Search PubMed⌕ Search

Biomedical subjects

J T Buckley

Publications and source records attributed to J T Buckley.

At least 73 records · Page 4Linked to original sources
PubMed1990

Crystallization of a proform of aerolysin, a hole-forming toxin from Aeromonas hydrophila.

Crystals of proaerolysin, the precursor of the hole-forming toxin from Aeromonas hydrophila, have been obtained. The mature form of this protein binds to a receptor on mammalian cells, aggregates and forms 30 A holes in the membrane. The crystals are tetragonal, space group P4(3)2(1)2, a = b = 104.00 A, c = 222.0 A. They contain a dimer in the asymmetric unit and diffract to a resolution of 2.6 A.

Aeromonas↗
PubMed1990

Site-directed mutagenesis of the hole-forming toxin aerolysin: studies on the roles of histidines in receptor binding and oligomerization of the monomer.

The six histidines of the channel-forming protein aerolysin have been replaced one at a time with asparagine by site-directed mutagenesis, and each of the modified proteins has been purified. Three proteins had the same hemolytic activity as native toxin, but the others, those changed at His107, His132, or His332, were less able to disrupt both human and rat erythrocytes. The largest reduction in activity, more than 100-fold, was observed with the His132 mutant protein. Studies with radioiodinated samples showed that it had approximately the same affinity as native aerolysin for the rat erythrocyte receptor. However, once bound to either rat or human erythrocytes, it was much less able to carry out the next essential step in hole formation, aggregation to form a stable oligomer. Aggregation was also reduced by replacing His107, but the contrast with native aerolysin and the effect on hemolytic activity were less pronounced. The protein modified at His332 behaved in a different way from those substituted at positions 107 and 132. Its affinity for the rat erythrocyte receptor was considerably lower than the affinity of the wild-type protein, but when bound it appeared to aggregate normally. The results suggest that His132 and perhaps His107 are involved in the aggregation of aerolysin whereas His332 may be at or near the receptor binding site.

Aeromonas↗
PubMed1990

Aerolysin, a hemolysin from Aeromonas hydrophila, forms voltage-gated channels in planar lipid bilayers.

The cytolytic toxin aerolysin was found to form ion channels which displayed slight anion selectivity in planar lipid bilayers. In voltage-clamp experiments the ion current flowing through the channels was homogeneous indicating a defined conformation and a uniform size. The channels remained open between -70 to +70 mV, but outside this range they underwent voltage-dependent inactivation which was observed as open-closed fluctuations at the single-channel level. Zinc ions not only prevented the formation of channels by inhibiting oligomerization of monomeric aerolysin but they also induced a closure of preformed channels in a voltage-dependent fashion. The results of a Hill plot indicated that 2-3 zinc ions bound to a site within the channel lumen. Proaerolysin, and a mutant of aerolysin in which histidine 132 was replaced by an asparagine, were both unable to oligomerize and neither could form channels. This is evidence that oligomerization is a necessary step in channel formation.

Aeromonas↗
PubMed1990

Cloned aerolysin of Aeromonas hydrophila is exported by a wild-type marine Vibrio strain but remains periplasmic in pleiotropic export mutants.

With a wide host range vector, the structural gene aerA for the hole-forming extracellular protein aerolysin of Aeromonas hydrophila was cloned into the marine Vibrio sp. strain 60 and into three pleiotropic export mutants (epr mutants). The parent strain and all of the mutants were able to express the protein with the aerA promoter in the plasmid. The parent strain exported proaerolysin into the medium, while all of the mutants accumulated the protoxin in their periplasms. Two of the mutants also accumulated protease; however, as we have found earlier with A. hydrophila, the periplasmic form of proaerolysin in the Vibrio sp. must somehow be protected from proteolysis because it was not converted to active toxin until the cells were shocked. Conversion could be prevented by adding o-phenanthroline to the solutions used in shocking. These results show that the export pathway in the marine Vibrio sp. is very similar to the pathway in A. hydrophila.

