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Abstract
Two membrane-active cationic proteins with potent bactericidal activity toward several gram-negative bacteria have been purified to near homogeneity from rabbit and human polymorphonuclear leukocytes (PMN). Both proteins are tightly associated with the membranes of subcellular granules. They are solubilized by acid extraction and isolated by ion-exchange and gel filtration chromatography.
The molecular and biological properties of the rabbit (R) and human (H) proteins are closely similar. These similarities include molecular size (M(,r) = 50K (R); 58-60K (H)); net charge (pI = > 9.5 (R); 9.8 (H)); amino acid composition and heat stability. The two proteins display partial immunological cross-reactivity.
The antimicrobial activity of both proteins appears directed specifically against gram-negative bacteria. Toward several rough strains of Escherichia coli and Salmonella typhimurium, the bactericidal potency of the two proteins is 5x greater (on a molar basis) than powerful antibiotics such as polymixin B (i.e. 10('-8)M (< 1 (mu)g/ml) protein produces > 90% killing of 10('7) bacteria). Higher protein concentrations (3- to 15-fold) are needed for similar killing of several smooth strains of E. coli, S. typhimurium and Pseudomonas aeruginosa. Both proteins, even at 160 (mu)g/ml, are inactive vs. all gram-positive bacteria and fungi examined.
The proteins' specific antibacterial action is closely correlated with their binding to the gram-negative bacterial outer membrane. Decreasing the polysaccharide chain length of outer membrane lipopolysaccharides favors protein binding and antibacterial activity. Divalent cations (Ca('2+), Mg('2+)) block protein binding and antibacterial activity. Neither protein binds to resistant microorganisms.
Killing of gram-negative bacteria is accompanied by two almost immediate alterations of the outer membrane: (1) increased permeability toward small hydrophobic molecules (i.e. actinomycin D); and (2) activation of bacterial phospholipase(s). In contrast, several aspects of inner membrane structure/function, including diffusion barriers to small hydrophilic molecules (o-nitrophenyl-(beta)-D-galactoside), active and passive K('+) fluxes and macromolecular biosynthesis, remain intact for at least 30-60 min. Addition of 40 mM Mg('2+) (or Ca('2+)) or trypsin (100 (mu)g/ml), even 1-2 h after PMN protein-bacterium interaction (at 37(DEGREES)C), releases essentially all surface-bound PMN protein and reverses the proteins' outer membrane effects, but does not rescue bacterial viability. At 37(DEGREES)C, the bactericidal action of the PMN proteins is initiated, irreversibly, within 15 sec. Binding also occurs rapidly (< 1 min) at 4(DEGREES)C, but even after 45 min most of the bacteria can be rescued by adding Mg('2+).
The bactericidal activity of the two purified proteins can account for nearly all the bactericidal activity of crude rabbit and human PMN fractions and of intact (rabbit) PMN toward several rough and smooth gram-negative bacteria. We therefore suggest that, toward these bacteria, these membrane-active cationic proteins are the principal bactericidal agents of rabbit and human PMN.
