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P Elsbach

Publications and source records attributed to P Elsbach.

At least 73 records · Page 4Linked to original sources
PubMed1985

Oxygen-independent intracellular and oxygen-dependent extracellular killing of Escherichia coli S15 by human polymorphonuclear leukocytes.

Effective killing of bacteria by polymorphonuclear leukocytes (PMN) is generally assumed to require intracellular sequestration and, depending on the bacterial species, can be both O2-dependent or O2-independent. Killing of several strains of Salmonella typhimurium and Escherichia coli by rabbit PMN does not require O2 and is apparently due to a granule-associated bactericidal/permeability-increasing protein (BPI) present in rabbit and human PMN. In this study we examined the O2 dependence of the killing of E. coli (S15) by human PMN. Ingested and noningested E. coli were separated by centrifugation after incubation with PMN in room air or under N2. In the presence of heat-treated serum approximately 50% of E. coli (10 bacteria/PMN) were taken up by PMN and rapidly (5-15 min) killed both in room air and under N2. The remaining extracellular bacteria (approximately 50%) were killed during 30-60 min of incubation in room air but not under N2. When uptake of E. coli by PMN was increased to approximately 80% by the use of C6-depleted serum (retaining heat-labile opsonins), bacterial survival under N2 was reduced from 54 +/- 7.6% to 13 +/- 5.5%. PMN from a patient with chronic granulomatous disease killed PMN-associated but not extracellular E. coli. BPI was detected, by indirect immunofluorescence, on the surface of PMN-associated E. coli within 5 min of incubation of E. coli with PMN both in room air and under N2. In contrast, at no time was BPI detected on the surface of extracellular E. coli, indicating that the non-PMN-associated E. coli had not been previously ingested. Thus, killing of ingested E. coli S15 by human as well as rabbit PMN does not require O2 and appears to be BPI-mediated. However, when ingestion is limited, extracellular bacteria can also be killed but principally by O2-dependent mechanisms.

Antimicrobial Cationic Peptides↗
PubMed1984

The role of lipopolysaccharides in the action of the bactericidal/permeability-increasing neutrophil protein on the bacterial envelope.

The killing of gram-negative bacteria by the bactericidal/permeability-increasing protein ( BPI ) of neutrophils requires surface binding, and is accompanied by a discrete increase in outer membrane permeability to small hydrophobic substances. This outer membrane alteration appears to be related to perturbation of outer membrane lipopolysaccharides (LPS). BPI causes extracellular release of LPS, but only at supra-saturating doses. Nevertheless, because the organization of LPS in the outer membrane is altered by pretreatment of bacteria with saturating doses of BPI (producing maximal bactericidal and permeability-increasing effects), the amount of LPS released during Tris-EDTA treatment is reduced by 80%. BPI markedly (approximately 50%) and selectively stimulates biosynthesis of LPS, suggesting an attempt by BPI -killed bacteria to repair outer membrane damage. The removal of surface-bound BPI by 40 mM Mg2+ initiates time- and temperature-dependent repair of the outer membrane permeability barrier and a further increase (approximately 170% of control) in LPS synthesis, even though the bacteria are no longer viable. Mg2+-induced repair is blocked when: 1) a temperature-sensitive mutant (Salmonella typhimurium HD50 ) with a conditional defect in LPS synthesis is incubated at the nonpermissive temperature (42 degrees C); and 2) LPS synthesis is selectively inhibited by a diazaborine derivative (Sandoz drug No. 84474). In contrast, repair is normal by the mutant at permissive temperatures (30 degrees C) and by the parent strain (S. typhimurium AG701 ) at both 30 degrees C and 42 degrees C. Inhibition (greater than 85%) of protein synthesis by chloramphenicol has little or no effect on repair. These findings indicate that the repair of the permeability barrier after the removal of BPI from the surface requires newly made LPS, but apparently no biosynthesis of other outer membrane constituents, which strongly suggests that the effects of BPI on LPS are mainly responsible for the break-down of the outer membrane permeability barrier.

Animals↗
PubMed1983

Role of charge and hydrophobic interactions in the action of the bactericidal/permeability-increasing protein of neutrophils on gram-negative bacteria.