Aeromonas↗
PubMed1990

Purification of cloned proaerolysin released by a low protease mutant of Aeromonas salmonicida.

The precursor to the hole-forming toxin aerolysin has been purified in high yield from culture supernatants of a mutant of Aeromonas salmonicida containing the cloned structural gene. The mutant strain was generated by Tn5 mutagenesis. It released little or no protease or other extracellular proteins, including phospholipase, suggesting that it is a regulatory mutant. The absence of protease allowed the isolation of protoxin free from contaminating aerolysin. Typically, more than 50 mg of pure proaerolysin was obtained from 2 L of culture supernatant. The purified protein was completely unable to lyse human erythrocytes without prior activation with trypsin.

Aeromonas↗
PubMed1989

Proton motive force involved in protein transport across the outer membrane of Aeromonas salmonicida.

Many Gram-negative bacteria export proteins to the exterior. Some of these proteins are first secreted into the periplasm and then cross the outer membrane in a separate step. The source of energy required for the translocation is unknown. Export of the extracellular protein proaerolysin from the periplasm through the outer membrane of Aeromonas salmonicida is inhibited by a proton ionophore and by low extracellular pH. One possible explanation of these results is that a proton gradient across the outer membrane is required for export.

Aeromonas↗
PubMed1989

Extracellular secretion of cloned aerolysin and phospholipase by Aeromonas salmonicida.

The promoterless structural genes for aerolysin and the extracellular phospholipase of Aeromonas hydrophila were inserted into a multi-host-range expression vector and transferred into Aeromonas salmonicida and Escherichia coli. In both species, gene expression was under the control of the inducible tac promoter of the vector. Neither the phospholipase nor the aerolysin was released by intact E. coli. Instead, both proteins accumulated in the periplasm, leading to reduced growth and eventual cell death. When the aerolysin gene inserted into the vector contained its own promoter, the toxin was expressed constitutively by A. salmonicida but not by E. coli. Production of aerolysin and the phospholipase by A. salmonicida did not affect cell growth, and the proteins were correctly processed and exported by intact cells. Both proteins could also be detected in the periplasm, where their concentrations were considerably higher then they were outside the cells. Periplasmic aerolysin was rapidly released when cells were transferred to fresh medium, indicating that this compartment is part of the normal export pathway and that the protein is not shunted there as a consequence of overproduction. Plasmid-coded aerolysin did not appear to compete with the cell proteins for export components, as even when very large quantities of aerolysin were being exported by A. salmonicida, there was no effect on chromosomal protease release and only a modest reduction in the export of chromosomal phospholipase.

Acyltransferases↗
PubMed1988

Molecular cloning of a phospholipid-cholesterol acyltransferase from Aeromonas hydrophila. Sequence homologies with lecithin-cholesterol acyltransferase and other lipases.

We have determined the nucleotide sequence of a gene encoding Aeromonas hydrophila phospholipid-cholesterol acyltransferase, an enzyme which shares many properties with mammalian lecithin:cholesterol acyltransferase. The derived amino acid sequence of the protein contains two regions which are homologous to the proposed active sites and binding sites of the plasma acyltransferase and to similar sequences in other interfacially acting lipolytic enzymes. The amino terminus is preceded by a typical 18 amino acid signal sequence. The protein, which is released into the culture supernatant by Aeromonas hydrophila, is confined to the periplasm of Escherichia coli.

Acyltransferases↗
PubMed1988

The cytolytic toxin aerolysin must aggregate to disrupt erythrocytes, and aggregation is stimulated by human glycophorin.

