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

Abstract

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...

Effect of lipopolysaccharide (LPS) chain length on interactions of bactericidal/permeability-increasing protein and its bioactive 23-kilodalton NH2-terminal fragment with isolated LPS and intact Proteus mirabilis and Escherichia coli

“…It has been proposed that this substitution may account for the resistance of this microorganism to cationic antibiotics, such as polymyxin B (12), and antibacterial proteins, such as BPI (also called CAP 57 [26,27] and BP 55 [9,31]).…”

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

“…Farley et al found that 11 times the amount of CAP57 required to kill the rough LPS mutant was required to kill the smooth LPS strain of S. typhimurium (6). This difference in sensitivity to PMN bactericidal proteins was not due to a difference in the proteins, for BP55, B/PI, and CAP57 have the same N-terminal amino acid sequence and are very likely the same protein (24,27,35). Furthermore, although we have not carried out a comprehensive comparison, we have previously established that killing of a smooth strain of S. typhimurium required 300-fold more BP55 than did killing of a rough strain of E. coli or of the smooth strain P. aeruginosa type 1 (15).…”

“…This 55,000-Da bactericidal protein (BP55) binds to the target bacteria and depolarizes the cytoplasmic membrane, which results in the cessation of amino acid transport and eventual cell death (16). Like the 57,000-Da cationic antimicrobial protein (CAP57) and the 59,000-Da bactericidal and permeabilityincreasing protein (B/PI), which bind to the lipopolysaccharides (LPS) from the outer membranes of Salmonella typhimurium and Escherichia coli (31, 37), respectively, BP55 binds to the LPS from P. aeruginosa (14,35).The outermost portion of the LPS, the O-polysaccharide side chain, plays an important role in the pathogenicity of most gram-negative bacteria, including P. aeruginosa (4, 23). O-polysaccharide side chains of smooth LPS can sterically hinder the formation of the complement membrane attack complex, thus conferring serum resistance to the bacteria (8, 17).…”

“…This 55,000-Da bactericidal protein (BP55) binds to the target bacteria and depolarizes the cytoplasmic membrane, which results in the cessation of amino acid transport and eventual cell death (16). Like the 57,000-Da cationic antimicrobial protein (CAP57) and the 59,000-Da bactericidal and permeabilityincreasing protein (B/PI), which bind to the lipopolysaccharides (LPS) from the outer membranes of Salmonella typhimurium and Escherichia coli (31,37), respectively, BP55 binds to the LPS from P. aeruginosa (14,35).…”

Bioactive peptides derived from the Limulus anti‐lipopolysaccharide factor: structure‐activity relationships and formation of mixed peptide/lipid complexes

“…LALF is a small basic protein that binds to and neutralises the effect of LPS in the coagulation cascade of the crab [12]. BPI binds to LPS and could participate in the homeostasis of LPSinduced response [13,14]. The structures of both, LALF and BPI, have been solved by X-ray crystallography 964 MORA, DE LA PAZ AND PÉREZ-PAYÁ and structural analysis has revealed putative LPSbinding sites located at solvent exposed amphipathic β-hairpin structures [12,15,16].…”