Pulmonary collectins selectively permeabilize model bacterial membranes containing rough lipopolysaccharide.
We have reported that Gram-negative organisms decorated with rough lipopolysaccharide (LPS) are particularly susceptible to the direct antimicrobial actions of the pulmonary collectins, surfactant proteins A (SP-A) and D (SP-D). In this study, we examined the lipid and LPS components required for the permeabilizing effects of the collectins on model bacterial membranes. Liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), with or without rough Escherichia coli LPS (J5), smooth E. coli LPS (B5), or cholesterol, were loaded with self-quenching probes and exposed to native or oxidatively modified SP-A. Fluorescence that resulted from permeabilization of liposomes and diffusion of dyes was assessed by microscopy or fluorimetry. Human SP-A and melittin increased the permeability of J5 LPS/POPE liposomes, but not B5 LPS/POPE liposomes or control (POPE only) liposomes. At a human SP-A concentration of 100 microg/mL, the permeability of the J5 LPS/POPE membranes increased 4.4-fold (p < 0.02) compared to the control with no added SP-A. Rat SP-A and SP-D also permeabilized the J5-containing liposomes. Incorporation of cholesterol into J5 LPS/POPE liposomes at a POPE:cholesterol molar ratio of 1:0.15 blocked human SP-A or melittin-induced permeability (p < 0.05) compared to cholesterol-free liposomes. Exposure of human SP-A to surfactant lipid peroxidation blocked the permeabilizing activity of the protein. We conclude that SP-A permeabilizes phospholipid membranes in an LPS-dependent and rough LPS-specific manner, that the effect is neither SP-A- nor species-specific, and that oxidative damage to SP-A abolishes its membrane destabilizing properties. Incorporation of cholesterol into the membrane enhances resistance to permeabilization by SP-A, most likely by increasing the packing density and membrane rigidity.
Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, Ohio 45267-0564, USA.
Alexander I
AI
Kuzmenko
Huixing
H
Wu
Francis X
FX
McCormack
D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002463:Cell Membrane Permeability / Q000187:drug effects*; D002784:Cholesterol / Q000378:metabolism / Q000494:pharmacology; D037222:Collectins / Q000378:metabolism / Q000494:pharmacology*; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D015227:Lipid Peroxidation / Q000187:drug effects; D008070:Lipopolysaccharides / Q000378:metabolism* / Q000494:pharmacology; D008081:Liposomes / Q000378:metabolism; D008168:Lung / Q000378:metabolism*; D008954:Models, Biological; D019081:O Antigens / Q000378:metabolism / Q000494:pharmacology; D010714:Phosphatidylethanolamines / Q000378:metabolism; D037662:Pulmonary Surfactant-Associated Protein A / Q000378:metabolism / Q000494:pharmacology; D037663:Pulmonary Surfactant-Associated Protein D / Q000378:metabolism / Q000494:pharmacology; D051381:Rats
D037222:Collectins; D005456:Fluorescent Dyes; D008070:Lipopolysaccharides; D008081:Liposomes; D019081:O Antigens; D010714:Phosphatidylethanolamines; D037662:Pulmonary Surfactant-Associated Protein A; D037663:Pulmonary Surfactant-Associated Protein D; C057561:1-palmitoyl-2-oleoylphosphatidylethanolamine; D002784:Cholesterol; D002118:Calcium