Bactericidal Properties of Human and Murine Groups I, II, V, X, and XII Secreted Phospholipases A2

Veli J.O Laine,Michael H Gelb,Gérard Lambeau,Juha O Grönroos,Timo J Nevalainen,Catherine Le Calvez,Rao S Koduri

doi:10.1074/jbc.m109699200

Veli J.O Laine, Michael H Gelb + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m109699200

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Abstract

Group IIA secreted phospholipase A(2) (sPLA2) is known to display potent Gram-positive bactericidal activity in vitro and in vivo. We have analyzed the bactericidal activity of the full set of recombinant murine and human groups I, II, V, X, and XII sPLA2s on Listeria monocytogenes, Staphylococcus aureus, and Escherichia coli. The rank order potency among human sPLA2s against Gram-positive bacteria is group IIA > X > V > XII > IIE > IB, IIF (for murine sPLA2s: IIA > IID > V > IIE > IIC, X > IB, IIF), and only human group XII displays detectable bactericidal activity against the Gram-negative bacterium E. coli. These studies show that highly basic sPLA2s display potent bactericidal activity with the exception of the ability of the acidic human group X sPLA2 to kill Gram-positive bacteria. By studying the Bacillus subtilis and S. aureus bactericidal potencies of a large panel of human group IIA mutants in which basic residues were mutated to acidic residues, it was found that: 1) the overall positive charge of the sPLA2 is the dominant factor in dictating bactericidal potency; 2) basic residues on the putative membrane binding surface of the sPLA2 are modestly more important for bactericidal activity than are other basic residues; 3) relative bactericidal potency tracks well with the ability of these mutants to degrade phospholipids in the bacterial membrane; and 4) exposure of the bacterial membrane of Gram-positive bacteria by disruption of the cell wall dramatically reduces the negative effect of charge reversal mutagenesis on bactericidal potency.

Highlights

Group IIA secreted phospholipase A2 is known to display potent Gram-positive bactericidal activity in vitro and in vivo
By studying the Bacillus subtilis and S. aureus bactericidal potencies of a large panel of human group IIA mutants in which basic residues were mutated to acidic residues, it was found that: 1) the overall positive charge of the sPLA2 is the dominant factor in dictating bactericidal potency; 2) basic residues on the putative membrane binding surface of the sPLA2 are modestly more important for bactericidal activity than are other basic residues; 3) relative bactericidal potency tracks well with the ability of these mutants to degrade phospholipids in the bacterial membrane; and 4) exposure of the bacterial membrane of Gram-positive bacteria by disruption of the cell wall dramatically reduces the negative effect of charge reversal mutagenesis on bactericidal potency
Murine sPLA2s toward two Gram-positive bacteria (L. monocytogenes and S. aureus) and against the Gram-negative bacterium E. coli were tested by measuring the number of colony-forming units (CFU) after incubating live bacteria with various concentrations of sPLA2 for various times

Summary

EXPERIMENTAL PROCEDURES

Materials—The hGIIA-N1A mutant of human group IIA sPLA2 (as well as all hGIIA-N1A mutants) was obtained by bacterial expression and in vitro refolding as described [21,22,23]. The sPLA2s mGIID, mGIIE, hGIIE, mGIIF, hGIIF, hGX, and hGXII were obtained by bacterial expression and in vitro refolding as described (10, 26 –29). The preparation of recombinant mGIB, hGIB, mGIIA, mGIIC, mGV, hGV, and mGX will be described elsewhere. To test the bactericidal effects of sPLA2s, dilutions of 100, 10, and 1 ␮g/ml in sterile water were prepared for each recombinant sPLA2 enzyme. The resulting solution contained 500 ␮g/ml sPLA2 and was diluted with sterile water to give the dilutions mentioned above. The sPLA2 negative control for hGV and mGV contained the same amount of acetonitrile and trifluoroacetic acid as the 50 ␮g/ml sPLA2 solutions. Studies of the bactericidal effect of hGIIA mutants on B. subtilus and S. aureus and release of [14C]oleic acid from [14C]oleic acid-labeled bacteria were carried out as described [12]. Bacteria were incubated with sPLA2 for 90 min at 37 °C prior to the analysis of CFU and phospholipid hydrolysis

RESULTS

Phosphatidylmethanol vesicle hydrolysis

DISCUSSION