Bacterial cell membrane hydrolysis by secreted phospholipases A 2: a major physiological role of human group IIa sPLA 2 involving both bacterial cell wall penetration and interfacial catalysis

Abstract

The ability of human group IIa secreted phospholipase A 2 (human sPLA 2) to hydrolyse the phospholipid membrane of whole cell suspensions of Gram-positive bacteria is demonstrated in real time using a continuous fluorescence displacement assay. Micrococcus luteus is used as a model system and demonstrates an almost absolute specificity for this human enzyme compared with porcine pancreatic and Naja naja venom sPLA 2s. This specificity is due to selective penetration of the highly cationic human sPLA 2 through the highly anionic bacterial cell wall. Disruption of the peptidoglycan cell wall by treatment with lysozyme allows all three enzymes to express similar hydrolytic activity against the anionic bacterial cell membrane. Extensive (>50%) phospholipid hydrolysis was observed and this was confirmed by electrospray mass spectrometry that allowed the identification of several molecular species of phosphatidylglycerol as the targets for hydrolysis. However, the bactericidal activity of the human enzyme under these assay conditions was low, highlighting the capacity of the organism to survive a major phospholipid insult. In addition to pure enzyme, the human sPLA 2 activity in tears was demonstrated using M. luteus as substrate. In comparison to M. luteus, cell suspensions of Staphylococcus aureus were highly resistant to hydrolysis by human sPLA 2 as well as to the pancreatic and venom enzymes. Treatment of this organism with the specific cell wall protease lysostaphin resulted in a dramatic enhancement in cell membrane phospholipid hydrolysis by all three sPLA 2s. Overall, the results highlight the potential of the human sPLA 2 as a selective antimicrobial agent against Gram-positive bacteria in vivo because this enzyme is essentially inactive against mammalian plasma membranes. However, the enzyme will be most effective in combination with other antimicrobial agents that enhance the permeability of the bacterial cell wall and where potentiation of the effectiveness of other antibiotics would be expected.