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Biomedical subjects

R E Hancock

Publications and source records attributed to R E Hancock.

At least 127 records · Page 7Linked to original sources
PubMed1994

Improvement of outer membrane-permeabilizing and lipopolysaccharide-binding activities of an antimicrobial cationic peptide by C-terminal modification.

Antimicrobial cationic peptides have been discovered in many different organisms and often possess a broad range of activity. In this study, we investigated the mechanisms of actions of melittin and two synthetic peptides, CEME (a cecropin-melittin hybrid) and CEMA, against gram-negative bacteria. CEMA was produced by recombinant DNA procedures and is an analog of CEME with a modified C terminus resulting in two additional positive charges. All three peptides showed good antimicrobial activity against four different gram-negative bacteria, but only CEMA was able to somewhat augment the activity of some conventional antibiotics in synergy studies. Studies using the bacteria Pseudomonas aeruginosa and Enterobacter cloacae showed that the peptides all possessed the ability to permeabilize bacterial outer membranes to the hydrophobic fluorophor 1-N-phenylnaphthylamine and the protein lysozyme, with CEMA being the most active. CEMA also had the strongest relative binding affinity for bacterial endotoxin (lipopolysaccharide). These data collectively indicated that these peptides all cross the outer membrane by the self-promoted uptake pathway and that CEMA is the peptide most effective at accessing this pathway.

Amino Acid Sequence↗
PubMed1994

Isolation of the outer membranes from Treponema pallidum and Treponema vincentii.

The outer membranes from Treponema pallidum subsp. pallidum and Treponema vincentii were isolated by a novel method. Purified outer membranes from T. pallidum and T. vincentii following sucrose gradient centrifugation banded at 7 and 31% (wt/wt) sucrose, respectively. Freeze fracture electron microscopy of purified membrane vesicles from T. pallidum and T. vincentii revealed an extremely low density of protein particles; the particle density of T. pallidum was approximately six times less than that of T. vincentii. The great majority of T. vincentii lipopolysaccharide was found in the outer membrane preparation. The T. vincentii outer membrane also contained proteins of 55 and 65 kDa. 125I-penicillin V labeling demonstrated that t. pallidum penicillin-binding proteins were found exclusively with the protoplasmic cylinders and were not detectable with purified outer membrane material, indicating the absence of inner membrane contamination. Isolated T. pallidum outer membrane was devoid of the 19-kDa 4D protein and the normally abundant 47-kDa lipoprotein known to be associated with the cytoplasmic membrane; only trace amounts of the periplasmic endoflagella were detected. Proteins associated with the T. pallidum outer membrane were identified by one- and two-dimensional electrophoretic analysis using gold staining and immunoblotting. Small amounts of strongly antigenic 17- and 45-kDa proteins were detected and shown to correspond to previously identified lipoproteins which are found principally with the cytoplasmic membrane. Less antigenic proteins of 65, 31 (acidic pI), 31 (basic pI), and 28 kDa were identified. Compared with whole-organism preparations, the 65- and the more basic 31-kDa proteins were found to be highly enriched in the outer membrane preparation, indicating that they may represent the T. pallidum rare outer membrane proteins. Reconstitution of solubilized T. pallidum outer membrane into lipid bilayer membranes revealed porin activity with two estimated channel diameters of 0.35 and 0.68 nm based on the measured single-channel conductances in 1 M KCl of 0.40 and 0.76 nS, respectively.

Animals↗
PubMed1993

Recombinant DNA procedures for producing small antimicrobial cationic peptides in bacteria.

