Pseudomonas aeruginosa are widespread in nature and thrive in a hospital environment. They often resist all but the most toxic antimicrobial drugs and cause the highest mortality of the opportunistic gram-negative bacteria. We have purified a 55,000 dalton protein from th cytoplasmic granules of human polymorphonuclear leukocytes which has remarkable bactericidal potency towards P. aeruginosa. Our studies show that this bactericidal protein (BP) is a single polypeptide which may be endowed with two functions: binding to specific sites on the outer membrane of susceptible bacteria and damage to the inner membrane of cells which is associated with bacterial killing. We will explore the possibility that the two functions, binding to and killing of P. aeruginosa, can be assigned to differet portions of the molecule. Fragments derived by limited proteolysis and cyanogen bromide cleavage of the bactericidal protein will be analyzed for ability to bind to P. aeruginosa without causing their demise. The capacity of the fragments to kill intact P. aeruginosa cells and carbenicillin-induced spheroplasts of bacteria will be determined. Monoclonal antibodies specific for the bactericidal protein will be characterized by their ability to inhibit binding to P. aeruginosa and to inhibit killing of intact bacteria or spheroplasts of bacteria prepared by growth in medium containing carbenicillin. A further analysis of the reaction of biologically active fragments and cyanogen bromide fragments of BP with individual monoclonal antibodies will contribute to our understanding of the organization of the BP molecule. The amino acid sequence of the bactericidal protein will be deduced from the nucleotide sequence of cDNA clones for BP. The clones will be selected from an expression library of an HL-60 promyelocytic cell line. Knowledge of a predicted amino acid sequence for BP wll facilitate alignment of biologically active and cyanogen bromide fragments of BP with the parent molecule.
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| Siefferman, C M; Regelmann, W E; Gray, B H (1991) Pseudomonas aeruginosa variants isolated from patients with cystic fibrosis are killed by a bactericidal protein from human polymorphonuclear leukocytes. Infect Immun 59:2152-7 |
