials produced coincident banding patterns, each with a high degree of O side-chain length heterogeneity. Densitometric scans of the autoradiograms were analyzed for possible differences in the distribution and relative abundance of lipopolysaccharide molecules with different O chain lengths. It was found that in SL696 the released materials were identical to the total lipopolysaccharide; in SH4247 subtle deviations from the total lipopolysaccharide were seen. We conclude from these results that lipopolysaccharide molecules with short and long O side chains are linked to and stabilized in the outer membrane by similar mechanisms equally susceptible to the effects of EDTA and polycations.

Edetic Acid↗
PubMed1986

Effect of bacterial lipopolysaccharide on serum lipids and on the development of aortic atherosclerosis in rabbits.

The effect of repeated intravenous administration of bacterial lipopolysaccharide (LPS) on serum lipids and on aortic atherosclerosis was studied in rabbits on basal diet and on hypercholesterolemic diets containing 0.15-1.0% cholesterol. LPS (10 or 100 ng/kg body weight) was administered 3 times per week for 3 or 6 weeks. No difference was observed in serum lipid levels or in aortic atherosclerosis between LPS- and saline-treated animals. These observations do not support the hypothesis that LPS has an effect on the progression of atherosclerosis.

Animals↗
PubMed1986

Susceptibility of gram-negative bacteria to the synergistic bactericidal action of serum and polymyxin B nonapeptide.

Polymyxin B nonapeptide was able to sensitize Escherichia coli strains and strains of Salmonella typhimurium, Klebsiella spp., Enterobacter cloacae, Pseudomonas aeruginosa, and Haemophilus influenzae to the bactericidal action of fresh normal human serum. The degree of sensitization varied significantly within the strains. Strains of Proteus mirabilis, Neisseria gonorrhoeae, and N. meningitidis remained resistant.

Ceftazidime↗
PubMed1985

Binding of polymyxin B nonapeptide to gram-negative bacteria.

The binding of the outer membrane-disorganizing peptide polymyxin B nonapeptide (PMBN) to gram-negative bacteria was studied by using tritium-labeled PMBN. Smooth Salmonella typhimurium had a binding capacity of ca. 6 nmol of PMBN per mg (dry weight) of bacteria, which corresponds to ca. 1 X 10(6) to 2 X 10(6) molecules of PMBN per single cell. The binding was of relatively high affinity (Kd, 1.3 microM). The isolated outer membrane of S. typhimurium bound ca. 100 nmol of PMBN per mg of outer membrane protein (Kd, 1.1 microM), whereas the cytoplasmic membrane bound 9 to 10 times less. Other bacteria which are susceptible to the action of PMBN (Escherichia coli strains, Pseudomonas aeruginosa, Haemophilus influenzae) also bound large amounts of PMBN. The S. typhimurium pmrA mutant, Neisseria gonorrhoeae, and Proteus mirabilis (all known as resistant to polymyxin and PMBN) bound 3.3, 4, and 12 times less than S. typhimurium, respectively. The binding of PMBN to S. typhimurium was effectively inhibited by low concentrations of polymyxin B, compound EM49 (octapeptin), polylysine, and protamine. Spermine, Ca2+, and Mg2+ also inhibited the PMBN binding although they were ca. 160, 700, and 2,400 times less active (based on molarity) than polymyxin B, respectively. No binding inhibition was found at the tested concentrations of streptomycin, tetralysine, spermidine, or cadaverine.

Cell Membrane↗
PubMed1984

Effect of bacterial lipopolysaccharide on serum high density lipoprotein cholesterol in rabbits.

The effect of bacterial lipopolysaccharide (LPS) on serum lipids was examined in rabbits. LPS was prepared from the smooth Salmonella typhimurium LT2 strain and given intravenously at a dose of 100 ng/kg b.wt. There were no significant changes in serum triglyceride or cholesterol levels in 1-3 days after the administration of LPS. There was, however, a decrease in serum high density lipoprotein (HDL) cholesterol, which was greatest after 2 days (P less than 0.001). Simultaneously, the HDL/total cholesterol ratio decreased (P less than 0.005).

