Mitochondria protect against an intracellular pathogen by restricting access to folate

Abstract

As major consumers of cellular metabolites, mitochondria are poised to compete with invading microbes for the nutrients that they need to grow. Whether cells exploit mitochondrial metabolism to protect from infection is unclear. In this work, we found that the activating transcription factor 4 (ATF4) activates a mitochondrial defense based on the essential B vitamin folate. During infection of cultured mammalian cells with the intracellular pathogen Toxoplasma gondii, ATF4 increased mitochondrial DNA levels by driving the one-carbon metabolism processes that use folate in mitochondria. Triggered by host detection of mitochondrial stress induced by parasite effectors, ATF4 limited Toxoplasma access to folates required for deoxythymidine monophosphate synthesis, thereby restricting parasite growth. Thus, ATF4 rewires mitochondrial metabolism to mount a folate-based metabolic defense against Toxoplasma.

Fig. 1.. ATF4 increases mtDNA levels during Toxoplasma infection.

(A) Schematic of human mitochondrial genome, genes analyzed by qPCR in red. (B) mtDNA levels monitored by qPCR (normalized to RUNX2) in ES-2s cells that were uninfected (uninf) or infected with Toxoplasma (Toxo) at a multiplicity of infection (MOI) of 4 at 12 and 24 hours post infection (hpi). Data are mean ± SD of n=7 independent cultures; *p<0.05; **p<0.01; ****p<0.0001 for uninfected versus infected by means of two-way ANOVA analysis. (C) Volcano plot of whole cell lysates from uninfected and infected HeLa cells at 24 hpi and analyzed by means of mass spectrometry with highlighted transcription factors. (D) ES-2 cells were uninfected or Toxo-infected (MOI: 4) for 6, 12, and 24 hours and analyzed by means of immunoblotting for ATF4, ~50 kDa; β-Actin (ACTB), ~45 kDa; and Toxoplasma GAP45 (TgGAP45), ~45 kDa. (E) Immunofluorescence images of uninf cells and cells infected with mCherry-expressing Toxoplasma at 24 hpi. Arrowhead indicates an uninfected bystander cell that is proximal to an infected cell; scale bar, 10 μm. (F) Quantification of mean fluorescence intensity (MFI) of ATF4 in the nuclei of cells in images as in (E); byst: bystander cell. ****p<0.001 for comparison to uninf by means of one-way ANOVA analysis (G) Immunoblot analyses of lysates from uninfected and Toxoplasma-infected (MOI: 4) WT and ATF4 KO ES-2 cells at 24hpi: ATF4, ~50 kDa; Vinculin (VCL), ~124 kDa; and Toxoplasma GAP45 (TgGap45), ~45 kDa. (H, I and J) mtDNA levels monitored by qPCR of DLOOP, mt-ND1 and mt-CYTB (normalized to RUNX2) in uninfected and Toxoplasma-infected (MOI: 4) ES-2 cells at 12 at 24 hpi. Data are mean ± SD of n=9 independent cultures; **p<0.01, ***p<0.001 for uninfected versus infected; #p<0.05; ##p<0.01 for WT vs. ATF4 KO by means of two-way ANOVA analysis. (B-J) ES-2 cells used for all experiments.

Fig. 2.. ATF4 requires mitochondrial one-carbon metabolism to increase mtDNA levels.

(A) Immunoblot (IB) analyses of WT and eIF2α^S51A AN3–12 cells that were uninfected (uninf), infected with Toxoplasma (Toxo), or tunicamycin (TUN)-treated (3 μg/ml) for 24 hours (h): ATF4; phospho-EIF2α (Ser51), ~38 kDa; α-Tubulin (TUBA), ~55 kDa; and TgGAP45. (B) Heatmap of mRNA levels of ATF4 target genes in uninf, Toxo, and TUN (3 μg/ml)-treated ES-2 cells; n=3 independent cultures. (C and D) WT and ATF4 KO ES-2 cells were uninf or Toxo for 24 h and analyzed by qPCR for SHMT2 and MTHFD2. Transcripts were normalized to ACTB and are relative to WT uninf. Data are mean ± SD of n=7 independent cultures. *p<0.05; ****p<0.0001 for uninf versus Toxo by two-way ANOVA analysis. (E) IB of ES-2 cells as indicated: MTHFD2, ~32 kDa and TUBA. (F, G and H) ES-2 cells as indicated were analyzed for mtDNA by qPCR for DLOOP, mt-ND1 and mt-CTYB normalized to RUNX2 levels and relative to WT uninfected. Data are mean ± SD of n=4 independent cultures. *p<0.05; **p<0.01 for uninf versus Toxo, and #p<0.05; ##p<0.01 for WT versus MTHFD2 KO by two-way ANOVA analysis. (I) IB analysis of ES-2 cells as indicated at 24 hpi (MOI: 4 for Toxo): ATF4, VCL, and TgGAP45. (J and K) Cells treated as in (I) were analyzed by qPCR as in (C and D). Data are mean ± SD of n=4 independent cultures, *p<0.05; **p<0.01 by one-way ANOVA analysis. (L) IB analysis of WT and HRI KO ES-2 cells that were uninf or Toxo at 24 hpi; Toxoplasma GRA7 (TgGRA7), ~32 kDa, (M) IB analysis of uninf and Toxo WT and OMA1 KO mouse embryonic fibroblasts (MEFs) at 24 hpi: OMA1, ~35 kDa, ATF4, ACTB, and TgGAP45. (N) Toxo WT and ATF4 KO ES-2 were treated ± ISRIB (200nM) and analyzed 24 hpi via flow cytometry for Toxoplasma burden (mCherry median FI). Data are mean ± SEM of n=3 biological experiments, **p< 0.01 for DMSO versus ISRIB, ####p < 0.0001 for WT versus ATF4 KO by two-way ANOVA analysis.

Fig. 3.. Mitochondrial 1C metabolism restricts parasite dTMP synthesis and replication.

(A) M+2 ²H-labelleled dTMP from cyto-1C (2 blue dots), M+1 ²H-labelled dTMP from mito-1C (1 red dot). (B and C) Labeled dTMP and dTTP in WT and ATF4 KO ES-2 cells uninfected (uninf) or infected (inf) with WT Toxo and ΔcpsII parasites. Data are mean ± SD of n=3; ****p<0.0001 for uninf vs. inf ####p<0.0001 for WT vs. ATF4 KO clones by two-way ANOVA. (D) ATF4, VCL, and TgGAP45 in ES-2 cells as indicated. (E) dTMP levels in Toxo from WT and ATF4 KO cells. