Search PubMed⌕ Search

Biomedical subjects

R E Hancock

Publications and source records attributed to R E Hancock.

At least 163 records · Page 9Linked to original sources

Determinants of the efficacy of tobramycin therapy against isogenic nonmucoid and mucoid derivatives of Pseudomonas aeruginosa PAO1 growing in peritoneal chambers in mice.

Mice which were supporting the growth of Pseudomonas aeruginosa in chambers implanted in their peritoneums were given two intramuscular injections of tobramycin (15 mg/kg of body weight each) at an interval of 8 h. Three hours later, chambers were removed and their contents were assessed for viable counts. Controls revealed that tobramycin levels in the chambers were 3.8 micrograms/ml 15 min after injection of 15 mg of tobramycin per kg and remained above the in vitro MICs (0.5 to 1 microgram/ml) for the tested strains for 8 h. It was demonstrated that tobramycin therapy was less effective with higher inocula and with longer delay before administration. Thus, in vivo, the concentration of bacteria in the chambers at the time of the first tobramycin injection had a profound effect on the bactericidal efficacy of tobramycin therapy. No such concentration dependence was observed in mock in vitro therapy experiments. A phage-selected mucoid derivative of P. aeruginosa PAO1 showed only a marginal increase in in vitro aminoglycoside susceptibility and no major alteration in in vivo susceptibility compared with its isogenic nonmucoid parent strain.

Animals↗

Pseudomonas aeruginosa pili as ligands for nonopsonic phagocytosis by fibronectin-stimulated macrophages.

Fibronectin is capable of activating macrophages for enhanced nonopsonic phagocytosis of Pseudomonas aeruginosa grown in vivo in rats or mice or in vitro on nutrient agar plates. In this study it was determined that while fibronectin was able to significantly increase phagocytosis of organisms grown in static broth, uptake of agitated bacteria could not be promoted. Agitated P. aeruginosa cultures were proven to lack surface pili expression, as assessed by electron microscopic studies. A pilus-deficient pilA::Tn501 mutant of P. aeruginosa PAO was constructed by gene replacement techniques. Phagocytosis of this mutant could not be enhanced by fibronectin regardless of growth conditions. Furthermore, 60 micrograms of exogenously added Pseudomonas pili per ml was capable of abrogating the enhanced phagocytosis of the wild-type strain observed with fibronectin-stimulated macrophages. It is concluded that Pseudomonas pili were the bacterial ligands required for attachment to fibronectin-stimulated macrophages in the initial stages of nonopsonic phagocytosis.

Animals↗

Surface characteristics of Pseudomonas aeruginosa grown in a chamber implant model in mice and rats.

Pseudomonas aeruginosa PAO1 was grown in vivo in chambers implanted into the peritoneums of mice and rats. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracts of bacterial cells taken from the chambers and washed to remove loosely bound host proteins revealed the presence of the major outer membrane proteins D2, E, F, G, and H2. Western immunoblotting with specific antisera confirmed the presence of porin protein F and lipoprotein H2. However, there was no apparent induction of the phosphate starvation-inducible porin P or the divalent cation starvation-inducible protein H1. Small amounts of proteins with molecular weights similar to those of the iron-regulated outer membrane proteins were found in cells grown in vivo; however, their presence could not be confirmed immunologically. The presence of pili and flagella on the cells grown in vivo was demonstrated by electron microscopy and Western immunoblotting. A consistent alteration in the lipopolysaccharide banding pattern was observed after growth in vivo. Compared with cells of strain PAO1 grown in vitro, cells grown in vivo appeared to lack a series of high-molecular-weight O-antigen-containing lipopolysaccharide bands and gained a new series of lower-molecular-weight lipopolysaccharide bands. This alteration in the lipopolysaccharide after growth in vivo did not affect the O-antigen serotype or the resistance of the bacteria to serum.

Animals↗

Stimulation by fibronectin of macrophage-mediated phagocytosis of Pseudomonas aeruginosa.

In a previous investigation it was determined that Pseudomonas aeruginosa cells taken directly from a mouse in vivo growth system were significantly more susceptible to nonopsonic phagocytosis by macrophages than were similar cells after being washed in buffer (N. M. Kelly, J. L. Battershill, S. Kuo, J. P. Arbuthnott, and R. E. W. Hancock, Infect. Immun. 55:2841-2843, 1987). It was demonstrated that a phagocytosis-promoting factor was found in the supernatant obtained from chambers incubated in the peritoneal cavities of laboratory mice or rats. The phagocytosis-promoting factor was effective with both strains of P. aeruginosa tested, using both unelicited mouse peritoneal macrophages and the P388D1 mouse macrophage cell line as the phagocytic cells. Phagocytosis enhancement was observed with in vivo-grown bacteria and with bacteria grown in vitro on agar plates, but not with bacteria grown in vitro with rapid agitation. Supernatants from mice and rats were fractionated using a fast pressure liquid chromatography gel exclusion column. The phagocytosis-promoting factor copurified with fibronectin. Furthermore, antifibronectin sera negated the phagocytosis-promoting activities of in vivo chamber supernatant, while commercial bovine fibronectin was itself capable of promoting phagocytosis. The concentrations of fibronectin increased in both rat and mouse peritoneal chambers with time, coincident with the ability of chamber supernatants to promote phagocytosis. It was concluded that fibronectin was the phagocytosis-promoting factor of chamber supernatants. Bacterial presence in the peritoneal chambers was not required to elicit fibronectin uptake into the chambers.

Animals↗

Pseudomonas aeruginosa cytotoxin: periplasmic localization and inhibition of macrophages.

Pseudomonas aeruginosa cytotoxin has been isolated previously from cell autolysates. Both purified cytotoxin and periplasmic contents (osmotic shock fluid) cross-reacted on Western immunoblots with antibodies specific for cytotoxin. In addition, both preparations caused a significant reduction in antibody-mediated phagocytosis of P. aeruginosa M2 by mouse macrophage cell line P388D1. Phagocytosis was restored in each case on preincubation of cytotoxin or periplasmic contents with anti-cytotoxin serum. Both cytotoxin and periplasmic contents caused depolarization of the P388D1 cell membrane, as demonstrated with a polarization-sensitive fluorescent probe. Similar correlations were not observed for other P. aeruginosa cell fractions or for osmotic shock fluid from Escherichia coli C600. These data indicate that P. aeruginosa cytotoxin is localized in the periplasm and has the potential to inhibit macrophage-mediated phagocytosis, possibly by perturbing ion gradients across the macrophage plasma membrane.

Animals↗

Fibronectin as an enhancer of nonopsonic phagocytosis of Pseudomonas aeruginosa by macrophages.

Fibronectin is capable of enhancing uptake by macrophages of Pseudomonas aeruginosa grown in vivo in rats or mice or in vitro on nutrient agar plates. It was demonstrated that concentrations as low as 27 nM fibronectin produced significant enhancement of macrophage phagocytosis. Washing of fibronectin-treated macrophages did not prevent phagocytosis enhancement, but washing of fibronectin-treated bacteria did. The tetrapeptide arginine-glycine-aspartic acid-serine, which comprises the eucaryotic cell-binding domain of fibronectin, was also capable of promoting bacterial uptake, whereas the control tetrapeptide tetraglycine was not. Fibronectin caused depolarization of the mouse macrophage cell line P388D1, plasma membrane, as demonstrated by using a polarization-sensitive fluorescent probe. These data indicate that promotion by fibronectin of nonopsonic phagocytosis is mediated by the action of fibronectin on the macrophages.

Adjuvants, Immunologic↗

Properties of a Pseudomonas stutzeri outer membrane channel-forming protein (NosA) required for production of copper-containing N2O reductase.

