Host glutathione is required for Rickettsia parkeri cell division and intracellular survival

Intracellular bacteria rely on eukaryotic metabolites for their fitness and pathogenesis. Yet, the mechanisms of how host metabolites promote bacterial physiology and immune evasion are often unclear. Here, we employed obligate cytosolic Rickettsia, which parasitizes over fifty host metabolites, to study bacterial utilization of host glutathione (GSH). We observed that GSH depletion strongly impaired R. parkeri intracellular survival. Super-resolution microscopy revealed that GSH depletion caused dramatic bacterial chaining in the host cytosol, prohibiting proper actin-based motility and cell-to-cell spread. GSH was especially critical for R. parkeri survival in primary macrophages, where it enabled R. parkeri to evade ubiquitylation and antibacterial autophagy. Cell division and survival defects were restored by supplementing N-acetylcysteine, suggesting that GSH serves as a cysteine source for R. parkeri. Together, these data suggest that Rickettsia requires GSH as a nutrient source t..., Data were collected in a variety of ways as described in the Material and Methods of the manuscript, including in vitro PFU assays, cell death (LDH and resazurin) assays, type I interferon (ISRE) assays, reactive oxygen species (ROS) assays, and quantification of microscopy assays, , # Host glutathione is required for Rickettsia parkeri cell division and intracellular survival

[https://doi.org/10.5061/dryad.905qfttvz](https://doi.org/10.5061/dryad.905qfttvz)

## Description of the data and file structure

Figure 1 - includes plaque assay (PFU) data; quantification of microscopy images for the number of bacteria per cell, and bacterial length measurements from microscopy images.

Figure 2: Contains quantification of microscopy images for a number of bacteria associated with actin and quantification of bacteria from PFU assays.

Figure 3: Contains quantification of bacterial abundance (PFU assays), cell death, IFN-I production, and NF-kB activity assay data.

Figure 4: Contains % colocalization data of bacteria with ubiquitin; PFU data in BMDMs for ATG5-/- cells, and PFU data upon addition of 3MA inhibitor

Figure 5: Contains PFU data in BMDMs; contains quantification of microscopy images for chained bacteria; contains fold change of ROS; and PFU data upon addition o...,