Quantitative proteomic analysis of extracellular membrane vesicles from Acinetobacter baumannii (AbOMVs) isolated under normoxic and hypoxic conditions.

ABSTRACT: Acinetobacter baumannii were cultured in Brain Heart Infusion medium (BD Bioscience). For normoxic condition, the cultures were shaken at 125 rpm for 24 h, whereas for hypoxic condition, the cultures were placed under static condition for 48 h. Outer membrane vesicles from A. baumannii (AbOMVs) in the culture supernatant from normoxic and hypoxic conditions (referred to nAbOMV and hAbOMVs, respectively) were harvested by ultracentrifugation at 100,000 × g, 4℃ for 90 min using a Himac CP80WX Preparative Ultracentrifuge (HITACHI, Tokyo, Japan). After removal of the supernatant, the pellet was washed twice and suspended in ice-cold sterile phosphate-buffered saline (PBS). AbOMVs were then isolated from other damaged membranes, aggregated proteins and non-membranous proteins by step-gradient ultracentrifugation using 15 to 45% OptiPrep™ Density Medium (Sigma Aldrich, St. Louis, MO). After centrifugation at 100,000 × g, 4℃ for 16 h, the substances in all six fractions (F1-F6) were collected, diluted in PBS, and harvested by ultracentrifugation at 100,000 × g, 4℃ for 3 h. The pellet from each fraction was washed twice and resuspended in a suitable volume of ice-cold sterile PBS.

ORGANISM(S): Acinetobacter Baumannii

SUBMITTER: Krisana Asano 

PROVIDER: PXD037014 | JPOST Repository | Thu Mar 16 00:00:00 GMT 2023

REPOSITORIES: jPOST