A protein (NosA) in the outer membrane of Pseudomonas stutzeri that is required for copper to be inserted into N2O reductase has been extracted and purified to homogeneity. The purified protein could form channels in black lipid bilayers. Like N2O reductase, NosA contained copper and was only made anaerobically. In contrast to N2O reductase, its synthesis was repressed by exogenous copper (but not by Mn, Co, Ni, Zn, or Fe). Also in contrast to N2O reductase, NosA homologs were not immunologically detectable in Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas alcaligenes, or other strains of P. stutzeri.

Bacterial Outer Membrane Proteins↗

Outer membrane protein H1 of Pseudomonas aeruginosa: purification of the protein and cloning and nucleotide sequence of the gene.

Overexpression of the divalent cation-regulated outer membrane protein H1 of Pseudomonas aeruginosa is associated with resistance to polymyxin B, aminoglycosides, and EDTA. Protein H1 is believed to act by replacing divalent cations at binding sites on lipopolysaccharide, thereby preventing disruption of the sites and subsequent self-promoted uptake of the antibiotics. Protein H1 purified by two cycles of anion-exchange chromatography was apparently associated with lipopolysaccharide. Lipopolysaccharide-free protein H1 was purified in high yield by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was subjected to N-terminal amino sequencing. Complementary oligodeoxyribonucleotides were used to clone the structural gene for protein H1, oprH, into Escherichia coli. Successful cloning was confirmed by nucleotide sequence analysis. Southern hybridization suggested that oprH was present as a single-copy gene in P. aeruginosa. The deduced amino acid sequence revealed that H1 was a slightly basic polypeptide of 178 residues, with a leader sequence typical of an exported procaryotic protein. It had little similarity, however, to other bacterial surface proteins for which sequence data were available. No expression of protein H1, from its own or the lac promoter, was detected in E. coli. We concluded that, as for some other regulated Pseudomonas genes, expression of oprH, at least under some conditions, is blocked in E. coli.

Amino Acid Sequence↗

Pseudomonas aeruginosa outer membrane protein F: structural role and relationship to the Escherichia coli OmpA protein.

A Pseudomonas aeruginosa outer membrane protein F-deficient omega-insertion mutant strain H636, in contrast to its protein F-sufficient parent strain H103, was unable to grow on unsupplemented Proteose Peptone no. 2 broth (Difco Laboratories, Detroit, Mich.). Addition of high concentrations of NaCl, KCl, glucose, sucrose, or potassium succinate permitted growth of strain H636 at rates approaching those of the parent strain H103. Strain H636 cells were 33% shorter and had a 46% smaller cross-sectional area than did the parent strain growing at similar rates on the same medium. These properties of the oprF::omega mutant were analogous to those previously observed for Escherichia coli ompA mutants in an lpp (Braun lipoprotein-deficient) mutant background. Therefore, we compared P. aeruginosa protein F and the E. coli OmpA protein. In addition to many similarities previously described, sequence alignment demonstrated substantial amino acid sequence homology throughout the carboxy-terminal 168 to 180 amino acids of the two proteins. Consistent with this observation, polyclonal antiserum specific for OmpA reacted on Western blots (immunoblots) with protein F. Expression of protein F from the cloned oprF gene in an E. coli ompA lpp double mutant resulted in a 1.7-fold increase in cell length and a 2.1-fold increase in cross-sectional area compared with values for the same mutant containing only the plasmid vector onto which the oprF gene had been cloned. These results favor a structural role for P. aeruginosa protein F and suggest that it is strongly related to the E. coli OmpA protein.

Amino Acid Sequence↗

Large-scale purification and biochemical characterization of crystallization-grade porin protein P from Pseudomonas aeruginosa.

