(459) Pseudomonas aeruginosa interaction with host-derived sphingosine

Background: One metabolic consequences of CF is age-dependent accumulation of ceramide in airway cells and the airway lumen at the cost of lower sphingosine in the lumen. Sphingosine is an antimicrobial lipid and Pseudomonas aeruginosa has a specific set of genes to deal with the growth-inhibiting effects of sphingosine. While host sphingosine is at lower concentrations in CF, P. aeruginosa can generate sphingosine from ceramide via the action of neutral ceramidase CerN, supporting the need for P. aeruginosa to manage both endogenous and self-generated sphingosine within the CF lung.

Methods: Here, we examined P. aeruginosa’s interactions with sphingosine, including (i) the function of the sphingosine metabolic genes in the sphBCD operon, (ii) localization of exogenously applied sphingosine within P. aeruginosa in the presence and absence of sphBCD, and (iii) the ability of sphingosine to directly induce the virulence factor PlcH.

Results: We show that the sphBCD operon encodes a cytochrome C family protein (SphB), a sphingosine oxidase (SphC), and an aldolase of unknown function (SphD). SphB and SphC play an important role in maintaining P. aeruginosa growth in the presence of sphingosine and in the presence of sphingomyelin and ceramide when the P. aeruginosa sphingosine generation system (PlcH & CerN) is intact. We have observed sub-cellular sphingosine localization using alkyne-labeled sphingosine exposure and are currently using confocal and super-resolution microscopy to examine differences in actively growing vs growth inhibited cells in the presence and absence of sphBCD. Finally, we have discovered sphingosine as a novel inducer of plcH/PlcH transcription and resultant enzyme activity. This induction is dependent on the sphingosine-sensitive regulator SphR, but current data suggests that this regulation is due to transcription of a conditional operon containing cerN and plcH.

Conclusions: Understanding the interaction of P. aeruginosa with host-derived sphingolipids could suggest novel ways of sensitizing P. aeruginosa to sphingosine with the potential to improve infectious outcomes.

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