The assessment of several extraction methods, purification, and characterization of Pseudomonas aeruginosa lipopolysaccharide (LPS) and studying its biological activity

Abstract

Many Gram-negative bacteria have LPS, a virulent component, on their outer membranes. LPS, a surface structural component protects the outside layer while intoxicating host cells. Research linked LPS to antibiotic tolerance and biofilm formation. LPS induces pathophysiological effects, LPS is pyrogenic, it causes endotoxin shock and induces the clinical manifestations of infections. Therefore, several methods for isolating and purifying LPS have been developed. Extraction, purification, characterization and yields of Lipopolysaccharide (LPS) were obtained from P. aeruginosa through various methods to determine the pyrogenic effect of extracted LPS (ELPS) in rabbits. The study included the extraction of LPS from P. aeruginosa using manual methods including the modified hot (phenol-water) method, the methanol/ chloroform extraction method and an LPS extraction kit. The extracted LPS was detected and characterized by SDS-PAGE with silver staining and purified with HPLC. The protein and nucleic acid content of Extracted LPS were detected using SDS-PAGE with Coomassie blue staining and gel electrophoresis respectively. The study also determined the pyrogenic effect of extracted LPS (ELPS) in rabbits. Extracted LPS, demonstrated a typical ladder pattern upon silver staining and no contaminating protein was observed by coomassie blue staining and contamination of nucleic acid. In contrast, the extracted LPS without the enzymatic treatments revealed lighter protein and nucleic acid contamination bands. The elevated body temperature of rabbits following intraperitoneal administration of purified LPSs confirmed the functional activity of extracted LPSs. It was concluded from the study that the LPS extracted with manual methods and with an extraction kit showed high purity and yield compared to standard LPS. The modified hot (phenol-water) method showed the highest yield among all three methods. Rabbit pyrogencity testing of extracted LPS demonstrated the endotoxin's biological activity and pyrogenic nature.