Ceramidase Enhances Phospholipase C-induced Hemolysis by Pseudomonas aeruginosa

Abstract

We previously reported the purification, molecular cloning, and characterization of a neutral ceramidase from Pseudomonas aeruginosa strain AN17 (Okino, N., Tani, M., Imayama, S., and Ito, M. (1998) J. Biol. Chem. 273, 14368 -14373; Okino, N., Ichinose, S., Omori, A., Imayama, S., Nakamura, T., and Ito, M. (1999) J. Biol. Chem. 274, 36616 -36622). Interestingly, the gene encoding the enzyme is adjacent to that encoding hemolytic phospholipase C (plcH) in the genome of Pseudomonas aeruginosa, which is a well known pathogen for opportunistic infections. We report here that simultaneous production of PlcH and ceramidase was induced by several lipids and PlcH-induced hemolysis was significantly enhanced by the action of the ceramidase. When the strain was cultured with sphingomyelin or phosphatidylcholine, production of both enzymes drastically increased, causing the increase of hemolytic activity in the cellfree culture supernatant. Ceramide and sphingosine were also effective in promoting the production of ceramidase but not that of PlcH. Furthermore, we found that the hemolytic activity of a Bacillus cereus sphingomyelinase was significantly enhanced by addition of a recombinant Pseudomonas ceramidase. TLC analysis of the erythrocytes showed that ceramide produced from sphingomyelin by the sphingomyelinase was partly converted to sphingosine by the ceramidase. A ceramidase-null mutant strain caused much less hemolysis of sheep erythrocytes than did the wild-type strain. Sphingosine was detected in the erythrocytes co-cultured with the wild-type strain but not the mutant strain. Finally, we found that the enhancement of PlcHinduced hemolysis by the ceramidase occurred in not only sheep but also human erythrocytes. These results may indicate that the ceramidase enhances the PlcH-induced cytotoxicity and provide new insights into the role of sphingolipid-degrading enzymes in the pathogenicity of P. aeruginosa.

Acid Sphingomyelinase Amplifies Redox Signaling in Pseudomonas aeruginosa-Induced Macrophage Apoptosis

“…2B). This decrease might be mediated by consumption of ceramide by host cell enzymes, but also by the ceramidase activity from P. aeruginosa (21). Furthermore, surface ceramide release was detected in WT macrophages, as determined by FACS using anti-ceramide mAbs.…”

Mechanistic Insights into the Hydrolysis and Synthesis of Ceramide by Neutral Ceramidase

“…1). The latter reaction strongly depends on the conversion of Cer to Sph in the plasma membranes (24). Hydrolysis of membrane-bound Cer will occur when the enzyme faces the membrane and a substrate penetrates into the remote binding site where GOL4 bound and inhibited the loop closure.…”

“…5B), but the enzyme is not likely to hydrolyze the cis-located membrane Cer, because the N-terminal anchor region is not long enough to allow the cavity direct access to the cis-located membrane Cer. Type III could represent the action of P. aeruginosa exotoxin (PaCD) on membrane Cer that is generated from membrane sphingomyelin by the action of P. aeruginosa sphingomyelinase (PlcH) (24). PaCD significantly enhanced the PlcH-induced hemolysis due to the conversion of Cer to Sph in the plasma membranes.…”

“…The hemolysis of human erythrocytes caused by PlcH was significantly enhanced by the nCDase. This is due to the conversion of Cer to Sph in the plasma membranes (24). Acharya et al (25)(26)(27) reported that the nCDase of Drosophila is integral to maintain photoreceptor functions and homeostasis, and a null mutation of the nCDase was lethal (28).…”

Bacterial Sphingomyelinases and Phospholipases as Virulence Factors

“…P. aeruginosa mutants lacking plcH have reduced virulence and result in decreased mortality in systemic experimental infections in mice (252,253). Interestingly, adjacent to plcH the P. aeruginosa genome encodes a neutral ceramidase which is secreted with PlcH/PlcS and enhances its hemolytic activity (254). Remarkably, PlcH/PlcS is also required for P. aeruginosa to elicit disease in Arabidopsis thaliana (131,255) and G. melonella (256) and has a role in killing Candida albicans filaments, suggesting that the antagonism with this fungus could have contributed to the evolution and maintenance of this virulence factor (257).…”