We have recently provided evidence suggesting that the action of purified cationic bactericidal/permeability-increasing protein (BPI) from neutrophils on susceptible gram-negative bacteria requires saturation binding to negatively charged surface sites (Weiss, J., S. Beckerdite-Quagliata, and P. Elsbach, 1980, J. Clin. Invest., 65: 619-628.)We now show that this charge interaction is necessary but not sufficient to produce the effects of BPI on the envelope and on viability. By altering the hydrophobic properties of the bacterial (outer) membrane, it is possible to separate saturation binding from the biological action of BPI, indicating that steps beyond surface binding are needed for the antibacterial action. Outer membrane properties were modified by (a) reducing temperature during BPI-Escherichia coli interaction; (b) growing E. coli at 42 degrees C to increase the saturated fatty acid content of membrane phospholipids; and/or (c) using smooth E. coli with a natively less fluid outer membrane. Hydrophobic interaction chromatography on phenyl-Sepharose and measurement of sensitivity to the hydrophobic antibiotic rifampicin were used to monitor the changes in hydrophobic properties of the bacterial outer membrane produced by these manipulations. Nearly all BPI can be removed from the bacterial surface by 80 mM MgCl(2) or by trypsin. At 37 degrees C, removal of BPI results in repair of the envelope alterations, but viability is irreversibly lost, even when Mg(2+) is added after only 15 s of exposure of the bacteria to BPI. However, under conditions of reduced outer membrane hydrophobicity, when saturation binding still occurs within 30 s, E. coli can be rescued by addition of Mg(2+) after up to 5-min exposure to BPI, indicating retardation of postbinding steps. We conclude that after initial binding BPI must enter into a hydrophobic interaction with the outer membrane in order to produce its antibacterial effects. These postbinding events reversibly mediate the membrane perturbations and irreversibly trigger the bactericidal action of BPI.

Animals↗
PubMed1982

The role of phospholipase A2 lysines in phospholipolysis of Escherichia coli killed by a membrane-active neutrophil protein.

Purified rabbit bactericidal/permeability-increasing protein at bactericidal concentrations is a membrane-perturbing agent that triggers hydrolysis of envelope phospholipids of a phospholipase A-less Escherichia coli (S17) mutant by a highly basic (pI greater than 10) phospholipase A2, purified from Agkistrodon halys blomhoffii snake venom. Most other purified phospholipases A2 do not degrade the phospholipids of E. coli killed by the bactericidal protein. To study the role of enzyme charge in bactericidal protein-dependent phospholipid hydrolysis, lysines of the Agkistrodon phospholipase A2 were modified, either by carbamylation (decreases net charge), or by reductive methylation (no delta charge). Incorporation of [14C]cyanate or [14C]formaldehyde and amino acid analysis served to monitor modification. Modification appears to be limited to epsilon-NH2 groups. Incorporation of up to 5 mol of cyanate or formaldehyde/mol of enzyme did not affect catalytic activity. In contrast, incorporation of, on average, 1 mol of either reagent/mol of protein reduced by 80% the activity of the enzyme toward E. coli S17 killed by the bactericidal protein. Since this loss is similar with carbamylation and reductive methylation, the role of the epsilon-NH2 group in the bactericidal protein-dependent hydrolysis seems independent of charge. Thus, the lysines in this phospholipase A2 are not essential for catalysis and substrate binding, but are essential for the action of this enzyme on E. coli killed by the bactericidal protein.

Animals↗
PubMed1982

Sensitivity of K1-encapsulated Escherichia coli to killing by the bactericidal/permeability-increasing protein of rabbit and human neutrophils.

The presence of K1 capsular polysaccharides increases the resistance of Escherichia coli to killing by serum and phagocytosis by polymorphonuclear leukocytes (PMNs). To determine whether K1 capsule impedes the action of intracellular bactericidal systems of PMNs, we compared the sensitivity of several K1-encapsulated and non-encapsulated strains of E. coli to killing by the bactericidal/permeability-increasing protein (BPI) isolated from rabbit and human PMNs. BPI appears to be the principal bactericidal agent of PMNs toward E. coli and other gram-negative bacteria (Weiss et al., J. Clin. Invest. 69:959-970, 1982). The presence of K1 capsule was monitored by sensitivity to K1-specific bacteriophages. The non-encapsulated strains used represent both random bacteremic isolates and non-encapsulated derivatives of K1-encapsulated strains obtained by selection for resistance to K1-specific phages. We found little or no difference in the sensitivity of K1-encapsulated and non-encapsulated E. coli to killing by neutralized acid extracts of rabbit PMNs. Bacterial killing by these crude fractions can be attributed to the action of BPI because: (i) bacterial killing was blocked by immune (anti-BPI) immunoglobulin but not by preimmune immunoglobulin and (ii) comparison of the dose-response curves of bacterial killing by crude extracts and by purified BPI showed that the bactericidal activity of crude fractions corresponded closely to the BPI content. Human and rabbit BPIs exhibited similar bactericidal potency toward K1-encapsulated E. coli; i.e., <5 mug of either protein killed >90% of 2.5 x 10(7) bacteria. Thus, the potent bactericidal action of BPI toward E. coli is not impeded by K1 capsule, suggesting that the virulence of K1-encapsulated E. coli is a consequence of extracellular survival but not of resistance to intracellular killing.

