Rühling, M.; Wagner, F.; Epprecht, T.; Köhling, P. F.; Schumacher, F.; Sachs, S.; Kersting, L.; Fink, J.; Girndt, L.; Kleuser, B.; Sauer, M.; Seibel, J.; Weiss, G. L.; Rudel, T.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen and a leading cause of sexually transmitted infections worldwide. During its biphasic developmental cycle, infectious, non-replicative elementary bodies alternate with replicative reticulate bodies within a membrane-bound intracellular niche known as the inclusion. C. trachomatis relies heavily on host-derived metabolites, including sphingolipids, which are essential for inclusion integrity, bacterial growth and production of infectious progeny. Here, using expansion microscopy, we uncover an unexpected localization of host sphingolipids within the highly condensed DNA nucleoids of elementary bodies. These sphingolipids are released from nucleoids prior to DNA decondensation during the elementary-to-reticulate body transition, the earliest phenotypic event in the complex developmental cycle of these bacteria. Thereafter, nucleoids undergo a characteristic DNA decondensation process that we visualized by expansion microscopy. By combining super-resolution imaging with a FRET-based metabolic tracking approach and lipidomics, we identified sphingomyelin as the sphingolipid species incorporated into the condensing nucleoids of elementary bodies. Notably, reticulate bodies arrested in their developmental stage fail to accumulate sphingomyelin, suggesting a role for this lipid in stage-specific DNA condensation. Together, our findings reveal an unanticipated role for sphingolipids in bacterial DNA organization and developmental regulation in C. trachomatis .