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Bacterial cell membrane hydrolysis by secreted phospholipases A2: a major physiological role of human group IIa sPLA2 involving both bacterial cell wall penetration and interfacial catalysis

Bacterial cell membrane hydrolysis by secreted phospholipases A2: a major physiological role of human group IIa sPLA2 involving both bacterial cell wall penetration and interfacial catalysis
Bacterial cell membrane hydrolysis by secreted phospholipases A2: a major physiological role of human group IIa sPLA2 involving both bacterial cell wall penetration and interfacial catalysis
The ability of human group IIa secreted phospholipase A2 (human sPLA2) 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 sPLA2s. This specificity is due to selective penetration of the highly cationic human sPLA2 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 sPLA2 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 sPLA2 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 sPLA2s. Overall, the results highlight the potential of the human sPLA2 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.
secreted phospholipase a2, membrane hydrolysis, antimicrobial, antibacterial, lysostaphin, lysozyme, micrococcus luteus, staphylococcus aureus
1388-1981
195-206
Buckland, Andrew G.
c0b3a024-9df7-4414-9853-11fc6e55f7d1
Heeley, Emma L.
a419a91d-b809-4dc6-af94-40052cb5be82
Wilton, David C.
4b995f66-ad6c-4d96-9179-c64f3b54466a
Buckland, Andrew G.
c0b3a024-9df7-4414-9853-11fc6e55f7d1
Heeley, Emma L.
a419a91d-b809-4dc6-af94-40052cb5be82
Wilton, David C.
4b995f66-ad6c-4d96-9179-c64f3b54466a

Buckland, Andrew G., Heeley, Emma L. and Wilton, David C. (2000) Bacterial cell membrane hydrolysis by secreted phospholipases A2: a major physiological role of human group IIa sPLA2 involving both bacterial cell wall penetration and interfacial catalysis. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 1484 (2-3), 195-206. (doi:10.1016/S1388-1981(00)00018-4).

Record type: Article

Abstract

The ability of human group IIa secreted phospholipase A2 (human sPLA2) 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 sPLA2s. This specificity is due to selective penetration of the highly cationic human sPLA2 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 sPLA2 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 sPLA2 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 sPLA2s. Overall, the results highlight the potential of the human sPLA2 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.

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More information

Published date: April 2000
Keywords: secreted phospholipase a2, membrane hydrolysis, antimicrobial, antibacterial, lysostaphin, lysozyme, micrococcus luteus, staphylococcus aureus
Organisations: Centre for Biological Sciences

Identifiers

Local EPrints ID: 372349
URI: http://eprints.soton.ac.uk/id/eprint/372349
ISSN: 1388-1981
PURE UUID: 71fdb5ce-cd17-44e4-897d-c1ba711b92dc

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Date deposited: 01 Dec 2014 13:32
Last modified: 15 Jul 2019 21:37

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