Animals↗
PubMed1982

Killing of gram-negative bacteria by polymorphonuclear leukocytes: role of an O2-independent bactericidal system.

Previous studies have suggested that a cationic bactericidal/permeability-increasing protein (BPI) present in both rabbit and human polymorphonuclear leukocytes is the principal O2-independent bactericidal agent of these cells toward several strains of Escherichia coli and Salmonella typhimurium (1978. J. Biol. Chem. 253: 2664--2672; 1979. J. Biol. Chem. 254: 11000--11009). To further evaluate the possible role of this protein in the killing of gram-negative bacteria by polymorphonuclear leukocytes, we have measured the bactericidal activity of intact rabbit peritoneal exudate leukocytes under aerobic or anaerobic conditions and of intact human leukocytes from a patient with chronic granulomatous disease. Anaerobic conditions were created by flushing the cells under a nitrogen stream. Effective removal of oxygen was demonstrated by the inability of nitrogen-flushed leukocytes to mount a respiratory burst (measured as increased conversion of 1-[14C]glucose leads to 14CO2 or by superoxide production) during bacterial ingestion. At a bacteria/leukocyte ratio of 10:1, killing of gram-positive, BPI-resistant, Staphylococcus epidermidis is markedly impaired in the absence of oxygen (76.4 +/- 3.3% killing in room air, 29.2 +/- 8.2% killing in nitrogen). Essentially all increased bacterial survival is intracellular. In contrast, both a nonopsonized rough strain (MR-10) and an opsonized smooth strain (MS) of S. typhimurium 395 are killed equally well in room air and nitrogen. A maximum of 70--80 MR-10 and 30--40 MS are killed per leukocyte either in the presence or absence of oxygen. There is no intracellular bacterial survival in either condition indicating that intracellular O2-independent bactericidal system(s) of rabbit polymorphonuclear leukocytes can at least match the leukocyte's ingestive capacity. Whole homogenates and crude acid extracts manifest similar bactericidal capacity toward S. typhimurium 395. This activity can be accounted for by the BPI content of these cell fractions and is virtually eliminated by immune (anti-BPI), but not by preimmune goat IgG-rich fractions. Opsonization of smooth MS, required for bacterial killing by intact leukocytes, does not alter bacterial sensitivity to BPI in crude or purified form. Leukocytes of a patient with chronic granulomatous disease killed ingested S. typhimurium 396 MS nearly as well as did normal leukocytes. The bactericidal activity toward E. coli (J5) of crude acid extracts of the CGD and normal human leukocytes was virtually the same and was nearly completely inhibited by anti-BPI IgG-rich fractions, but not by preimmune IgG-rich fractions. These findings suggest that the killing of gram-negative bacteria such as S. typhimurium by intact polymorphonuclear leukocytes may also be attributed to the action of BPI.

Aerobiosis↗
PubMed1981

Heparin inhibits phagocytosis by polymorphonuclear leukocytes.

Phagocytosis of unopsonized Salmonella typhimurium 395, MR-10, opsonized Salmonella typhimurium 395 MS, and Staphylococcus epidermidis by rabbit polymorphonuclear leukocytes was inhibited by heparin at concentrations as low as 0.5 U/ml. Inhibition was dose dependent and nearly complete at 20 U/ml. Provided that heparin concentrations did not exceed 100 U/ml, inhibition could be largely reversed by washing. Heparin also reversibly inhibited the adherence of polymorphonuclear leukocytes to glass. In contrast, hexose monophosphate shunt activity of polymorphonuclear leukocytes stimulated by noningested S. typhimurium MR-10 or Streptococcus pyogenes B14 was not inhibited by heparin at concentrations as high as 100 U/ml.

Animals↗
PubMed1980

Resistance of gram-negative bacteria to purified bactericidal leukocyte proteins: relation to binding and bacterial lipopolysaccharide structure.