A large-scale purification scheme was developed for lipopolysaccharide-free protein P, the phosphate-starvation-inducible outer-membrane porin from Pseudomonas aeruginosa. This highly purified protein P was used to successfully form hexagonal crystals in the presence of n-octyl-beta-glucopyranoside. Amino-acid analysis indicated that protein P had a similar composition to other bacterial outer membrane proteins, containing a high percentage (50%) of hydrophilic residues. The amino-terminal sequence of this protein, although not homologous to either outer membrane protein, PhoE or OmpF, of Escherichia coli, was found to have an analogous protein-folding pattern. Protein P in the native trimer form was capable of maintaining a stable functional trimer after proteinase cleavage. This suggested the existence of a strongly associated tertiary and quaternary structure. Circular dichroism studies confirmed these results in that a large proportion of the protein structure was determined to be beta-sheet and resistant to acid pH and heating in 0.1% sodium dodecyl sulphate.

Amino Acid Sequence↗

Antibiotic uptake into gram-negative bacteria.

Antibiotics taken up into gram-negative bacteria face two major diffusion barriers, the outer and cytoplasmic membranes. Of these, the former has been most studied and is discussed in detail here. Evidence from antibiotic MIC studies on porin-deficient mutants compared with their porin-sufficient parent strains has provided strong support for the proposal that some antibiotics, particularly beta-lactams, pass across the outer membrane through the water-filled channels of a class of proteins called porins. Nevertheless substantial evidence has accumulated for the importance of non-porin pathways of antibiotic uptake across the outer membranes of gram-negative bacteria. Examples discussed include the uptake of polycationic antibiotics via the self-promoted pathway, the uptake of hydrophobic antibiotics in some bacterial species and in mutants of others via the hydrophobic pathway, and the possible importance of poorly understood non-porin pathways of uptake of a variety of antibiotics. Other potential barriers to diffusion, including the cytoplasmic membrane, are briefly discussed.

Anti-Bacterial Agents↗

Regulation of components of the Pseudomonas aeruginosa phosphate-starvation-inducible regulon in Escherichia coli.

Plasmids pPBP and pRS-XP containing the cloned genes for the Pseudomonas aeruginosa phosphate-starvation-inducible periplasmic phosphate-binding protein and outer membrane porin P (oprP), respectively, were introduced into various Escherichia coli Pho-regulon regulatory mutants. Using Western immunoblots and specific antisera, the production of both gene products was observed to be under the control of regulatory elements of the E. coli Pho regulon. Sequencing of the region upstream of the translational start site of the oprP gene revealed a 'Pho box' with strong homology to the E. coli consensus 'Pho box', the putative binding site of the PhoB activator. Since P. aeruginosa and E. coli belong to different families and have quite different GC contents, these data suggest strong evolutionary conservation of regulatory elements of the Pho regulon.

Bacterial Outer Membrane Proteins↗

Enhanced binding of polycationic antibiotics to lipopolysaccharide from an aminoglycoside-supersusceptible, tolA mutant strain of Pseudomonas aeruginosa.

The lipopolysaccharide (LPS) of the aminoglycoside-supersusceptible Pseudomonas aeruginosa tolA mutant PAO1715 was compared with its parent strain PAO1670 and tol+ revertant PAO1716. Electrophoretic separation of purified LPSs from the three isolates showed similar LPS banding patterns. Analysis of the Western blots of these LPSs from the three isolates with O-antigen-specific monoclonal antibody indicated that the ladder pattern consisted of doublet bands, which presumably reflected a modification of core or lipid A; the level of one of the bands in the doublet was in much lower amounts in the isolate from the tolA mutant than in that from the parent or revertant. Results of competitive displacement experiments, in which the cationic spin probe 4-dodecyldimethylammonium-1-oxyl-2,2,6,6-tetramethylpiperidine bromide was displaced from its LPS-binding site by polycations, revealed that the tolA mutant had a much higher affinity for gentamicin, polymyxin, Ca2+, and Mg2+ than did the parent or revertant. The order of affinity for all samples was polymyxin B much greater than gentamicin C much greater than Ca2+ greater than Mg2+. Both gentamicin and polymyxin induced rigidification of all of the LPS samples, but for the sample from the tolA mutant, rigidification occurred at substantially lower concentrations. Dansyl polymyxin titration experiments with intact cells demonstrated that the increased affinity of the LPS from the tolA mutant for polycations was reflected in an increase in the affinity of binding to the cell. Together these data suggest that the tolA mutant is supersusceptible to aminoglycosides by virtue of an LPS change which increases the binding affinity of the LPS for polycations, including gentamicin.

