title: Molecular mechanism for sphingosine-induced Pseudomonas ceramidase expression through the transcriptional regulator SphR.
abstract: 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.
authors.affiliation: The Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
authors.forename: Nozomu
authors.initials: N
authors.lastname: Okino
authors.affiliation: The Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Ito
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D055572:Ceramidases / Q000096:biosynthesis* / Q000235:genetics; D002518:Ceramides / Q000494:pharmacology; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005798:Genes, Bacterial; D005797:Genes, araC; D006461:Hemolysis; D005810:Multigene Family; D011401:Promoter Regions, Genetic; D040081:Protein Array Analysis; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D012329:RNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013109:Sphingomyelins / Q000378:metabolism; D013110:Sphingosine / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity; D014157:Transcription Factors / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D001426:Bacterial Proteins; D002518:Ceramides; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D013109:Sphingomyelins; D014157:Transcription Factors; D055572:Ceramidases; D013110:Sphingosine