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.

A comprehensive multi-omics approach uncovers adaptations for growth and survival of Pseudomonas aeruginosa on n-alkanes

“…In addition to being induced intracellularly by ATCC 33988, the CerN protein was also present at high levels in the extracellular media of the environmental isolate (data not shown). These genes are all transcriptionally induced by sphingosine [ 36 , 37 ], but their roles in alkane degradation pathways are not known. No sphingosine was present in the extracellular media of any cultures.…”

“…9d ). Little is known about the SphR regulon, other than the fact that SphR binds, and is responsive to, exogenous sphingosine, dihydrosphingosine and phytosphingosine [ 36 , 37 ]. Interestingly, sphingosine is a component of human lung surfactants, and while ATCC 33988 does not produce rhamnolipids, it is possible that it synthesizes a yet unidentified surfactant species in the presence of n -alkanes, and that this compound may be structurally similar to sphingosine, and more compatible with ATCC 33988 than rhamnolipids.…”

An integrated genomic regulatory network of virulence-related transcriptional factors in Pseudomonas aeruginosa

“…To normalise the growth conditions (OD 600 = 0.6 in LB) for all mutants, we performed 10 RNA-seq analyses (AlgR 58 , ExsA 61 , GacA 62 , MvfR 45,71 , RsaL 63 , VqsM 72 , VqsR 60 , PhoB 73 , GbdR 74 and SphR 75 ) for which the regulons had been published previously in other strains and under other conditions. Compared with the previous results, the present study significantly extended the regulons of AlgR, ExsA, GacA, RsaL, PhoB, GbdR and SphR (see Supplementary Table 3 for a comparison of the results).…”

A Comprehensive Review on the Manipulation of the Sphingolipid Pathway by Pathogenic Bacteria

“…Pseudomonas species are also known to express and secrete sphingolipid-metabolizing enzymes. P. aeruginosa encodes a sphingomyelin synthase, PlcH, which specifically recognizes the choline head-group of sphingomyelin as well as the primary hydroxyl group of ceramide (Luberto et al, 2003) and its gene expression is strictly regulated by cellular amounts of sphingolipids (Okino and Ito, 2016). Furthermore, P. aeruginosa seems also to be able to hydrolyze ceramide as an alkaline ceramidase (Cdase) has been characterized from the P. aeruginosa strain AN17 isolated from the skin of a patient with atopic dermatitis (Okino et al, 1998).…”