The hole-forming toxin aerolysin was shown to aggregate after binding to erythrocytes at 37 degrees C. Although the protein also bound and aggregated at 4 degrees C, hole formation was not observed, indicating that aggregation preceded penetration of the lipid bilayer. Aggregation, but not binding, could be blocked by pretreatment of the toxin with diethyl pyrocarbonate, a histidine-reactive reagent. This resulted in inactivation of the toxin. Incubation of aerolysin with glycophorin purified from human erythrocytes caused aggregation and complete inactivation. Erythrocytes which lacked glycophorin were less sensitive to the toxin. Proaerolysin, the inactive precursor of aerolysin, also bound to erythrocytes; however, it did not aggregate, nor did it aggregate when preincubated with glycophorin. The protoxin could be activated by treatment with trypsin even after it had bound to erythrocytes. Activation could also be achieved by reaction of proaerolysin with a variety of other proteases, each of which brought about a similar reduction in protein molecular weight. The activated protein was resistant to further proteolysis. These results indicate that aggregation is a necessary step in hole formation and that the sites on aerolysin required for binding and for aggregation and hole formation are separate.

Bacterial Toxins↗
PubMed1986

Molecular cloning and expression in Escherichia coli of the structural gene for the hemolytic toxin aerolysin from Aeromonas hydrophila.

The structural gene for the hemolytic toxin aerolysin has been cloned into the plasmid vectors pBR322 and pEMBL8+. The gene was localized on the hybrid plasmids by analysis of plasmids generated by transposon mutagenesis. The sequence of the first 683 bases of an insert in pEMBL8+ was determined and shown to encode the amino terminus of the protein as well as a typical signal sequence of 23 amino acids. Aerolysin is produced by E. coli cells containing the cloned aerolysin gene and it is processed normally by removal of the signal sequence, however it is not released from the cell. The protein appears to be translocated across the inner membrane of E. coli as its signal sequence is removed and the processed protein can be released by osmotic shock.

Aeromonas↗
PubMed1985

Phospholipids and lipopolysaccharide of Aeromonas hydrophila.

The phospholipids and lipopolysaccharide of Aeromonas hydrophila were characterized. Phosphatidylethanolamine and phosphatidylglycerol were the major phospholipid components. The outer membrane contained more phosphatidylethanolamine and less phosphatidylglycerol than the inner membrane, and the phospholipids of the outer membrane contained a higher proportion of saturated fatty acids. Only four fatty acids (C14:0, C16:0, C16:1, and C18:1) were found in the phospholipids. The lipopolysaccharide of A. hydrophila did not contain the eight-carbon sugar 3-deoxyoctulosonic acid nor did it contain C16:0, both of which are typical constituents of the lipopolysaccharide of many other species.

Aeromonas↗
PubMed1985

Protein export by a gram-negative bacterium: production of aerolysin by Aeromonas hydrophila.

The synthesis and export of aerolysin, an extracellular protein toxin released by the gram-negative bacterium Aeromonas hydrophila, was studied by pulse-labeling with [35S]methionine. The toxin was synthesized as a higher-molecular-weight precursor. This was processed cotranslationally, resulting in the appearance within the cell of the mature protein, which was then exported to the supernatant. Precursor aerolysin accumulated in cells incubated in the presence of carbonyl cyanide m-chlorophenyl hydrazone, a substance which also inhibited the export of mature aerolysin from the cell. The entrapped mature toxin could not be shocked from the cells, although it could be digested by protease applied to shocked cells. The toxin was processed and translocated across the inner membrane of pleiotropic export mutants and accumulated in the periplasm. The results indicate that more than one step is required for the export of the protein and that aerolysin does not cross the inner and outer membranes simultaneously.

Aeromonas↗
PubMed1985

Activation of the hole-forming toxin aerolysin by extracellular processing.

A precursor-product relationship between aerolysin and a protein with a higher molecular weight was observed in culture supernatants of Aeromonas hydrophila. The larger protein was isolated by ammonium sulfate precipitation and ion-exchange and hydroxyapatite chromatography and compared with purified aerolysin. It was at least 250 times less hemolytic than aerolysin. Both proteins had the same amino acid sequence at the amino terminus. Cyanogen bromide fragments obtained from the two were identical except that each protein contained one unique fragment, and the fragment from the larger protein was 2,500 daltons larger than the fragment obtained from aerolysin. Treatment with trypsin or with an extracellular Aeromonas protease resulted in rapid conversion of the larger protein to a form corresponding in molecular weight and activity to aerolysin. The results indicate that aerolysin is exported to the culture supernatant as a protoxin which is later activated by proteolytic removal of a peptide from the C terminus.