Natural polycationic antibiotic peptides have been found in many different species of animals and insects and shown to have broad antimicrobial activity. To permit further studies on these peptides, bacterial expression systems were developed. Attempts to produce these peptides with an N-terminal signal sequence were unsuccessful due to the lability of the basic peptides. Therefore, a number of different fusion protein systems were tested, including fusions to glutathione-S-transferase (GST) (on plasmid pGEX-KP), Pseudomonas aeruginosa outer membrane protein OprF (on plasmid pRW5), Staphylococcus aureus protein A (on plasmid pRIT5), and the duplicated IgG-binding domains of protein A (on plasmid pEZZ18). In the first three cases, stable fusion proteins with the defensin, human neutrophil peptide 1 (HNP-1), and/or a synthetic cecropin/melittin hybrid (CEME) were obtained. In the course of these studies, we developed a novel method of purifying inclusion bodies, using the detergent octyl-polyoxyethylene (octyl-POE), as well as establishing methods for preventing fusion protein proteolytic breakdown. Cationic peptides could be successfully released from the carrier protein with high efficiency by chemical means (CNBr cleavage) and with low efficiency by enzymatic cleavage (using factor Xa protease). Fusions of protein A to cationic peptides were secreted into the culture supernatant of S. aureus clones and after affinity purification, CNBr digestion and column chromatography, pure cationic peptide was obtained. CEME produced by this procedure had the same amino acid (aa) content, aa sequence, gel electrophoretic mobility and antibacterial activity as CEME produced by protein chemical procedures.

Amino Acid Sequence↗
PubMed1993

Conservation of surface epitopes in Pseudomonas aeruginosa outer membrane porin protein OprF.

The outer membrane proteins of several prominent bacterial pathogens demonstrate substantial variation in their surface antigenic epitopes. To determine if this was also true for Pseudomonas aeruginosa outer membrane protein OprF, gene sequencing of a serotype 5 isolate was performed to permit comparison with the published serotype 12 oprF gene sequence. Only 16 nucleotide substitutions in the 1053 nucleotide coding region were observed; none of these changed the amino acid sequence. A panel of 10 monoclonal antibodies (mAbs) reacted with each of 46 P. aeruginosa strains representing all 17 serotype strains, 12 clinical isolates, 15 environmental isolates and 2 laboratory isolates. Between two and eight of these mAbs also reacted with proteins from representatives of the rRNA homology group I of the Pseudomonadaceae. Nine of the ten mAbs recognized surface antigenic epitopes as determined by indirect immunofluorescence techniques and their ability to opsonize P. aeruginosa for phagocytosis. These epitopes were partially masked by lipopolysaccharide side chains as revealed using a side chain-deficient mutant. It is concluded that OprF is a highly conserved protein with several conserved surface antigenic epitopes.

Antigens, Bacterial↗
PubMed1993

Anion transport through the phosphate-specific OprP-channel of the Pseudomonas aeruginosa outer membrane: effects of phosphate, di- and tribasic anions and of negatively-charged lipids.

The mechanism of anion transport through the phosphate-starvation inducible OprP-channel of Pseudomonas aeruginosa outer membrane was studied in planar lipid bilayer membranes. The single-channel conductance of OprP was 160 pS in 100 mM chloride solution. Addition of other anions, in particular of phosphate, di and tribasic anions lead to a strong decrease of the chloride conductance. The decrease was used to calculate the stability constants for the binding of these ions to the binding site of the channel on the basis of a one-site two-barrier model. The stability constant of the binding of phosphate to the site was 11,000 M-1 at neutral pH. Surprisingly, di- and tribasic anions, such as sulfate and citrate had a much lower affinity to the binding site inside the channel. Although the single-channel conductance was dependent on the external pH, the stability constants for phosphate binding decrease only slightly for increasing the pH. The use of negatively-charged lipids instead of neutral ones in the planar lipid bilayers had no influence on the single-channel conductance of the OprP-channel, suggesting that the channel is shielded from the influence of surrounding molecules. Its permeability properties are probably not influenced by negatively-charged lipopolysaccharide molecules.

Anions↗
PubMed1993

Mechanism of uptake of deglucoteicoplanin amide derivatives across outer membranes of Escherichia coli and Pseudomonas aeruginosa.