Anti-Bacterial Agents↗

Interaction of macrophage cationic proteins with the outer membrane of Pseudomonas aeruginosa.

The interaction of the polycationic rabbit alveolar macrophage cationic proteins MCP-1 and MCP-2 (or their identical neutrophil equivalents NP-1 and NP-2) with the surface of Pseudomonas aeruginosa was investigated. Both proteins bound avidly to purified lipopolysaccharide, as judged by their ability to competitively displace the probe dansyl polymyxin with 50% inhibition (I50) values of 2 to 3 microM. Similar I50 were measured with dansyl polymyxin as a probe for cell surface binding, suggesting that the initial binding site for MCP-1 and MCP-2 on the surface of cells was lipopolysaccharide. Both MCP-1 and MCP-2 permeabilized outer membranes to the hydrophobic fluorescent probe 1-N-phenylnaphthylamine (NPN). The initial rate of NPN uptake plotted against the concentration of MCP-1 or MCP-2 gave sigmoidal curves, suggesting cooperative permeabilization of the outer membrane. Replotting the data as a Hill plot gave an affinity parameter, S0.5, the concentration of MCP giving a half-maximal increase in the rate of NPN uptake, of 5 and 25 microM for MCP-1 and MCP-2, respectively, and thus subsequent studies concentrated on the more active permeabilizer MCP-1. Permeabilization of outer membranes to NPN was a function of buffer pH, with lower pH considerably favoring the permeabilizing effects of MCP-1. Thin-section electron microscopic visualization of MCP-1-treated cells showed production of extended blebs. Further evidence of an altered cell surface after MCP-1 treatment was obtained by demonstrating that treated unopsonized cells were more efficiently phagocytosed by unelicited rabbit alveolar macrophages. The data overall suggest that macrophage cationic proteins interact with the P. aeruginosa outer membrane in a manner typical of other polycations and suggest that one of their major functions may be to permeabilize the outer membrane.

Antimicrobial Cationic Peptides↗

Heterogeneity of lipopolysaccharides from Pseudomonas aeruginosa: analysis of lipopolysaccharide chain length.

Lipopolysaccharide (LPS) from smooth strains of Pseudomonas aeruginosa 503, PAZ1, PAO1715, PAO1716, and Z61 was fractionated by gel filtration chromatography. LPS samples from the first four strains, all PAO1 derivatives, separated into three major size populations, whereas LPS from strain Z61, a Pac K799/WT mutant strain, separated into two size populations. When column fractions were applied to sodium dodecyl sulfate-polyacrylamide gels in their order of elution, molecules of decreasing size were resolved, and the ladder of molecules with different-length O antigens formed a diagonal across the gel. The LPS from the PAO1 derivatives contained two distinct sets of bands, distinguished on the gels as two sets of diagonals. The set of bands with the faster mobility, the B bands, was found in column fractions comprising the three major amino sugar-containing peaks. In the sample from strain 503, a fourth minor peak which contained B bands was resolved. The slower-moving set of bands, the A bands, were recovered in a minor peak. LPS from strain Z61 contained only one set of bands, with the higher-molecular-weight molecules eluting from the column in a volume similar to that of the B bands of the PAO1 strains. Analysis of the fractions of LPS from all strains indicated that less than 8% of the LPS molecules had a long, attached O antigen. Analysis of the peak that contained mainly A bands indicated a lack of reactive amino sugar and phosphate, although heptose and 2-keto-3-deoxyoctulosonic acid were detected. Reaction of isolated fractions with monoclonal antibody specific for the PAO1 O-antigen side chain indicated that only the B bands from the PAO1 strains were antigenically reactive. The bands from strain Z61 showed no reactivity. The data suggest that the A and B bands from the PAO1 strains are antigenically distinct. We propose that PAO1 strains synthesize two types of molecules that are antigenically different.

