DOI: 10.5061/dryad.905qfttvz
title: Host glutathione is required for Rickettsia parkeri cell division and intracellular survival

description: 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 to
 promote cell division, actin-based motility, evasion of antibacterial
 autophagy, and intracellular survival. This knowledge contributes to the
 expanding paradigm that GSH plays diverse roles in the pathogenesis of
 intracellular bacteria and represents a promising target for host-acting
 therapeutics.
description: 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
description: # 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 of L-NIL Sup Fig. 1: Measurement of bacterial abundance (PFU assays). Sup Fig. 2: Measurement of bacterial abundance (PFU assays). Sup Fig. 4: contains P value data ### Files and variables #### File: Source\_Data\_Sun\_et\_al\_Nat\_Communications\_for\_repository.xlsx **Description:** The Excel file has tabs for each figure. The figures are described below. GSH=Glutathione; R=Rickettsia ##### Variables * Fig. 1: GSH is required for cell division and growth of R. parkeri in epithelial cells. * Fig. 2: Host GSH is required for proper actin-based motility and cell-to-cell spread of *R. parkeri* * Fig. 3: Host GSH is critical for R. parkeri survival in macrophages via a non-bacteriolytic restriction mechanism. * Fig. 4: Host GSH is required for R. parkeri to avoid ubiquitylation and antibacterial autophagy in macrophages. * Fig. 5: GSH serves as a cysteine source for R. parkeri cell division and survival. * Supplementary Figure 1: GSH, CysGly and cystine do not rescue survival or chaining defects of *R. parkeri in BSO-treated cells * Supplementary Figure 2: Inhibiting iNOS does not rescue R. parkeri survival upon BSO treatment in the absence or presence of ATG5. * Supplementary Figure 4: p value for all figures. **Abbreviations and units of measure:** * BSO = buthionine sulfoximine * Rp or R. parkeri = Rickettsia parkeri * GSH = glutathione * h = hour, hpi = hours postinfection, d = day, dpi = days postinfection * tail refers to actin tails * LDH = lactate dehydrogenase * BMDM = bone marrow-derived macrophage * RLUs = relative light units * 3MA = 3 methyl adenine * NAC = N-acetylcysteine * GSHee = glutathione ethyl ester * L-NIL is an inhibitor of inducible nitric oxide synthase (iNOS) * Cys-Gly = cysteinyl glycine **Code/software** No software or unique code is needed to analyze the data **Access information** Other publicly accessible locations of the data: * NA Data was derived from the following sources: * NA