Teicoplanin is a glycopeptide antibiotic which is ineffective against gram-negative bacteria because of its inability to penetrate the outer membrane. Removal of the sugar residues and attachment of polyamines to carbon 63 yielded two dibasic deglucoteicoplanin amides, MDL 62,766 (766) and MDL 62,934 (934), with moderate MICs for Escherichia coli (2 to 4 micrograms/ml) and Pseudomonas aeruginosa (8 to 32 micrograms/ml) compared with those of the monobasic teicoplanin aglycone (16 and > 1,024 micrograms/ml, respectively). MICs were increased 16- to 32-fold by Mg2+ supplementation of Luria broth but not by Na+ supplementation at an equivalent ionic strength. Both 766 and 934 were capable of binding to P. aeruginosa lipopolysaccharide (LPS) at Mg(2+)-binding sites, as assessed by dansyl polymyxin displacement experiments. Furthermore, both compounds increased E. coli and P. aeruginosa outer membrane permeability to the hydrophobic fluorescent probe 1-N-phenylnaphthylamine (NPN), whereas the parent compounds teicoplanin aglycone and teicoplanin and the beta-lactam ceftazidime were totally ineffective. Addition of 1 mM Mg2+ blocked the increase in outer membrane permeability. Compound 766 had a lower MIC than 934 for both bacteria tested, bound to LPS with a higher affinity, and permeabilized outer membranes to NPN at lower concentrations. We propose that both deglucoteicoplanin amides exhibit increased gram-negative activity by virtue of their ability to access the self-promoted uptake pathway across the outer membrane.

Binding Sites↗
PubMed1993

Pore-forming properties of the major 53-kilodalton surface antigen from the outer sheath of Treponema denticola.

A 53-kDa protein from the outer sheath of the oral spirochete Treponema denticola was purified to homogeneity and shown to reconstitute channels in black lipid bilayer model membranes. The channel had a single-channel conductance of 1.8 nS in 0.1 M KCl, making this the largest porin channel observed to date (estimated diameter, 3.4 nm). Electron micrographs of 53-kDa-protein-containing outer sheaths of T. denticola showed a regular hexagonal array of darker staining pits.

Antigens, Bacterial↗
PubMed1993

Mechanisms of nonopsonic phagocytosis of Pseudomonas aeruginosa.

The interaction of the macrophage cell line P388D1 with Pseudomonas aeruginosa in the absence of stimulators or opsonins led to substantial association of bacteria, as judged by visual counting and FACScan assays. This association was observable within 5 min of addition of bacteria, could not be disturbed by exhaustive washing, and occurred with pilus- or flagellum-deficient mutants but not with rpoN mutants, which have been proposed to lack a secondary adhesin. In contrast, specific antibody was capable of causing similar enhancement of bacterial uptake regardless of the rpoN phenotype. Fibronectin stimulated uptake of bacteria with the pilus as an adhesin, and stimulation was observable within 5 min. Both fibronectin-enhanced and antibody-opsonized uptake were susceptible to inhibition by pertussis toxin but not by cholera toxin. The influence of fibronectin on P388D1 cells was distinguishable from that of lipopolysaccharide, which caused substantial morphological changes in cells. Although lipopolysaccharide stimulated bacterial uptake, it actually suppressed fibronectin-mediated enhancement of uptake at high concentrations.

Adhesins, Bacterial↗
PubMed1993

Genetic definition of the substrate selectivity of outer membrane porin protein OprD of Pseudomonas aeruginosa.