Antigens, Bacterial↗

Cloning of the Pseudomonas aeruginosa outer membrane porin protein P gene: evidence for a linked region of DNA homology.

The gene encoding the outer membrane phosphate-selective porin protein P from Pseudomonas aeruginosa was cloned into Escherichia coli. The protein product was expressed and transported to the outer membrane of an E. coli phoE mutant and assembled into functional trimers. Expression of a product of the correct molecular weight was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis, using polyclonal antibodies to protein P monomer and trimer forms. Protein P trimers were partially purified from the E. coli clone and shown to form channels with the same conductance as those formed by protein P from P. aeruginosa. The location and orientation of the protein P-encoding (oprP) gene on the cloned DNA was identified by three methods: (i) mapping the insertion point of transposon Tn501 in a previously isolated P. aeruginosa protein P-deficient mutant; (ii) hybridization of restriction fragments from the cloned DNA to an oligonucleotide pool synthesized on the basis of the amino-terminal protein sequence of protein P; and (iii) fusion of a PstI fragment of the cloned DNA to the amino terminus of the beta-galactosidase gene of pUC8, producing a fusion protein that contained protein P-antigenic epitopes. Structural analysis of the cloned DNA and P. aeruginosa chromosomal DNA revealed the presence of two adjacent PstI fragments which cross-hybridized, suggesting a possible gene duplication. The P-related (PR) region hybridized to the oligonucleotide pool described above. When the PstI fragment which contained the PR region was fused to the beta-galactosidase gene of pUC8, a fusion protein was produced which reacted with a protein P-specific antiserum. However, the restriction endonuclease patterns of the PR region and the oprP gene differed significantly beyond the amino-terminal one-third of the two genes.

Bacterial Outer Membrane Proteins↗

Construction and characterization of Pseudomonas aeruginosa protein F-deficient mutants after in vitro and in vivo insertion mutagenesis of the cloned gene.

Mutants with insertion mutations in the Pseudomonas aeruginosa protein F (oprF) gene were created in vivo by Tn1 mutagenesis of the cloned gene in Escherichia coli and in vitro by insertion of the streptomycin resistance-encoding omega fragment into the cloned gene, followed by transfer of the mutated protein F gene back to P. aeruginosa. Homologous recombination into the P. aeruginosa chromosome was driven by a bacteriophage F116L transduction method in the oprF::Tn1 mutants or Tn5-instability in the oprF::omega mutants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting demonstrated that the resultant oprF insertion mutants had lost protein F, whereas restriction digestion and Southern blotting experiments proved that the mutants contained a single chromosomal oprF gene with either Tn1 or omega inserted into it. It has been proposed that protein F has a role in antibiotic uptake in P. aeruginosa. Measurement of antibiotic resistance levels showed small to marginal increases in resistance, compared with that of the parent P. aeruginosa strain, to a variety of beta-lactam antibiotics. Protein F-deficient mutants had altered barrier properties as revealed by a three- to fivefold increase in the uptake of the hydrophobic fluorescent probe 1-N-phenylnaphthylamine.

Anti-Bacterial Agents↗

Outer membranes of environmental isolates of Pseudomonas aeruginosa.

The outer membrane composition of 30 environmental isolates of Pseudomonas aeruginosa was examined. Other than variations in the amounts of lipoprotein H2, there were no major differences in the outer membrane protein or lipopolysaccharide patterns when compared with those of previously studied clinical isolates.

Bacterial Outer Membrane Proteins↗