Downloaded from www.microbiologyresearch.org by IP: 54.90.45.205 On: Thu, 28 Jan 2016 12:03:28 Journal of Medical Microbiology (2003), 52, 1039–1045 DOI 10.1099/jmm.0.05303-0 05303 & 2003 SGM Printed in Great Britain 1039 Correspondence Anne Dubouix dubouix.a@chu-toulouse.fr Received 25 April 2003 Accepted 11 August 2003 Bactericidal properties of group IIa secreted phospholipase A 2 against Pseudomonas aeruginosa clinical isolates Anne Dubouix, 1,2 Catherine Campanac, 3 Josette Fauvel, 1 Marie-Franc ¸ oise Simon, 4 Jean-Pierre Salles, 1 Christine Roques, 3 Hugues Chap 1 and Nicole Marty 2 1 INSERM CPTP-U563, CHU Purpan, 1 Place du Dr Baylac, TSA 40031, 31059 Toulouse Cedex 9, France 2,4 Laboratoire de Bacte ´ riologie-Hygie `ne 2 and INSERM U583 4 , CHU Rangueil, 1 Avenue Jean Poulhes, TSA 50032, 31059 Toulouse Cedex 9, France 3 Laboratoire de Microbiologie, Faculte ´ de Pharmacie, 27 Chemin des Maraı ˆchers, 31400 Toulouse, France It has been shown that human group IIa secreted phospholipase A 2 (sPLA 2 ), found at high levels in inflammatory fluids, displays direct bactericidal properties against Gram-positive bacteria, while activity against Gram-negative bacteria requires the complement system or additional co-factors produced by neutrophils. Pseudomonas aeruginosa, an increasingly prevalent opportunistic human pathogen, is the most common Gram-negative rod found in cystic fibrosis lung infections, where it is associated with an inflammatory environment. Because murine intestinal group II sPLA 2 produced by Paneth cells has been shown to be directly bactericidal against Gram-negative bacteria, IIa sPLA 2 activity against P. aeruginosa clinical isolates was evaluated and provides the first evidence that the enzyme can be fully bactericidal in a concentration- and time-dependent manner against Gram- negative rods. Furthermore, it was demonstrated that these bactericidal properties were unaffected by high protein and salt concentrations, as observed in cystic fibrosis secretions, and that bacterial killing paralleled phospholipid hydrolysis. Finally, no cytotoxicity was observed when IIa sPLA 2 was incubated with human pulmonary cells, highlighting its potential use to synergize bactericidal antibiotics by promoting sublethal alterations of the bacterial cell wall. INTRODUCTION Phospholipases A 2 (PLA 2 ) cleave fatty acids from the sn-2 position of phospholipids from bilayers or micelles, yielding free fatty acids and lysophospholipids. They have been classified into several groups based on their secretory or cytosolic nature and their molecular structure. Group IIa secreted PLA 2 (IIa sPLA 2 ) has been attracting interest for almost two decades, since its discovery in platelets (Murakami et al., 1997) and synovial fluids and its asso- ciation with inflammatory disorders (Valdas et al., 1993). Much effort has been expended to determine the precise role of sPLA 2 in physiological mechanisms. Interestingly, Weinrauch et al. (1996) demonstrated that, in infectious contexts, inflammatory exudates could exhibit potent anti- bacterial properties against both Gram-positive and Gram- negative bacteria. Furthermore, it has been demonstrated that, during systemic bacterial challenge, sPLA 2 is fully mobilized, conferring on plasma a potent bactericidal activity against Escherichia coli and Staphylococcus aureus (Weinrauch et al., 1998). In non-pathological circumstances, physiological fluids such as tears are naturally enriched in sPLA 2 : concentrations as high as 60 ìg ml 1 are frequently observed and are able to kill bacteria of the local flora such as Micrococcus species in vitro (Qu & Lehrer, 1998). More recently, studies involving transgenic mice expressing human group II sPLA 2 gene (PLA 2 þ mice) and group II PLA 2 - deficient mice (PLA 2  mice) allowed an accurate assessment of the antimicrobial role of group II sPLA 2 in vivo. For this purpose, Laine et al. (1999) evaluated the response of PLA 2  mice versus PLA 2 þ mice when challenged by Staphylococcus aureus and noted an increased morbidity and mortality in the first group. In contrast, expression of sPLA 2 resulted in improved resistance of the animals by increased killing of bacteria, as indicated by the small number present in the tissues (Laine et al., 1999). Similar results were obtained Abbreviations: BPI, bactericidal/permeability-increasing protein; CF, cystic fibrosis; sPLA 2 , secreted phospholipase A 2 .