Earlier studies proved that Pseudomonas aeruginosa OprD is a specific porin for basic amino acids and imipenem. It was also considered to function as a nonspecific porin that allowed the size-dependent uptake of monosaccharides and facilitation of the uptake of quinolone and other antibiotics. In the present study, we utilized P. aeruginosa strains with genetically defined levels of OprD to characterize the in vivo substrate selectivity of this porin. An oprD::omega interposon mutant was constructed by gene replacement utilizing an in vitro mutagenized cloned oprD gene. In addition, OprD was overexpressed from the lac promoter by cloning the oprD gene into the broad-host-range plasmid pUCP19. To test the substrate selectivity, strains were grown in minimal medium with limiting concentrations of the carbon sources glucose, gluconate, or pyruvate. In minimal medium with 0.5 mM gluconate, the growth rates of the parent strain H103 and its oprD::omega mutant H729 were only 60 and 20%, respectively, of that of the OprD-overexpressing strain H103(pXH2). In contrast, no significant differences were observed in the growth rates of these three strains on glucose or pyruvate, indicating that OprD selectively facilitated the transport of gluconate. To determine the role of OprD in antibiotic uptake, nine strains representing different levels of OprD and OprF were used to determine the MICs of different antibiotics. The results clearly demonstrated that OprD could be utilized by imipenem and meropenem but that, even when substantially overexpressed, it could not be significantly utilized by other beta-lactams, quinolones, or aminoglycosides. In addition, competition experiments confirmed that imipenem had common binding sites with basic amino acids in the OprD channel, but not with gluconate or glucose.

Amino Acids, Diamino↗
PubMed1993

Linker-insertion mutagenesis of Pseudomonas aeruginosa outer membrane protein OprF.

The oprF gene, expressing Pseudomonas aeruginosa major outer membrane protein OprF, was subjected to semi-random linker mutagenesis by insertion of a 1.3 kb HincII kanamycin-resistance fragment from plasmid pUC4KAPA into multiple blunt-ended restriction sites in the oprF gene. The kanamycin-resistance gene was then removed by PstI digestion, which left a 12 nucleotide pair linker residue. Nine unique clones were identified that contained such linkers at different locations within the oprF gene and were permissive for the production of full-length OprF variants. In addition, one permissive site-directed insertion, one non-permissive insertion and one carboxyterminal insertion leading to proteolytic truncation were also identified. These mutants were characterized by DNA sequencing and reactivity of the OprF variants with a bank of 10 OprF-specific monoclonal antibodies. Permissive clones produced OprF variants that were shown to be reactive with the majority of these monoclonal antibodies, except where the insertion was suspected of interrupting the epitope for the specific monoclonal antibody. In addition, these variants were shown to be 2-mercaptoethanol modifiable, to be resistant to trypsin cleavage in intact cells and partly cleaved to a high-molecular-weight core fragment in outer membranes and , where studied, to be accessible to indirect immunofluorescence labelling in intact cells by monoclonal antibodies specific for surface epitopes. Based on these data, a revised structural model for OprF is proposed.

Amino Acid Sequence↗
PubMed1993

Canadian Bacterial Diseases Network: a new approach to university-industry relationships.

Bacterial diseases are a substantial problem worldwide. Their diagnosis and therapy form the basis of a multibillion dollar industry. This industry is dynamic and is continuously revitalized by research. The Canadian Bacterial Diseases Network (CBDN) was established to capitalize on the enhanced opportunities that now exist for the rapid progression of an idea from conceptualization to implementation and, ultimately, commercialization. CBDN is one of 15 Networks of Centres of Excellence, a Federal Government initiative whose intention is to improve Canada's economic competitiveness in the global market. CBDN research involves fundamental science, is broadly-based, and encompasses all aspects of bacterial diseases. Current projects include the investigations of strategies to block Pseudomonas aeruginosa binding to epithelial cells and a novel anti-toxin approach for Escherichia coli. Also CBDN is investigating the basis for antibiotic resistance in Gram-negative bacteria, and is devising improved procedures for overcoming such resistance mechanisms. CBDN is only 28 months into its first 4-year mandate; however, considerable successes have been enjoyed to date.

Academies and Institutes↗
PubMed1992

Analysis of two gene regions involved in the expression of the imipenem-specific, outer membrane porin protein OprD of Pseudomonas aeruginosa.

