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Bactericidal Properties of Group IIA and Group V Phospholipases A₂¹

Juha O. Grönroos^*, Veli J. O. Laine^*, Marcel J. W. Janssen, Maarten R. Egmond and Timo J. Nevalainen^2,*

^* Department of Pathology, University of Turku and Turku University Hospital, Turku, Finland; and Department of Enzymology and Protein Engineering, CBLE Institute of Biomembranes, University of Utrecht, Utrecht, The Netherlands

	Abstract

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Group V phospholipase A₂ (PLA₂) is a recently characterized 14-kDa secretory PLA₂ of mammalian heart and macrophage-derived cells. Group IIA PLA₂, which is structurally close to group V PLA₂, has been shown to kill Gram-positive bacteria in vitro and to prevent symptoms of Gram-positive infection in vivo. We studied the antibacterial properties of fully active recombinant rat group IIA and V PLA₂s. Both group IIA and V PLA₂s were highly bactericidal against Gram-positive bacteria, including methicillin-resistant staphylococci and vancomycin-resistant enterococci. Only high concentrations of group IIA PLA₂ showed some bactericidal effect against the Gram-negative bacterium Escherichia coli. Our results confirm that group IIA PLA₂ is a potent antibacterial enzyme against Gram-positive bacteria. Moreover, we show here that group V PLA₂ is a novel antibacterial mammalian protein, but is less potent than group IIA PLA₂. Both enzymes may be considered as future therapeutic agents against bacterial infections.

	Introduction

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Phospholipases A₂ (PLA₂;³ EC 3.1.1.4.) cleave fatty acids from the sn-2 position of phospholipids, yielding free fatty acids and lysophospholipid. PLA₂s have been classified into various groups based on the molecular structure of the enzyme protein (1). Group IIA PLA₂ is a small molecular mass (14 kDa) secretory enzyme and is highly bactericidal against Gram-positive bacteria in vitro (2). Overexpression of group IIA PLA₂ in transgenic mice results in decreased mortality in experimental Staphylococcus aureus infection and improved clearance of bacteria from organs and body fluids of the experimental animals, emphasizing the role of this enzyme in innate immunity (3). High concentrations of group IIA PLA₂ are found in tears (4), where the majority of bactericidal action is due to group IIA PLA₂ (5). The bactericidal potency of rabbit (2), mouse (6), and human (3) group IIA PLA₂ is basically directed against Gram-positive bacteria. However, Harwig and coworkers showed that murine intestinal group IIA PLA₂ is bactericidal against some Gram-negative bacteria, such as Escherichia coli and Salmonella typhimurium (6).

Group V PLA₂ is a novel type of PLA₂ expressed in, e.g., mouse heart (7) and macrophage-derived cells (8), where the enzyme seems to have a role in mobilization of arachidonic acid (8). Moreover, LPS and platelet-activating factor-induced sphingomyelin synthesis is dependent on group V PLA₂ in the latter cells (9). Lately, the enzymatic properties of group V PLA₂ have been thoroughly characterized (7, 10, 11).

Macrophages phagocytize and kill invading bacteria at the sites of bacterial infection, but the mechanisms of bacterial killing by these cells are poorly defined. The purpose of the present study was to evaluate the possible role of group V PLA₂ in killing various antibiotic-resistant and sensitive bacterial strains. For this purpose we used recombinant rat group IIA and V PLA₂s that were folded in vitro to obtain full enzymatic activity (11) and measured their bactericidal properties in vitro. Our results show that rat group IIA PLA₂ is highly bactericidal against Staphylococcus aureus, methicillin-resistant S. aureus, and Listeria monocytogenes, moderately bactericidal against Enterococcus faecalis and Enterococcus faecium and shows a weak bactericidal activity against E. coli. Group V PLA₂ is bactericidal against L. monocytogenes, methicillin-sensitive S. aureus, E. faecalis, and E. faecium. Thus, group V PLA₂ seems to be a novel antibacterial enzyme, but weaker in antibacterial potency than group IIA PLA₂.

	Materials and Methods

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Production of rat group IIA and group V PLA₂s

Rat group IIA and V PLA₂s were produced by recombinant expression in E. coli and were folded in vitro as described previously (11). The isoelectric points of the group IIA and group V PLA₂s were 9.1 and 8.3, respectively. For the studies of antibacterial potency, both group IIA and V PLA₂s were diluted in sterile deionized water to an initial concentration of 61 µg/ml.

