Molecular mechanism for sphingosine-induced Pseudomonas ceramidase expression through the transcriptional regulator SphR

• Published in: Scientific reports Vol. 6; no. 1; p. 38797
• Main Authors: Okino, Nozomu, Ito, Makoto
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 12.12.2016; Nature Publishing Group
• Subjects: 38/23; 38/39; 38/43; 38/61; 38/70; 38/77; 45/41; 631/326/1320; 631/45/287/1196; 82/16; Bacteria; Bacterial Proteins - biosynthesis; Bacterial Proteins - genetics; Base Sequence; Ceramidase; Ceramidases - biosynthesis; Ceramidases - genetics; Ceramide; Ceramides - pharmacology; Cytotoxicity; DNA microarrays; Electrophoretic mobility; Fatty acids; Gene Deletion; Gene expression; Gene Expression Regulation, Bacterial - drug effects; Genes, araC; Genes, Bacterial; Genomes; Hemolysis; Humanities and Social Sciences; Kinases; Lipids; Mammals; Metabolism; Metabolites; multidisciplinary; Multidrug resistance; Multidrug resistant organisms; Multigene Family; Nosocomial infections; Opportunist infection; Pathogens; Phospholipase C; Polypeptides; Prokaryotes; Promoter Regions, Genetic; Protein Array Analysis; Pseudomonas aeruginosa - enzymology; Pseudomonas aeruginosa - genetics; Pseudomonas aeruginosa - growth & development; Recombinant Fusion Proteins - metabolism; RNA, Bacterial - biosynthesis; RNA, Bacterial - genetics; RNA, Messenger - biosynthesis; RNA, Messenger - genetics; Roles; Science; Secretion; Sequence Alignment; Sequence Homology, Nucleic Acid; Sphinganine; Sphingolipids; Sphingomyelin; Sphingomyelins - metabolism; Sphingosine - metabolism; Sphingosine - pharmacology; Substrate Specificity; Transcription; Transcription Factors - deficiency; Transcription Factors - genetics; Transcription Factors - physiology; Transcription, Genetic - drug effects; Virulence; β-Galactosidase
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep38797

Pseudomonas aeruginosa, an opportunistic, but serious multidrug-resistant pathogen, secretes a ceramidase capable of cleaving the N -acyl linkage of ceramide to generate fatty acids and sphingosine. We previously reported that the secretion of P. aeruginosa ceramidase was induced by host-derived sphingolipids, through which phospholipase C-induced hemolysis was significantly enhanced. We herein investigated the gene(s) regulating sphingolipid-induced ceramidase expression and identified SphR, which encodes a putative AraC family transcriptional regulator. Disruption of the sphR gene in P. aeruginosa markedly decreased the sphingomyelin-induced secretion of ceramidase, reduced hemolytic activity, and resulted in the loss of sphingomyelin-induced ceramidase expression. A microarray analysis confirmed that sphingomyelin significantly induced ceramidase expression in P. aeruginosa . Furthermore, an electrophoretic mobility shift assay revealed that SphR specifically bound free sphingoid bases such as sphingosine, dihydrosphingosine, and phytosphingosine, but not sphingomyelin or ceramide. A β-galactosidase-assisted promoter assay showed that sphingosine activated ceramidase expression through SphR at a concentration of 100 nM. Collectively, these results demonstrated that sphingosine induces the secretion of ceramidase by promoting the mRNA expression of ceramidase through SphR, thereby enhancing hemolytic phospholipase C-induced cytotoxicity. These results facilitate understanding of the physiological role of bacterial ceramidase in host cells.