Fig 4.

Inhibition of P. aeruginosa phospholipase A₂ activity prevents sphingosine-induced death of the bacteria. (A, B) P. aeruginosa strains ATCC 27853 (A) and 762 (B) were grown to the early log phase and washed; 2 × 10⁷ CFU/sample was labeled with 40 µCi [¹⁴C]acetate/100 mL TSB, washed, and incubated with the PLA₂ inhibitors AACOCF3 (25 µM), VU0364739 (25 µM), OBAA (25 µM), FIPI (50 µM), darapladip (50 µM), or aristolochic acid (5 µM) or the PLC inhibitor U73122 (10 µM), which served as negative control, or the samples were left untreated for 10 min. We then added sphingosine at 1, 5, or 10 µM or left the samples untreated. Bacteria were incubated for 15 min, and cardiolipin was determined by TLC after lipid extraction. Given are the mean ± SD of five independent experiments; ^***P <0.001, ANOVA, post hoc Tukey test. (C, D) P. aeruginosa strains ATCC27853 (C) and 762 (D) were grown to the early log phase and washed, and 10,000 CFU/sample were incubated with the inhibitors as above or left untreated for 10 min. We then added sphingosine at 1, 5, or 10 µM or left the samples untreated. Bacteria were incubated for 45 min, aliquots were spotted on TSA plates, grown o/n, and the CFU were counted. Shown are the mean ± SD of five independent experiments; ^***P <0.001, ANOVA, post hoc Tukey test. (E) Complementation of cardiolipin in ATCC 27853 or 762 P. aeruginosa reduced the bactericidal effects of 1 or 5 µM sphingosine. Bacteria were treated for 30 min with 10 µM cardiolipin prior to a 30 min treatment with 1 or 5 µM sphingosine in H/S, pH 7.0. Aliquots were then plated, and CFU were counted grown after o/n growth. Shown are the mean ± SD of four independent experiments; ^***P <0.001, ANOVA, post hoc Tukey test.