The sensitivity or resistance of gram-negative bacteria to antibacterial systems appears to be related to the length of the saccharide chain of the bacterial envelope lipopolysaccharides (LPS). To explore this relationship further, we made use of two bactericidal, membrane-active cationic proteins, recently purified to near homogeneity, one from human and one from rabbit polymorphonuclear leukocytes (PMN). We have studied the effects of these two closely similar proteins on strains of Salmonella typhimurium and Escherichia coli, each separate strain differing in the saccharide chain length of its outer membrane LPS. Binding of these proteins to the bacterial outer membrane is required for killing, and is accompanied by an almost immediate increase in outer membrane permeability to normally impermeant actinomycin D. Sensitivity to the bactericidal and permeability-increasing activities of the human and rabbit proteins increases with decreasing LPS-saccharide chain length (chemotype: [S < Ra < Rb(3) < Rc < Rd(1)]). S. typhimurium G-30 and E. coli J5, mutant strains lacking UDP-galactose-4-epimerase, synthesize incomplete LPS (chemotype Rc) when grown without galactose, and are then as sensitive to both PMN proteins as the S. typhimurium strains 395 R10 (Rd(1)) and R5 (Rb(3)). However, when these mutants are grown with galactose, they synthesize complete LPS (chemotype S) and exhibit nearly the same relative insensitivity as the smooth strains S. typhimurium 395 MS and E. coli 0111:B4. The differences among strains in sensitivity to the effects of the proteins on bacterial viability and permeability correspond to differences in bacterial binding of these PMN proteins. Thus, at protein concentrations that produce maximal antibacterial activity toward the rough bacteria, but little or no activity toward the smooth strains, rough bacteria bind from 3- to 10-fold more protein (S. typhimurium 395 R10; S. typhimurium G-30, and E. coli J5 [grown without galactose]) than do the smooth bacteria (S. typhimurium 395 MS; E. coli 0111:B4; S. typhimurium G-30 and E. coli J5 [grown with galactose]). These findings suggest that bacterial sensitivity or resistance to these purified bactericidal PMN proteins is determined by the binding properties of the outer membrane, which in turn depends upon the LPS-saccharide chain length.

Animals↗
PubMed1980

The action of human and rabbit serum phospholipase A2 on Escherichia coli phospholipids.

We have compared the properties of phospholipase A (E.C. 3.1.1.4) activity in whole human and rabbit serum toward the phospholipids of Escherichia coli. Using as substrate E. coli labeled during growth with either [1-(14)C]-palmitic acid or [1-(14)C]oleic acid, and then autoclaved to inactivate E. coli phospholipases and to render the labeled phospholipids accessible to exogenous phospholipases, we show that the deacylating activity in both human and rabbit serum is almost exclusively of the A(2) type. Rabbit serum is at least 20-fold more active than human serum. Activity in both sera is maximal at physiological Ca(2+) concentrations (2 mM) and is abolished by ethylenediaminetetraacetic acid. To examine hydrolysis of intact (unautoclaved) E. coli treated with 25% serum, use was made of a phospholipase A-deficient E. coli strain (E. coli S17), thereby eliminating the possible contribution of bacterial phospholipases to degradation. Human and rabbit serum are about equally bactericidal toward E. coli and cause comparable structural damage. However, only rabbit serum produces substantial hydrolysis of the phospholipids of intact E. coli S17. Heated (56 degrees C, 30 min) rabbit serum is non-bactericidal and retains phospholipase A(2) activity toward autoclaved, but not intact E. coli. The ability of heated serum to degrade phospholipids of intact E. coli S17 is restored, however, by adding 25% normal human serum, which is bactericidal. In this combination, doses of heated rabbit serum containing as much phospholipase A(2) activity (toward autoclaved E. coli) as is present in 25% unheated rabbit serum, produce roughly the same extent of hydrolysis of intact E. coli as does normal rabbit serum alone. Low doses with a phospholipase A(2) activity comparable to that of normal human serum elicit little or no hydrolysis. These findings indicate that hydrolysis of the phospholipids of intact E. coli S17 by serum occurs when: 1) the serum is bactericidal, and 2) when sufficient phospholipase A(2) is present. The difference in phospholipid hydrolysis that accompanies killing of E. coli by human or rabbit serum appears to reflect, therefore, the different amounts of phospholipase A(2) activity in the two sera. Phospholipid degradation is not required for the bactericidal action of serum. Bacterial phospholipid breakdown may be important, however, in the overall destruction and digestion of invading bacteria by the host.-Kaplan-Harris, L., J. Weiss, C. Mooney, S. Beckerdite-Quagliata, and P. Elsbach. The action of human and rabbit serum phospholipase A(2) on Escherichia coli phospholipids.

Animals↗
PubMed1979

Separation and purification of a potent bactericidal/permeability-increasing protein and a closely associated phospholipase A2 from rabbit polymorphonuclear leukocytes. Observations on their relationship.

