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.
Highlights
Detected in yeasts and mammals, but not in prokaryotes
We previously reported that the release of CerN into a culture medium of P. aeruginosa was strongly enhanced by the addition of sphingolipids such as SM, Cer, and Sph[21]
We initially generated a P. aeruginosa mutant (AN17-BGT), in which the cerN gene was replaced with the E. coli β-galactosidase gene (Fig. 1)
Summary
Detected in yeasts and mammals, but not in prokaryotes. On the other hand, the genetic information on neutral CDase is conserved from bacteria to humans. P. aeruginosa is known to produce multiple virulence factors including proteases, lipases, phospholipases, and exotoxins. These virulence factors play important roles when P. aeruginosa invades host cells and causes severe cellular damage[15,16]. Among these virulence factors, hemolytic phospholipase C (PlcH) hydrolyzes phosphatidylcholine (PC) and SM to generate diacylglycerol (DG) and Cer, respectively, and phosphorylcholine. This study provides the molecular basis for understanding the mechanisms responsible for the transcriptional regulation of CerN in bacteria that do not possess sphingolipids and facilitates understanding on how pathogens incorporate sphingolipids as a nutrient to survive under severe conditions including the host environment
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