Comparison of granule proteins from human polymorphonuclear leukocytes which are bactericidal toward Pseudomonas aeruginosa.
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 micrograms of protein per 5 x 10(6) bacteria per ml killed P. aeruginosa and was far more active than BP 30 and cathepsin G. The LD50s of BP 30 and cathepsin G were 16.9 and 28.3 micrograms of protein per 5 x 10(6) 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.
Wasiluk
K R
KR
Department of Microbiology, University of Minnesota, Minneapolis 55455.
Skubitz
K M
KM
Gray
B H
BH
D000595:Amino Acid Sequence; D023181:Antimicrobial Cationic Peptides; D001770:Blood Bactericidal Activity; D001798:Blood Proteins / Q000032:analysis*; D015153:Blotting, Western; D002352:Carrier Proteins; D056649:Cathepsin G; D002403:Cathepsins / Q000032:analysis / Q000378:metabolism; D006801:Humans; D008070:Lipopolysaccharides / Q000378:metabolism; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009504:Neutrophils / Q000737:chemistry*; D011550:Pseudomonas aeruginosa / Q000276:immunology*; D012697:Serine Endopeptidases; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology
C062991:AZU1 protein, human; D023181:Antimicrobial Cationic Peptides; D001798:Blood Proteins; D002352:Carrier Proteins; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D015842:Serine Proteinase Inhibitors; C021623:bactericidal permeability increasing protein; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G