Chlamydia-like bacterium Simkania negevensis exploits host sphingolipids for infection and progeny formation

The obligate intracellular pathogen Simkania negevensis (Sne) resides within a Simkania-Containing Vacuole (SnCV), establishing close interactions with the endoplasmic reticulum (ER) and mitochondria. Due to limited metabolic capacity, Sne relies on host sphingolipids for survival. Previous findings showed that Sne does not depend on the de novo ceramide synthesis or ceramide transport by CERT. To investigate the role of salvage pathway in sphingolipid acquisition, we studied the importance of acid ceramidase (AC) and sphingomyelin synthases (SMS1 and SMS2) in modulating Simkania infection.

We first inhibited AC with a fluoxetine derivative inhibitor AKS466, which both reduced Sne infection rates and altered SnCV morphology. In contrast, AC knockdown or knockout in HeLa229 cells showed no significant effect on infection. Lipidomic analysis showed accumulation of ceramide and dihydroceramide, indicating an off-target inhibition of dihydroceramide desaturase (DEGS) by AKS466. This was further confirmed through DEGS activity assay, suggesting that a combined inhibition of AC and DEGS might be necessary to reduce infection.

The Sne infection of SMS1/2 double knockout (DKO) HeLa229 cells was significantly reduced. SMS1 is localized to Golgi and SMS2 is a plasma membrane/Golgi distributed enzyme. Using cell lines that inducibly expressed either the mutated, miss localized SMS2-M64A-HA or the wild type SMS2-HA, we observed that only the expression of the wildtype enzyme restored the infection rates of Sne, pointing to the importance of SMS2 localization for Sne infection. Currently, we are analyzing in which way SMS1 contributes to infection.

Finally, plasma membrane sphingomyelin depletion using Staphylococcus aureus β-toxin did not affect infection rates, indicating that sphingomyelin itself may not be the sole cause of infection reduction, but that the function and cellular localization of SMS1 and SMS2 may be critical. Together, our findings highlight that Sne utilizes both lysosomal- and Golgi-associated sphingolipid pathways for infection. We are currently examining whether SnCV surface similar to Chlamydia, serves as a platform for sphingomyelin or ceramide synthesis within the host. Additionally, we aim to explore the role of cholesterol trafficking and modifications in Sne infection.