Killing of bacteria in vitro

The bacteria used were S. aureus (American Type Culture Collection, Manassas, VA; ATCC 25923 and MRSA 27R), L. monocytogenes (RHD 1343), E. faecalis (Van B, ATCC 51299), E. faecium (VRE, Van A, World Health Organization, Geneva, Switzerland), and E. coli (ATCC 25922). Bacteria were first incubated in 100 ml of brain heart infusion broth (BHIB; Difco, Detroit, MI) at 37°C for 210 min. Thereafter, bacteria were centrifuged at 6°C for 10 min at 2000 x g in an Eppendorf centrifuge 5810R. The pellets were suspended in 10 ml of sterile saline and centrifuged. The washing step was repeated twice. Then, the pellets were suspended in saline, and the OD at 650 nm (OD₆₅₀) of the suspension was adjusted to 0.500 by diluting with steriline saline. This value is equivalent with 3 x 10⁸ bacteria/ml. OD₆₅₀ was measured with an Ultrospec III spectrophotometer (Pharmacia, Piscataway, NJ). One microliter of the suspension was added to 500 µl of HEPES buffer (20 mmol/L HEPES, 2.0 mmol/L Ca²⁺, and 10 mg/ml BSA, pH 7.4) and shaken. The 0 min sample was taken from this dilution. Twenty microliters of this suspension were added to four tubes with 20 µl of sterile saline containing 61, 6.1, 0.61, and 0.061 µg/ml PLA₂ and one tube with 20 µl sterile saline only. The resulting medium thus contained 10 mmol/L HEPES, 1 mmol/L Ca²⁺, 5 mg/ml BSA, and 30.5, 3.05, 0.305, or 0.0305 µg/ml PLA₂, respectively. Bacteria were then incubated at 37°C, and 5-µl samples were taken at 20, 60, and 120 min. The samples taken were diluted in sterile saline and grown on brain heart infusion agar (Difco) at 37°C. The number of live bacteria in growth medium at each time point was determined by measuring the CFU at 24 h. All bactericidity tests for group IIA and group V PLA₂s were performed twice with each bacterium.

Determining the effect of nutrition and salt concentration on bactericidal effect of PLA₂s

To determine the possible effects of the composition of culture medium on antibacterial potency of the two PLA₂s, S. aureus was cultured as described above in medium containing 10 mmol/L HEPES and 1 mmol/L Ca²⁺ (pH 7.4) with varying amounts of NaCl (0.0–0.9%), BSA (0–5 mg/ml), and BHIB (0–5%). Control medium contained 10 mmol/L HEPES and 1 mmol/L Ca²⁺ (pH 7.4) only. Solutions containing 3.05–30.5 µg/ml of group IIA or group V PLA₂ were prepared for each medium.

Cytotoxicity test

To test the possible cytotoxic effects of group IIA and group V PLA₂s on eukaryotic cell, a trypan blue exclusion test was performed using HEK cells (human embryonic kidney cells, ATCC CRL-1573). Solutions containing 10 µg/ml of group IIA or group V PLA₂ in DMEM were prepared by mixing 200 µl of PLA₂ solution (61 µg/ml in sterile saline) or saline (negative control) with 1 ml of DMEM. A 10% (v/v) solution of ethanol in DMEM was used as a positive control for cytotoxicity. Approximately 1 x 10⁴ cells were added to the wells of a 96-well cell culture cluster (Costar/Corning, Corning, NY). The cells were first incubated in 100 µl of DMEM for 24 h; thereafter the medium was removed, and 100 µl of the test solution was pipetted into each well. After 4, 12, and 24 h of incubation at 37°C under 5% CO₂, the medium containing detached cells was transferred from the wells into test tubes. The remaining cells were detached from the wells by trypsin treatment and added to the test tubes. Forty microliters of 0.5% trypan blue dye solution was added to the tube, and after 5 min the cells were centrifuged at 100 x g using an Eppendorf 5417R centrifuge. The supernatant was carefully removed, and the cells were resuspended in 100 µl of DMEM. The ratio between viable (nondyed) and nonviable (dyed) cells was determined by counting 200 cells in a Bürker chamber. The cytotoxicity test was performed three times for each treatment.

