Pseudomonas aeruginosa variants isolated from patients with cystic fibrosis are killed by a bactericidal protein from human polymorphonuclear leukocytes

Siefferman, C M; Regelmann, Warren E.; Gray, Beulah H.

doi:10.1128/iai.59.6.2152-2157.1991

Cited by 12 publications

(3 citation statements)

References 34 publications

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“…We investigated the possibility that the active site of cathepsin G binds to the bacterial cell surface by studying the influence of free LPS from P. aeruginosa ATCC 27312 on enzyme and bactericidal activity toward P. aeruginosa type I. Both type I and ATCC 27312 are smooth strains (42). a One bactericidal unit of BP 55, BP 30, and cathepsin G was preincubated with the inhibitor for 15 min at room temperature before the addition of 5 x 106 P. aeruginosa cells.…”

Section: Resultsmentioning

confidence: 99%

Comparison of granule proteins from human polymorphonuclear leukocytes which are bactericidal toward Pseudomonas aeruginosa

1991

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Killing of Pseudomonas aeruginosa by a 55-kDa bactericidal protein (BP 55), a 30-kDa protein (BP 30), cathepsin G, elastase, and proteinase 3 has been compared. P. aeruginosa was resistant to killing by elastase and proteinase 3. BP 55 at a 50% lethal dose (LD50) of 0.23 ,ug of protein per 5 x 106 bacteria per ml killed P. aeruginosa and was far more active than BP 30 and cathepsin G. The LD5s of BP 30 and cathepsin G were 16.9 and 28.3 ,ug of protein per 5 x 106 bacteria per ml, respectively. Preincubation of BP 55 or BP 30 with lipopolysaccharide (LPS) from P. aeruginosa inhibited bactericidal activity. The N-terminal amino acid sequence of BP 55 and BP 30 revealed no relationship between the two proteins. However, a monoclonal antibody (AHN-15) reacted with both proteins by Western immunoblot. The bactericidal activity of cathepsin G toward P. aeruginosa appeared to be dependent on the availability of the active site of the enzyme; bactericidal activity was inhibited by phenylmethylsulfonyl fluoride (PMSF) and by the specific cathepsin G inhibitor, Z-Gly-Leu-Phe-CH2Cl. The enzyme and bactericidal activities of cathepsin G were also inhibited by LPS from P. aeruginosa. LPS from P. aeruginosa was shown to be a competitive inhibitor of the enzyme activity of cathepsin G. Elastase enzyme activity was also inhibited noncompetitively by LPS, but the enzyme was not bactericidal. We have concluded that all three bactericidal proteins (BP 55, BP 30, and cathepsin G) may bind to the LPS of the outer membrane of P. aeruginosa. It appears that the enzyme active site must be available for cathepsin G to kill P. aeruginosa and that the active site may be involved in the binding of cathepsin G to P. aeruginosa.Polymorphonuclear leukocytes (PMNL) occupy a central role in host defense and destroy intracellular microorganisms by both oxygen-dependent and oxygen-independent mechanisms. As reviewed by Spitznagel (45) and Thomas et al. (47), several proteins are implicated in oxygen-independent killing of microorganisms. Prominent among the cationic bactericidal proteins thus far purified from human PMNL is a 58to 60-kDa bactericidal/permeability-increasing protein (B/PI) described by Weiss et al. (51). B/PT is bactericidal toward Salmonella typhimurium and Escherichia coli. Shafer et al. have described two cationic antibacterial proteins (CAP) of 57 kDa (CAP57) and 37 kDa (CAP37) that are bactericidal toward S. typhimurium (37). We have described a 55-kDa glycoprotein (BP 55) that was isolated from normal human PMNL and was bactericidal toward Pseudomonas aeruginosa (19,20). The smaller cationic defensins (3.5 to 4.0 kDa) kill a spectrum of microorganisms, including P. aeruginosa (10, 16).More recently, reports by Gabay et al. (9) and Campanelli et al. (6) of a 30-kDa protein, called azurocidin, which is bactericidal for E. coli and Streptococcus faecalis and fungicidal for Candida albicans, have generated a great deal of interest. These same investigators have recalled attention to the bactericidal activity of serine proteinase...

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Section: Resultsmentioning

confidence: 99%

Comparison of granule proteins from human polymorphonuclear leukocytes which are bactericidal toward Pseudomonas aeruginosa

1991

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“…In order to do so, technical issues, such as delivery and quantitation of agents and the parameter of injury for study, will need to be resolved. Nevertheless, given clinical (34) and recent in vitro (29,32,35) data indicating that host defense against this organism does not require neutrophil oxidant formation, investigations into the possible therapeutic benefit of antioxidant agents which would potentially decrease airway susceptibility to HO ⅐ and other oxidants may be warranted. a Results are the means Ϯ standard errors of the means for A549 cells exposed to 10 M ferripyochelin plus either (i) H 2 O 2 (2.5 mM), (ii) PMA-stimulated neutrophils (10 7 /ml), or (iii) pyocyanin (80 M) in the presence of either SOD (30 U/ml), catalase (5,000 U/ml), DMTU (10 mM), or DMSO (140 mM), expressed as a percentage of the 51 Cr release observed in their absence.…”