Aeromonas↗
PubMed1985

Cone-shaped lipids increase the susceptibility of phospholipids in bilayers to the action of phospholipases.

Addition of cardiolipin or diacylglycerol to dispersions of phosphatidylcholine greatly increased hydrolysis by snake venom or pancreatic phospholipase A2, as well as by a microbial phospholipase. Monogalactosyl diglyceride which, like cardiolipin and diacylglycerol, will form nonbilayer hexagonal II structures also caused an increase in the breakdown of phosphatidylcholine. Addition of digalactosyl diglyceride, a bilayer lipid from the same source, had a much smaller effect on the three phospholipases, indicating that stimulation by the nonbilayer lipids was not due to their fatty acid compositions. Stimulation of the microbial phospholipase by cardiolipin did not require the presence of calcium, leading to the conclusion that the formation of nonbilayer structures was not necessary. The results suggest that cone-shaped lipids increase the accessibility of lipids in bilayers to phospholipases by decreasing the packing of the polar head groups.

Animals↗
PubMed1984

Action of a microbial glycerophospholipid:cholesterol acyltransferase on plasma from normal and LCAT-deficient subjects.

The action of a bacterial acyltransferase similar in overall reaction mechanism to the plasma enzyme lecithin:cholesterol acyltransferase (LCAT) has been studied using normal plasma and lipoproteins and plasma from LCAT-deficient patients. The microbial enzyme (GCAT) catalyzed acyl transfer using phosphatidylcholine and cholesterol in all of the lipoprotein fractions, presumably because it has no apolipoprotein cofactor. In addition, the enzyme was capable of hydrolyzing cholesteryl ester in lipoproteins but not in small unilamellar vesicles nor in micellar dispersions containing low amounts of Triton X-100. This suggests that cholesteryl ester is exposed on the surface of lipoprotein particles or that it may be transferred there quickly from the interior. Although considerable interconversion of radiolabeled cholesterol and cholesteryl ester could be demonstrated upon treatment of normal plasma or lipoproteins with the enzyme, there was little change in the actual amount of either steroid. This indicates that the rate of cholesteryl ester formation is very similar to the rate of hydrolysis. The relative proportions of cholesterol and cholesteryl ester in normal plasma are therefore near the equilibrium ratio for the reaction carried out by GCAT, or the ratio is controlled by the properties of the lipoproteins themselves. During reaction with the microbial acyltransferase, the ratio of cholesterol to cholesteryl ester in plasma from LCAT-deficient patients was reduced substantially, suggesting that the enzyme may have some practical applications.

Acyltransferases↗
PubMed1983

Intracellular accumulation of extracellular proteins by pleiotropic export mutants of Aeromonas hydrophila.

Pleiotropic export mutants of Aeromonas hydrophila were obtained which are unable to release protease, hemolysin, and glycerophospholipid:cholesterol acyltransferase. The synthesis of the proteins was not impaired; they were accumulated in active forms inside the mutant cells. The hemolysin could be isolated from cell contents by immunoprecipitation in a form with the same apparent molecular weight as the wild-type extracellular product, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Because both the protease and the hemolysin could be released from the mutant cells by osmotic shock, it was concluded that they were accumulated in the periplasmic space. Some mutants were missing two major outer membrane proteins, both of which reappeared in revertants with the wild-type excretory phenotype. Another mutant class had a normal outer membrane protein profile. That two different mutant classes could be obtained indicates that at least two gene products may be needed for export after protein translocation through the inner membrane. The accumulation of proteins which can be released by osmotic shock suggests that the periplasm may be part of the normal route for protein export.

Aeromonas↗