Susceptibility of Lipopolysaccharide Mutants to the Bactericidal Action of Human Neutrophil Lysosomal Fractions

Abstract

Acetate extracts of purified human neutrophil granules (a mixed population containing specific and azurophil granules) were dialyzed against phosphate-buffered saline (pH 7.0) and tested for bactericidal activity against smooth parent and rough mutant, gram-negative bacteria. Rough (Re) mutants of Escherichia coli, Salmonella typhimurium, and Salmonella minnesota were exquisitely more sensitive to extracts of human polymorphonuclear leukocyte granules than were their smooth (S) parents. The mean lethal dose (LD 50 ) for the parent strains was 25 to 50 μg of granule protein per ml. As much as 500 μg of extract per ml failed to kill 100% of the S parents. The LD 50 for the rough mutants was 1.5 to 2.0 μg of the same granule extract per ml; 100% killing occurred with 5 to 10 μg of lysosomal protein per ml. Conditions affecting the growth of the bacteria greatly affected their sensitivity to the granule extracts. Granule extract killed bacteria grown with aeration to log phase 10 to 15 times more efficiently than the same bacteria grown to stationary phase under static conditions. The bactericidal incubation mixture also influenced results, in that greater killing occurred with tryptone than with phosphate or N -2-hydroxyethyl piperazine- N ′-2-ethanesulfonic acid-buffered saline. Bactericidal activity depended on lysosomal protein concentration, time, and temperature. Boiled lysosomal fractions failed to kill the S parents but retained 20 to 50% of their ability to kill the Re mutants. Parents (smooth) were killed more efficiently at pH 5 to 6, whereas their Re mutants were killed more efficiently at pH 7 to 8.