Staphylococcus aureus exerts its pathogenicity partly by the secreted pore-forming alpha-toxin. The formation of pores in the plasma membrane of the host cell leads to lysosome exocytosis and thus the trafficking of the lysosomal-resident acid sphingomyelinase (ASM) to the plasma membrane. ASM catalyzes the conversion of sphingomyelin on the outer membrane layer to ceramide, which induces rearrangement of lipid microdomains and the internalization of the damaged membrane in small endocytic vesicles. Whether alpha-toxin or the whole pathogen is engulfed during this process can vary among the infection models and it is not clear whether this effort to repair the membrane is contributing to the cell toxicity of S. aureus in vivo. Caenorhabditis elegans encodes three ASM: ASM-1, ASM-2 and ASM-3 that are all homologues of the human ASM. Here, we use deletion alleles of the three asm genes to investigate the role of sphingomyelinases and their product ceramide during S. aureus infection in the model host C. elegans. We infected control worms as well as single or double asm mutants with S. aureus and compared their survival rate using Kaplan Maier plots. After 72 hours of feeding on S. aureus, the survival rate of the control strain was 0%. In contrast, survival of the asm mutants ranged between 40% to 60%, implying that ASM activity or their product ceramide is required for the pathogenicity. Searching for the cellular basis of the observed resistance of the asm mutants, we prepared non infected young adult hermaphrodites for Transmission Electron Microscopy by high pressure freezing and freeze substitution. Looking for altered cell or organelle morphology, we observed highly electron dense membranous organelles that resemble the multilamellar bodies described in various tissues in human Niemann Pick Disease, a sphingolipid-storage disease. While such organelles were rarely observed in N2 controls, various cell types in asm mutants contained these structures with
asm-1/asm-3 double mutants showing the highest count. Based on the high electron density, we predict that this phenotype is caused by accumulation of osmophilic unsaturated lipids, including sphingomyelin, that are inefficiently metabolized. Our findings show that the host ASM is required for full S. aureus virulence. Since the increase in multilamellar organelles in the asm mutants is also described in human Niemann Pick Disease, molecular mechanisms of ASM function may be conserved in nematodes and mammals. In the next steps we aim to reveal the mechanisms of increased survival of the asm mutants after S. aureus infection.