	Results

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Bactericidal properties of rat recombinant group IIA and V PLA₂s in vitro

Group IIA PLA₂ was bactericidal against all the bacteria tested (Fig. 1). Group V PLA₂ showed antibacterial activity against the Gram-positive bacteria (Fig. 2). With the highest doses of PLA₂s tested (30.5 µg/ml of either group IIA or V PLA₂), the most dramatic effect was seen on L. monocytogenes and E. faecium; after 20 min, >99.5% of the bacteria died in the presence of either PLA₂s. The antibacterial effect of group IIA PLA₂ was remarkable against E. faecalis and S. aureus. Over 98% of the bacteria died at 20 min at the highest doses of group IIA PLA₂. In the presence of group V PLA₂, 90% of S. aureus and 99.8% of E. faecalis died in 2 h. Over 99% of MRSA died in 1 h in the presence of group IIA PLA₂. The antibacterial potency of group V PLA₂ against MRSA was somewhat weaker than that of group IIA PLA₂; 85% of MRSA died in 2 h at a corresponding dose of the enzyme. Group IIA PLA₂ had a bacteriostatic effect against E. coli: 91% of the bacteria were alive at 120 min, whereas in the absence of group IIA PLA₂, the number of the bacteria increased almost 3-fold. Group V PLA₂ had neither bacteriostatic nor bactericidal effect against E. coli. The dose responses of the bactericidal activity of both PLA₂s against all microbes tested are presented in Table I.

View larger version (27K): [in this window] [in a new window]   FIGURE 1. Killing of bacteria in vitro by group IIA PLA2. Samples were taken after incubation for 20, 60, and 120 min and cultivated on agar. CFUs were counted after 24 h. The bactericidal effect of group IIA PLA2 was strongest against L. monocytogenes (A). A clear effect was also seen against methicillin-sensitive S. aureus strain (B), methicillin resistant S. aureus strain (C), E. faecium (D), and E. faecalis (E). Against E. coli, a bacteriostatic effect was seen with high concentration of group IIA PLA2 (F).

View larger version (25K): [in this window] [in a new window]   FIGURE 2. Killing of bacteria in vitro by group V PLA2. Samples were taken after incubation for 20, 60, and 120 min and cultivated on agar. CFUs were counted after 24 h. The bactericidal effect of group V PLA2 was strong against L. monocytogenes (A), E. faecium (D), and Enterococcus faecalis (E). Weaker effects were seen against methicillin sensitive (B) and methicillin-resistant S. aureus (C). No effect was seen against E. coli (F).

The presence or the absence of isotonic saline did not change the bactericidal effect of group IIA and group V PLA₂s against S. aureus (data not shown). No difference in the bactericidal activity was observed between culture medium containing 5 mg/ml and 0.5 mg/ml of BSA (data not shown). The bacterial viability in the medium depended on the presence of BSA in the medium (presumably the bacteria used BSA for their nutrition). When BSA was omitted from the culture medium, the bactericidal effect of group IIA PLA₂ decreased somewhat (Fig. 3A). On the contrary, the bactericidal effect of group V PLA₂ increased when BSA was omitted from the reaction mixture (Fig. 3B). BHIB increased the growth of the bacteria significantly. Without BHIB, the antibacterial effects of group IIA and group V PLA₂ were observed with the concentrations of 3.05 µg/ml of the enzyme. In the presence of BHIB, group IIA and group V PLA₂s were antibacterial only at the highest concentrations of the enzymes (30.5 µg/ml; Fig. 3, C and D).

View larger version (36K): [in this window] [in a new window]   FIGURE 3. The bactericidal effect of the two PLA2s against S. aureus in different culture media. Samples were taken after incubation for 20, 60, and 120 min and cultivated on agar. CFUs were counted after 24 h. The bactericidal effect of group IIA was diminished when no albumin was present (A). The effect of albumin on the bactericidity of group V PLA2 was minor (B). In the presence of BHIB, a higher concentration of group IIA PLA2 was needed to gain the bactericidal effect (C). Same observation was made with group V PLA2 (D). Culture medium contained 10 mmol/L HEPES and 1 mmol/L Ca2+.

The percentage of viable HEK cells in treatment medium containing group IIA PLA₂, group V PLA₂, or sterile saline was 95% at 24 h. No difference was seen among these three groups. In the treatment medium containing 10% (v/v) ethanol (positive control for cytotoxicity), 60% of the cells died in 24 h. The results show that neither group IIA PLA₂ nor group V PLA₂ is cytotoxic to mammalian cells.

