Secretory phospholipase A2 is the principal bactericide for staphylococci and other gram-positive bacteria in human tears - PubMed (original) (raw)

Secretory phospholipase A2 is the principal bactericide for staphylococci and other gram-positive bacteria in human tears

X D Qu et al. Infect Immun. 1998 Jun.

Abstract

We examined human tears for molecules that killed gram-positive bacteria. The principal mediator of bactericidal activity against staphylococci proved to be a calcium-dependent enzyme, secretory phospholipase A2. Whereas the concentration of secretory phospholipase A2 in the normal tear film exceeded 30 microg/ml, only 1.1 ng (<0.1 nM) of the enzyme per ml sufficed to kill Listeria monocytogenes and 15 to 80 ng/ml killed Staphylococcus aureus. Despite its efficacy against gram-positive bacteria, secretory phospholipase A2 lacked bactericidal activity against gram-negative organisms (Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa) when tested in the ionic environment of tears. Given the presence of secretory phospholipase A2 in tears, intestinal secretions, and leukocytes, this enzyme may play a substantial role in innate mucosal and systemic bactericidal defenses against gram-positive bacteria.

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Figures

FIG. 1

Purification of secretory PLA2 from tears. The left panel shows an SDS-PAGE gel (16.5% polyacrylamide) run under reducing conditions and stained with Coomassie blue. Its left lane contained molecular mass standards (STD.) with masses of 3.0 (a), 6.2 (b), 14.3 (c), 18.4 (d), 29.0 (e), and 43.0 (f) kDa. Lane A (5 μl of pooled tears) displays three major bands, which have been numbered. They correspond to the following proteins: 1, lysozyme; 2, tear-specific prealbumin (also called lipocalin); and 3, lactoferrin. Lane B shows the PLA2-containing fractions after the first stage of RP-HPLC purification. Lane C shows highly purified PLA2 after the second stage of RP-HPLC purification, with an acetonitrile (ACN) gradient in 0.13% HFBA. The panel on the right shows the sPLA2 peak, monitored at 230 nm, and is from the second HPLC purification step.

FIG. 2

Activity of HPLC fractions. Antimicrobial activity was tested against L. monocytogenes with radial diffusion assays by using low-salt (□) or high-salt (○) underlay gels that contained 10 mM sodium phosphate ± 100 mM NaCl. Enzymatic PLA2 activity (▴) was measured with 14C-labeled E. coli. Lysozyme activity (▾) was tested with a lysoplate assay.

FIG. 3

ECL assay. A standard curve is shown for sPLA2 purified from human tears.

FIG. 4

Antibacterial activity of purified sPLA2. Two-stage radial diffusion assays were performed with highly purified tear sPLA2 and five gram-positive bacteria: L. monocytogenes (•), S. aureus 67395 (○), S. aureus GM-1 (□), group B streptococcus (▵), and E. faecium 94.132 (▴). Each symbol represents a mean value derived from three separate experiments with each organism. The regression lines were fit by the method of least mean squares. The _x_-intercepts indicate the minimal effective concentration.

FIG. 5

Susceptibility of S. epidermidis and M. luteus. Radial diffusion assays were performed with purified human tear sPLA2, human tear lysozyme, and human milk lactoferrin. Note that the MIC (_x_-intercept) of sPLA2 for M. luteus (•) was approximately 0.3 μg/ml, whereas that of lysozyme was 13 μg/ml. The MIC of sPLA2 for S. epidermidis (□) was approximately 0.15 μg/ml, whereas lysozyme was not effective (MIC of >1.5 mg/ml).

FIG. 6

Colony count assay. Approximately 106 CFU per ml of L. monocytogenes (L. mono.) EGD or S. aureus 67395 was incubated for 15 min at 37°C with the indicated concentrations of tear sPLA2.

FIG. 7

Calcium dependence of antibacterial activity. Tear sPLA2 was tested against two gram-positive bacteria, L. monocytogenes and S. aureus. The underlay gels contained ATS without Ca2+ or Mg2+ and were supplemented with divalent cations (0.7 mM) or EGTA (2 mM), as indicated by the inset. Each symbol depicts a mean value derived from four separate experiments.

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Secretory phospholipase A2 is the principal bactericide for staphylococci and other gram-positive bacteria in human tears - PubMed (original) (raw)