Sphingosine induction of the Pseudomonas aeruginosa hemolytic phospholipase C/sphingomyelinase, PlcH

The hemolytic phospholipase C, PlcH, is an important virulence factor for Pseudomonas aeruginosa. PlcH preferentially hydrolyzes sphingomyelin and phosphatidylcholine and this hydrolysis activity can drives tissue damage, inflammation, and interferes with the oxidative burst of immune cells. Among other contributors, transcription of plcH was previously shown to be induced by phosphate starvation via PhoB and by the choline metabolite, glycine betaine, via GbdR. Here, we show that sphingosine can induce plcH transcription and resultant secreted PlcH enzyme activity. This induction is dependent on the sphingosine-sensing transcription regulator SphR. The SphR induction of plcH occurs from the promoter for the gene upstream of plcH that encodes the neutral ceramidase, CerN, and transcriptional read-through of the cerN transcription terminator. Evidence for these conclusions come from mutation of the SphR binding site in the cerN promoter, mutation of the cerN terminator, enhancement of cerN termination by adding the rrnB terminator, and RT-PCR showing that the intergenic region between cerN and plcH is made as RNA during sphingosine, but not choline, induction. We also observe that, like glycine betaine induction, sphingosine induction of plcH is under catabolite repression control, which likely explains why such induction was not seen in other studies using sphingosine in rich media. The addition of sphingosine as a novel inducer for PlcH points to regulation of plcH transcription as a site for integration of multiple host-derived signals.