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.
Kuzmenko
Alexander I
AI
Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, Ohio 45267-0564, USA.
Wu
Huixing
H
McCormack
Francis X
FX
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