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confidence: 99%

Augmentation of oxidant injury to human pulmonary epithelial cells by the Pseudomonas aeruginosa siderophore pyochelin

1997

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Pseudomonas aeruginosa causes acute and chronic infections of the human lung, with resultant tissue injury. We have previously shown that iron bound to pyochelin, a siderophore secreted by the organism to acquire iron, is an efficient catalyst for hydroxyl radical (HO ⅐) formation and augments injury to pulmonary artery endothelial cells resulting from their exposure to superoxide (O 2 ؊ ⅐) and/or H 2 O 2. Sources for O 2 ؊ ⅐ and H 2 O 2 included phorbol myristate acetate (PMA)-stimulated neutrophils and pyocyanin. Pyocyanin, another P. aeruginosa secretory product, undergoes cell-mediated redox, thereby forming O 2 ؊ ⅐ and H 2 O 2. In P. aeruginosa lung infections, damage to airway epithelial cells is probably more extensive than that to endothelial cells. Therefore, we examined whether ferripyochelin also augments oxidant-mediated damage to airway epithelial cells. A549 cells, a human type II alveolar epithelial cell line, was exposed to H 2 O 2 , PMA-stimulated neutrophils, or pyocyanin, and injury was determined by release of 51 Cr from prelabeled cells. Ferripyochelin significantly increased (>10-fold) oxidant-mediated cell injury regardless of whether H 2 O 2 , neutrophils, or pyocyanin was employed. Apo-pyochelin was not effective, and ferripyochelin was not toxic by itself at the concentrations employed. Spin trapping with ␣-(4-pyrridyl-1-oxide)-N-t-butyl-nitrone-ethanol confirmed the generation of HO ⅐ , and injury was decreased by a variety of antioxidants, including superoxide dismutase, catalase, and dimethylthiourea. These data are consistent with the hypothesis that the presence of ferripyochelin at sites of P. aeruginosa lung infection could contribute to tissue injury through its ability to promote HO ⅐-mediated damage to airway epithelial cells.

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“…βpep-19 is potently bactericidal in the 100 nM range [1]. βpep peptides function like Limulus (horseshoe crab) anti-LPS factor (LALF) and the homologous bactericidal\permeability-increasing protein (B\PI) [4] in that all appear to express activity through an amphipathic β-sheet structural motif having a cationic β-sheet face [5][6][7][8].…”

Section: Introductionmentioning

confidence: 99%

Structure–function relationships in novel peptide dodecamers with broad-spectrum bactericidal and endotoxin-neutralizing activities

Mayo

Haseman

Young

et al. 2000

Biochemical Journal

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A series of designed peptide 33-mers (betapep peptides) areknown to be bactericidal [Mayo, Haseman, Ilyina and Gray (1998)Biochim. Biophys. Acta 1425, 81-92]. Here dodecapeptides (SC-1-SC-8), which 'walk through' the sequence ofbetapep-25, were investigated for their ability to kill Gram-negativeand -positive bacteria and to neutralize endotoxin. SC-4 (KLFKRHLKWKI I-NH(2); the -NH(2) at the right of each sequenceindicates amidation of the C-terminal carboxylate group) is the mosteffective, more so than betapep-25, at killing Gram-negative bacteriawith nanomolar LD(50) values. Against Gram-positive bacteria,SC-4 also shows good activity with submicromolar LD(50)values. Leakage studies indicate rapid bacterial membrane permeability,with t(1/2) valuesof 10-15 min. SC-4 in the micromolar range also effectivelyneutralizes endotoxin and is not haemolytic below 10(-4)M. For all SC peptides, CD and NMR data indicate the presence of both 3(10)- and alpha-helix. For SC-4, nuclear-Overhauser-effect-based computational modelling yields an amphipathic helix with K1, K4,R5, and K8 arrayed on the same face (K is lysine, R is arginine). Activity differences among SC peptides and single-site variants of SC-4allow some structure-function relationships to be deduced. Relative to other known bactericidal peptides in the linear peptide,helix-forming category, SC-4 is the most potent broad-spectrumantibacterial identified to date. The present study contributes to thedevelopment of agents involved in combating the ever-recurring problemof drug-resistant micro-organisms.

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Pseudomonas aeruginosa variants isolated from patients with cystic fibrosis are killed by a bactericidal protein from human polymorphonuclear leukocytes

Cited by 12 publications

References 34 publications

Comparison of granule proteins from human polymorphonuclear leukocytes which are bactericidal toward Pseudomonas aeruginosa

Comparison of granule proteins from human polymorphonuclear leukocytes which are bactericidal toward Pseudomonas aeruginosa

Augmentation of oxidant injury to human pulmonary epithelial cells by the Pseudomonas aeruginosa siderophore pyochelin

Structure–function relationships in novel peptide dodecamers with broad-spectrum bactericidal and endotoxin-neutralizing activities

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