Antibacterial properties of chicken intestinal phospholipase A2

Abstract

Background: The presence of chicken group-IIA PLA2 (ChPLA2-IIA) in the intestinal secretion suggests that this enzyme plays an important role in systemic bactericidal defence. We have analyzed the bactericidal activity of purified ChPLA2-IIA, on several gram-positive and gram-negative bacteria by using the diffusion well and dilution methods.

Results: ChPLA2-IIA displays potent bactericidal activity against gram-positive bacteria but lacks bactericidal activity against gram negative ones. We have also demonstrated a synergic action of ChPLA2-IIA with lysozyme when added to the bacteria culture prior to ChPLA2-IIA. The bactericidal efficiency of ChPLA2-IIA was shown to be dependent upon the presence of calcium ions and then a correlation could be made to its hydrolytic activity of membrane phospholipids. Interestingly ChPLA2-IIA displays a higher dependence to Ca²+ ions than to Mg²+ ions.

Conclusion: We conclude that the main physiological role of ChPLA2-IIA could be the defence of the intestine against bacterial invasions.

Figure 1

In vitro bactericidal activity of chicken sPLA2-IIA. Samples of gram positive bacterial suspensions were taken after incubation with chPLA2-IIA at various concentrations for 20, 60 and 120 min and thereafter cultured on agar. Bacterial viability was assessed by measuring colony forming ability as described in "Materials and Methods". The results shown in the figure are means of two independent tests. The sPLA2 final concentrations were follows: white circle = 15 μg/ml sPLA2-IIA, white triangle = 30 μg/ml sPLA2-IIA, white square = 45 μg/ml sPLA2-IIA, black square = 50 μg/ml sPLA2-IB.

Figure 2

In vitro bactericidal activity of chicken sPLA2-IIA. Samples of gram negative bacterial suspensions were taken after incubation with sPLA2 at various concentrations for 20, 60 and 120 min and thereafter cultured on agar. Bacterial viability was assessed by measuring colony forming ability as described in "experimental procedures". The results shown in the figure are means of two independent tests. The sPLA2 final concentrations were follows: white circle = 15 μg/ml sPLA2-IIA, white triangle = 30 μg/ml sPLA2-IIA, white square = 45 μg/ml sPLA2-IIA, black square = 50 μg/ml sPLA2-IB.

Figure 3

Calcium dependency of the antibacterial activity. ChPLA2-IIA was tested against ML and BS bacteria. Strains were incubated with 0.5 and 10 μg/ml (final concentrations) of sPLA2-IIA without or with 0.7 mM divalent cations as indicated. Each symbol represents a mean value from three separate experiments. Results are given as mean values of duplicate determination of CFU.

Figure 4

Initial rates of hydrolysis of lysozyme against several lyophilized bacteria. Substrate suspensions (10⁷ cells/ml) were incubated with 2.5 mg/ml of lysozyme. One enzyme unit was defined as the amount causing decrease of 0.1 absorbance units at 540 nm in the reaction for 1 min at 25°C. The essay was performed in triplicate per sample.

Figure 5

Effect of lysozyme pre-treatment on the ability of ChPLA2-IIA to kill cell suspensions of BS and ML. Cell suspensions of BS and ML (10⁷/ml) were incubated with 2.5 mg/ml of lysozyme for 2 min prior to then addition of ChPLA2-IIA (45 μg/ml). Samples were taken at 20, 60 and 120 min and plated on BHI agar and grown for 18 h.