The Effect of Porphyromonas gingivalis Extracellular Vesicles on Neutrophil Membrane Fluidity

The purpose of this investigation was to determine the effect of Porphyromonas gingivalis extracellular vesicles (ECVs) on membrane fluidity and superoxide production in neutrophils. The ECVs were extracted from culture supernatant using the ammonium sulfate precipitation procedure. Transmission electron microscopic observations, SDS-PAGE profiles, and chemical composition analysis identified the ECVs prepared from the outer membrane of P. gingivalis ATCC 33277. Neutrophils were isolated from the fresh heparinized venous blood of healthy volunteers by the combined dextran/Ficoll sedimentation method. Membrane fluidity and superoxide production of isolated neutrophils were assayed in the increasing concentrations of the ECVs. The membrane fluidity of neutrophils was measured by spin-labeling neutrophils with stearic acids, consisting of nitroxide-free radicals bound to the 5, 16 position of stearic acids (5-, 16-SAL), to detect reductions of the nitroxide radicals at different positions in the membrane. Superoxide production by neutrophils was evaluated by the cytocrome C reduction procedure. ECVs significantly depressed membrane fluidity in a shallow portion (5-SAL) of lipid bilayers, but had no effect on membrane fluidity in the deep portion (16-SAL) of lipid bilayers. Superoxide production by neutrophils after stimulation with n -formyl-methionyl-leucyl-phenylalanine or phorbol myristate acetate was significantly suppressed by increasing concentrations of ECVs. These results indicate that the membrane fluidity and cell function of neutrophils are impaired by ECVs from periodontal pathogens in the periodontal pockets or periodontal tissues.