The interaction of the polycationic rabbit alveolar macrophage cationic proteins MCP-1 and MCP-2 (or their identical neutrophil equivalents NP-1 and NP-2) with the surface of Pseudomonas aeruginosa was investigated. Both proteins bound avidly to purified lipopolysaccharide, as judged by their ability to competitively displace the probe dansyl polymyxin with 50% inhibition (In) values of 2 to 3 ,uM. Similar 1.s were measured with dansyl polymyxin as a probe for cell surface binding, suggesting that the initial binding site for MCP-1 and MCP-2 on the surface of cells was llpopolysaccharide. Both MCP-1 and MCP-2 permeabilized outer membranes to the hydrophobic fluorescent probe l-N-phenylnaphthylamine (NPN). The initial rate of NPN uptake plotted against the concentration of MCP-1 or MCP-2 gave sigmoidal curves, suggesting cooperative permeabilization of the outer membrane. Replotting the data as a Hill plot gave an affinity parameter, S0.5, the concentration of MCP giving a half-maximal increase in the rate of NPN uptake, of 5 and 25 ,uM for MCP-1 and MCP-2, respectively, and thus subsequent studies concentrated on the more active permeabilizer MCP-1. Permeabilization of outer membranes to NPN was a function of buffer pH, with lower pH considerably favoring the permeabilizing effects of MCP-1. Thin-section electron microscopic visualization of MCP-1-treated cells showed production of extended blebs. Further evidence of an altered cell surface after MCP-1 treatment was obtained by demonstrating that treated unopsonized cells were more efficientiy phagocytosed by unelicited rabbit alveolar macrophages. The data overall suggest that macrophage cationic proteins interact with the P. aeruginosa outer membrane in a manner typical of other polycations and suggest that one of their major functions may be to permeabilize the outer membrane.

The lipopolysaccharide (LPS) of the aminoglycoside-supersusceptible Pseudomonas aeruginosa tolA mutant PA01715 was compared with its parent strain PA01670 and tol+ revertant PA01716. Electrophoretic separation of purified LPSs frotn the three isolates showed similar LPS banding patterns. Analysis of the Western blots of these LPSs from the three isolates with 0-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 thbe bands in the doublet was in much lower amounts in the isolate from the toLA mutant than in that from the pareht or revertant. Results of competitive displacement experiments, in which the cationic spin probe 4-dodecyldimethylammonium-i-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 >> gentamicin C >> Ca2+ > Mg2+. Both gentamicin 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.

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