A Tn501 mutant of Pseudomonas aeruginosa resistant to imipenem and lacking the imipenem-specific outer membrane porin protein OprD was isolated. The mutation could be complemented to imipenem susceptibility and OprD-sufficiency by a cloned 6-kb EcoRI-PstI fragment of DNA from the region of chromosome of the wild-type strain surrounding the site of Tn501 insertion. However, this fragment did not contain the oprD structural gene as judged by its inability to hybridize with an oligonucleotide corresponding to the N-terminal amino acid sequence of OprD. DNA sequencing of 3.9 kb of the region surrounding the Tn501 insertion site revealed three large open reading frames, one of which would be interrupted by the Tn501 insertion in the mutant. This latter open reading frame, named opdE (for putative regulator of oprD expression), predicted a hydrophobic protein of M(r) 41,592. Using the above-mentioned oligonucleotide, the oprD structural gene was cloned and expressed in Escherichia coli on a 2.1-kb Bam HI-KpnI fragment. DNA sequencing predicted a 420 amino acid mature OprD protein with a 23 amino acid signal sequence.

Bacterial Outer Membrane Proteins↗
PubMed1992

Extracellular lipase from Pseudomonas aeruginosa is an amphiphilic protein.

Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) secreted by Pseudomonas aeruginosa PAC1R was purified from cell-free growth medium by preparative isoelectric focusing. After blotting the N-terminal amino acid sequence and the amino acid composition were determined and compared to P. fragi and P. cepacia lipases yielding significant homology between all three species. Additionally, a consensus sequence K-Y-P-i-v-l-V-H-G was identified residing at the N-terminus of Pseudomonas lipases and in the central part of Staphylococcus lipases. Treatment of lipase with the serine-specific inhibitor diethyl p-nitrophenyl phosphate caused a rapid and complete inhibition of enzyme activity indicating the presence of a serine at the catalytic site as expected from lipase consensus sequences. Upon charge-shift electrophoresis the electrophoretic mobility of purified lipase was shifted either anodally or cathodally in the presence of sodium deoxycholate and cetyltrimethylammoniumbromide, respectively. This result demonstrates that extracellular lipase of P. aeruginosa exhibits an amphiphilic character like intrinsic membrane proteins.

Amino Acid Sequence↗
PubMed1992

Characterization of lipid A from Pseudomonas aeruginosa O-antigenic B band lipopolysaccharide by 1D and 2D NMR and mass spectral analysis.

The Lipid A from the lipopolysaccharide of Pseudomonas aeruginosa was examined by high-field nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS). The backbone structure and the position of phosphate substituents were unambiguously established by one- and two-dimensional 1H, 13C, and 31P NMR techniques on a de-O-acylated Lipid A sample. The Lipid A has a beta-(1,6)-glucosamine disaccharide structure which is substituted by phosphomonoesters through glycosidic bonds at C-1 and at C-4'. The configuration of the glycosidically linked phosphate at position C-1 was identified as alpha which is the same as that of Enterobacterial Lipid A. Chemical analysis revealed that the Lipid A contained 2-hydroxydodecanoic, 3-hydroxydodecanoic, dodecanoic, 3-hydroxydecanoic, and hexadecanoic acids in the approximate molar ratios 2.2:2.0:0.2:0.8:0.4. From 1H NMR and fast atom bombardment (FAB) mass spectrometry on the de-O-acylated Lipid A, it was established that both glucosamine residues were N-acylated by 3-hydroxydodecanoic acid. The identity and positions of the ester bound fatty acids in the intact Lipid A were investigated by negative ion FAB-MS. In addition to the hexaacyl and pentaacyl Lipid A species, a tetraacyl species was identified. Heterogeneity due to hydroxylated and nonhydroxylated dodecanoic acid esters could be uniquely localized to the nonreducing beta-glucosamine residue from the fragmentation pattern observed in the negative ion FAB-MS. The complete structure of the Lipid A from P. aeruginosa will be useful in understanding the determinants responsible for the endotoxin activity of this molecule.

