Acid sphingomyelinase deletion mutant C. elegans show formation of electron dense multilamellar bodies, accumulation of various sphingolipid species and increased resistance to Staphylococcus aureus infections

Abstract text (incl. figure legends and references)

Acid sphingomyelinase (ASM; SMPD1 gene in humans) is a lysosomal enzyme that catalyses the conversion of sphingomyelin to ceramide. Harmful mutations in this gene lead to the lipid storage disease Niemann Pick Disease Types A or B, which are characterized by abnormal accumulation of sphingomyelin and cholesterol in lysosomes of various tissues. Ultrastructural analysis of patient tissues or cell lines shows the lipids arranged as multilamellar structures within the lysosome.

Through its activity, ASM is contributing to lipid homeostasis. Stresses like pathogens or oxidative stress can induce the relocation of ASM from the lysosome to the plasma membrane, where it produces ceramide and thus leads to a rearrangement of ceramide-enriched domains. This process is important in many signalling cascades of the immune system. C. elegans has three ASM isoforms, ASM-1, ASM-2 and ASM-3, that are all homologous to the human ASM. Here, we used deletion mutants of the three asm genes to characterize ASM in the C. elegans sphingolipidome context and investigate their role in S. aureus infections. C. elegans is susceptible to the human pathogen S. aureus and a well-established model to study its pathogenicity.

We fed single, double and triple asm mutant worms with S. aureus and quantified their survival. None of the control worms, but 40% of the asm-1 and asm-3 single or double mutants survived after 72 hours of feeding on S. aureus. asm-2 mutation did not protect the worms against S. aureus infection, and even nullified the protective effect of asm-1 and asm-3 in double or triple mutants, implying divergent roles of ASM-2 vs. ASM-1 and ASM-3 in the pathogenicity of S. aureus.

Ultrastructural analysis of high-pressure frozen specimens revealed excessive lipid accumulation in asm-1 and asm-3 mutants, probably in lysosomes, which resemble the multilamellar bodies described in human Niemann-Pick patients. They were rarely observed in controls and asm-2.

To confirm that this phenotype is due to a disturbed sphingolipid metabolism, we analysed the sphingolipidome of C. elegans using liquid chromatography tandem-mass spectrometry. We found that all ASMs have an impact on the levels of various sphingomyelin and ceramide species. The changes induced by asm-2 and asm-3 are very similar to each other, whereas asm-1 seems to have different roles.

Our findings show that host ASM activities shape the outcome of experimental infection of C. elegans with S. aureus and that all three C. elegans ASMs have an impact on the sphingolipid homeostasis. In the next step, we want to characterize and localize the lipid species contained in the electron dense multilamellar bodies by using a combination of correlative light and electron microscopy and liquid chromatography tandem-mass spectrometry with labelled C. elegans specific iso-branched sphingolipids.