	Discussion

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Group V PLA₂ is a novel type of 14-kDa secretory PLA₂. The amino acid sequence of group V has significant homology with that of group IIA PLA₂ (12, 13). It has been proposed that the genes of groups V, IIA, and IIC share a common origin (14). The bactericidal properties of group V PLA₂ have not been tested previously. Rat recombinant group V PLA₂ has a strong affinity to phosphatidylcholine (PC) and displays activity on PC micelles and liposomes. These properties make group V PLA₂ suitable for action on biological membranes (11). The same kinds of results have been published for human group V PLA₂ (10). Human group V PLA₂ hydrolyses phosphatidylethanolamine (PE) vesicles more effectively than PC vesicles. However, in mixed vesicles, the hydrolysis of PE and PC by group V PLA₂ is equivalent (7). Human group V PLA₂ catalyzes the hydrolysis of PC over 100 times more effectively than human group IIA PLA₂ (10). Rat group V PLA₂ hydrolyses PC more effectively than rat group IIA PLA₂ (11). Thus, it seems that rat and human group V PLA₂s share some biochemical and functional properties. The major structural components of the cell walls of Gram-negative and Gram-positive bacteria are PE and phosphatidylglycerol (PG), respectively. The activities of rat recombinant group IIA and group V PLA₂s have not been tested on these two phospholipids. However, they have been tested on phosphatidylglycol (PGlo), which has the same charge, size, and interfacial properties as PG (11). The maximal velocity of group IIA PLA₂ on PGlo is almost 20-fold higher than that of group V PLA₂. Han and coworkers have calculated the specificity constants (k_cat/K_m) of human group IIA and V PLA₂s (10). The value for group V PLA₂ on PE is almost 10-fold higher than that for group IIA PLA₂. It is possible that group IIA PLA₂ hydrolyzes bacterial PG, whereas the main target of group V PLA₂ may be bacterial PE and eukaryotic membrane phospholipids, such as PC. The bactericidal properties of group V PLA₂ and its localization in murine macrophages (8) suggest an involvement of group V PLA₂ in immunity against bacteria. The actual role of group V PLA₂ in macrophages requires further investigation.

The bactericidal effect of group IIA PLA₂ is highest against bacteria in the phase of logarithmic growth (15). This probably reflects the increased vulnerability of dividing bacteria and the ability of group IIA PLA₂ to reach the bacterial plasma membrane through the dividing cell wall. Whether the effect of group V PLA₂ is dependent on the phase of the bacterial growth cycle remains to be established. The finding that the bactericidal effect of group IIA PLA₂ was diminished in culture medium containing no BSA correlates to the earlier results of Foreman-Wykert and coworkers (16), but the rational of this effect is not fully understood.

The MBC values for group IIA and V PLA₂s against the microbes tested here are 1–50 µg/ml. The serum PLA₂ concentrations in severe inflammatory conditions such as typhoid fever (17) and systemic fungal infections (18) have been reported to be 1–5 µg/ml. In normal human tears the concentration of group IIA PLA₂ exceeds 30 µg/ml (4). In the present study L. monocytogenes and S. aureus were the most sensitive microbes to group IIA PLA₂; 3.05 µg/ml of group IIA PLA₂ was bactericidal against these bacteria. These observations suggest that the concentrations of group IIA PLA₂ present in human serum in severe inflammatory diseases may kill bacteria. Secretory group IIA PLA₂ has been shown not to be cytotoxic to otherwise healthy cells (19, 20), whereas the cytosolic group IV PLA₂ may have cytotoxic properties (21). The present results indicate that neither group IIA PLA₂ nor group V PLA₂ have cytotoxic properties. We propose that both group IIA and group V PLA₂s may be considered future therapeutic agents against bacterial infections.

	Acknowledgments

 
The bacterial strains were provided by Drs. Pentti Huovinen and Jaana Vuopio-Varkila of the Finnish National Public Health Institute. HEK cells were provided by Dr. Klaus Elenius.

	Footnotes

 
¹ This work was supported by The University of Turku Foundation, The Finnish Medical Foundation, Turku University Hospital, The Maud Kuistila Foundation, and The Emil and Blida Maunula Foundation.

² Address correspondence and reprint requests to Dr. Timo J. Nevalainen, Department of Pathology, University of Turku, Kiinamyllynkatu 10, 20520 Turku 52, Finland.

³ Abbreviations used in this paper: PLA₂, phospholipase A₂; BHIB, brain heart infusion broth; HEK, human embryonic kidney cell; MBC, minimum bactericidal concentration; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PGlo, phosphatidylglycol.

Received for publication May 30, 2000. Accepted for publication January 2, 2001.

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