Lipid A↗
PubMed1992

Antibiotic screening of medicinal plants of the British Columbian native peoples.

One hundred methanolic plant extracts, 96 of which had documented medicinal uses by British Columbian native peoples, were screened for antibiotic activity against 11 bacterial strains. Eighty-five percent were found to have significant antibiotic activity against at least two of the bacteria tested. Ninety-five percent of the plants categorized as potential antibiotics based on their ethnobotanical usage were found to exhibit significant antibiotic activity. Seventy-five were found to be active against methicillin-resistant Staphylococcus aureus, 46 were active against an antibiotic supersusceptible strain of Pseudomonas aeruginosa and 18 of these were also active against a wild type strain. The extracts with the broadest spectra of activity were prepared from: Alnus rubra bark and catkins, Fragaria chiloensis leaves, Moneses uniflora aerial parts, and Rhus glabra branches.

Anti-Bacterial Agents↗
PubMed1992

Influence of outer membrane mutations on susceptibility of Escherichia coli to the dibasic macrolide azithromycin.

Azithromycin differs chemically from erythromycin by having an extra positive charge created by the presence of a methyl-substituted nitrogen in the 15-membered macrolide ring. This results in substantially increased potency against Gram-negative bacteria. Therefore, the possibility was considered that azithromycin was taken across the outer membrane of Escherichia coli by the self-promoted uptake route, which is utilized by other cationic antibiotics including polymyxins and aminoglycosides. Azithromycin, like polymyxin B and gentamicin, demonstrated equal activity against porin-sufficient and porin-deficient E. coli strains but its MIC was increased eight-fold by magnesium supplementation. Nevertheless, an outer membrane-altered mutant DC2 was eight-fold more susceptible than its parent strain UB1005 to azithromycin, indicating that the outer membrane was a permeability barrier to this macrolide. A mutant SC9252 which had an alteration in the self-promoted uptake of polymyxin and gentamicin, was more resistant to azithromycin, polymyxin and gentamicin compared to its parent SC9251. Further azithromycin, like polymyxin B and gentamicin, was capable of weakly permeabilizing cells to the hydrophobic fluorophor 1-N-phenyl-naphthylamine, a process antagonized by Mg2+. The monobasic macrolide erythromycin on the other hand was less affected by the SC9252 mutation, less effectively antagonized by Mg2+, and was a far less effective permeabilizer than dibasic azithromycin. These data are consistent with the hypothesis that the improved efficacy of azithromycin compared to erythromycin against E. coli reflects its better access to the self-promoted uptake pathway due to its additional positive charge.

Azithromycin↗
PubMed1992

Factors involved in the enhanced efficacy against gram-negative bacteria of fourth generation cephalosporins.

The fourth generation cephalosporins, cefpirome and cefepime, demonstrate better activity against strains of Enterobacter cloacae with derepressed beta-lactamase than the third generation compounds cefotaxime and ceftriaxone. Several methodological refinements were used to measure the parameters, predicted by the Zimmermann-Rosselet equation to be important in the efficacy of beta-lactams. Outer membrane permeability was measured by a novel HPLC method. The kinetics of interaction of purified beta-lactamase with beta-lactams were estimated to calculate the inhibition and catalytic constants. The periplasmic concentration of beta-lactams leading to growth inhibition of cells was determined by substituting the above parameters into the Zimmermann-Rosselet equation. Consideration of these three factors allowed accurate prediction of MICs in isogenic E. cloacae strains with differing porin or beta-lactamase contents. The fourth generation cephalosporins had markedly reduced affinity for beta-lactamase and increased outer membrane permeability when compared to the third generation cephalosporins. Such advantages were only partly offset by a lower stability of complexes with beta-lactamase and reduced affinity for their targets.

Anti-Bacterial Agents↗