Animals↗
PubMed1984

Susceptibility of gram-negative bacteria to polymyxin B nonapeptide.

Subinhibitory concentrations of polymyxin B nonapeptide sensitized all 21 polymyxin-susceptible gram-negative bacterial strains studied to hydrophobic antibiotics such as fusidic acid, novobiocin, and erythromycin. The susceptibility increases were usually 30- to 300-fold. The strains included representatives of Escherichia coli with different O- and K-antigens, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter agglomerans, Salmonella typhimurium, Acinetobacter calcoaceticus, Pseudomonas aeruginosa, and Pseudomonas maltophilia. In contrast, polymyxin-resistant strains (Proteus mirabilis, Proteus vulgaris, Morganella morganii, Providencia stuartii, and Serratia marcescens) were resistant to the action of polymyxin B nonapeptide.

Acinetobacter↗
PubMed1984

New Salmonella typhimurium mutants with altered outer membrane permeability.

We describe three new classes of Salmonella typhimurium mutants with increased sensitivity to hydrophobic agents. In contrast to many previously described mutants, the phage sensitivity pattern of these mutants did not give any indication of defective lipopolysaccharide. Furthermore, they had no detectable changes in their phospholipid or outer membrane protein composition, and their growth rate and cell morphology were normal. Class B mutants were nearly as sensitive to novobiocin, fusidic acid, erythromycin, rifampin, and clindamycin as are deep rough (heptoseless) mutants; in addition they were sensitive to methicillin, penicillin (to which heptoseless mutants are resistant), gentian violet, and anionic and cationic detergents. Class A and C mutants had less sensitive, but characteristic phenotypes. None of the three classes were sensitive to serum bactericidal action. The class B mutation mapped between map positions 7 and 11 on the S. typhimurium chromosome, and the class C mutation mapped between positions 5 and 7. The map position for the class A mutation remained undefined, but it was separate from the class B and C mutations and, like those, did not correspond to any gene loci known to participate in the synthesis of major outer membrane constituents.

Bacterial Outer Membrane Proteins↗
PubMed1984

An outer membrane-disorganizing peptide PMBN sensitizes E. coli strains to serum bactericidal action.

The small cationic outer membrane-disorganizing peptide PMBN sensitized four smooth, encapsulated strains of Escherichia coli (serotypes 02:K1, 04:K12, 018:K1, and 018:K5) to the lethal action of serum. The concentrations of PMBN required were low (0.3 to 1.0 microgram/ml). One E. coli strain (IH 11030; 075:K5) remained virtually resistant to serum and also to anti-075 hyperimmune serum plus complement (C) even in the presence of PMBN. This strain was nevertheless sensitive to the outer membrane permeability-increasing action of PMBN. In the bactericidal system, PMBN could be replaced by high concentrations of lysine20 or protamine but not lysine4. The PMBN-dependent bactericidal activity of GPS was abolished by heating or zymosan treatment that inactivate its C but not by lack of the action of the classical pathway of the C in C4-deficient GPS. PMBN formed a bactericidal system also with normal rabbit, rat, and human serum but not with mouse serum. The bactericidal system against E. coli 018:K1 and its derivative EH 817 (018:K1-) was found to require a factor that can be removed from normal sera by absorption with a rough E. coli strain. This factor could be replaced by specific anti-018 antibodies. The bactericidal activity of fetal calf serum plus PMBN against E. coli 018:K1 was enhanced by normal rabbit or anti-E. coli 018 hyperimmune serum. We suggest that PMBN unshields the deep structures and the hydrophobic membrane milieu of the outer membrane and facilitates the insertion of the membrane attack complex of the C into this milieu.

Animals↗