Enhanced binding of polycationic antibiotics to lipopolysaccharide from an aminoglycoside-supersusceptible, tolA mutant strain of Pseudomonas aeruginosa

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Abstract
The lipopolysaccharide (LPS) of the aminoglycoside-supersusceptible Pseudomonas aeruginosa tolA mutant PAO1715 was compared with its parent strain PAO1670 and tol+ revertant PAO1716. Electrophoretic separation of purified LPSs from the three isolates showed similar LPS banding patterns. Analysis of the Western blots of these LPSs from the three isolates with O-antigen-specific monoclonal antibody indicated that the ladder pattern consisted of doublet bands, which presumably reflected a modification of core or lipid A; the level of one of the bands in the doublet was in much lower amounts in the isolate from the tolA mutant than in that from the parent or revertant. Results of competitive displacement experiments, in which the cationic spin probe 4-dodecyldimethylammonium-1-oxyl-2,2,6,6-tetramethylpiperidine bromide was displaced from its LPS-binding site by polycations, revealed that the tolA mutant had a much higher affinity for gentamicin, polymyxin, Ca2+, and Mg2+ than did the parent or revertant. The order of affinity for all samples was polymyxin B .mchgt. gentamicin C .mchgt. Ca2+ > Mg2+. Both gentamcin and polymyxin induced rigidification of all of the LPS samples, but for the sample from the tolA mutant, rigidification occurred at substantially lower concentrations. Dansyl polymyxin titration experiments with intact cells demonstrated that the increased affinity of the LPS from the tolA mutant for polycations was reflected in an increase in the affinity of binding to the cell. Together these data suggest that the tolA mutant is supersusceptible to aminoglycosides by virtue of an LPS change which increases the binding affinity of the LPS for polycations, including gentamicin.