Two antibacterial proteins from rabbit polymorphonuclear leukocytes, a potent bactericidal cationic protein that increases the envelope permeability of susceptible gram-negative bacteria and a phospholipase A2, have been purified to near homogeneity by ion exchange, gel filtration, and hydrophobic interaction chromatography. The apparently noncatalytic bactericidal/permeability-increasing protein has an approximate molecular weight of 50,000 and is isoelectric at pH 9.5 to 10.0. The molecular properties, including amino acid composition, and the antibacterial potency and specificity of this rabbit leukocyte protein and of the bactericidal/permeability-increasing protein from human granulocytes that we have recently purified (J. Biol. Chem. 253, 2664-2672, 1978) are closely similar. Both proteins kill several strains of Escherichia coli and Salmonella typhimurium. Rough strains are more sensitive than smooth strains. All gram-positive bacterial species tested are insensitive to high concentrations of either rabbit or human protein. The phospholipase A2, purified by hydrophobic interaction chromatography on phenyl-Sepharose, ran as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 14,000 and had a specific enzymatic activity comparable to that of purified phospholipases A2 from other sources. Separation of the phospholipase A2 from the bactericidal/permeability-increasing protein has no noticeable effect on the bactericidal and permeability-increasing activities of the purified bactericidal protein, but removes the ability of the phospholipase A2 to hydrolyze the phospholipids of intact Escherichia coli. Upon recombination of the phospholipase A2 with the bactericidal/permeability-increasing protein, the phospholipase A2 regains its activity toward the phospholipids of intact E. coli suggesting that these two antibacterial leukocyte proteins act in concert.

Amino Acids↗
PubMed1978

Low concentrations of indomethacin inhibit phospholipase A2 of rabbit polymorphonuclear leukocytes.

Inhibition of prostaglandin synthesis by indomethacin, a drug with anti-inflammatory properties, has been attributed to its action on fatty acid cyclooxygenase. However, prostaglandin synthesis would also be inhibited if precursor fatty acids were not supplied. We find that indomethacin inhibits phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) of rabbit polymorphonuclear leukocytes in dose-dependent fashion. Inhibition is immediate and readily detected at 1 micrometer. The extent of inhibition is the same over a 10-fold range of substrate concentration and over a 500-fold range of enzyme purification. Inhibition is of the noncompetitive type, with an apparent Ki of 12 micrometer. Four other phospholipases A2--from venoms of Russell viper, Crotalus adamanteus, and bee, and from pig pancreas--are unaffected by 50 micrometer indomethacin, which inhibits leukocyte phospholipase A2 by 70%. This inhibition at low concentrations may well be important in the effects of the drug on protaglandin synthesis and inflammatory responses.

Animals↗
PubMed1978

Isolation and characterization of a phospholipase A2 from an inflammatory exudate.

Sterile peritoneal exudates produced in rabbits injected with 1% glycogen contain a phospholipase A activity in a cell-free supernatant fraction that hydrolyzed a synthetic phospholipid (1,2-diacyl-sn-glycero-3-phospho-ethanolamine) and phospholipids of autoclaved Escherichia coli. This phospholipase activity (phosphatidylacylhydrolase EC 3.1.1.4) exhibited an apparent bimodal pH optimum (pH 6.0 and pH 7.5) and was Ca(2+)-dependent; Mg(2+) and monovalent cations (Na(+) and K(+)) did not substitute for Ca(2+) in the reaction; EDTA was a potent inhibitor. The phospholipase hydrolyzed 1-[1-(14)C]palmitoyl-2-acyl-sn-glycero-3-phosphoethanolamine to form only radio-active lysophosphatidylethanolamine as the product, indicating that the enzyme had phospholipase A(2) specificity. The phospholipase A(2) was purified 302-fold by two successive chromatographic steps on carboxymethyl Sephadex. Gel filtration (Sephadex G75) of the purified enzyme resulted in a single peak of biological activity with a molecular weight of approximately 14,800. The same estimate of molecular weight was obtained by SDS-polyacrylamide gel electrophoresis, which yielded a single band. Polyacrylamide gel electrophoresis of this fraction at pH 4.3 revealed a single protein band migrating beyond lysozyme, with the dye front, suggesting that this protein was more basic than lysozyme (pI 10.5). The enzymatic and physical-chemical characteristics of this soluble enzyme were remarkably similar to a recently described phospholipase A(2) of rabbit polymorphonuclear leukocytes derived from glycogen-induced peritoneal exudates. The possible origin and physiological role of this soluble enzyme are discussed.

Animals↗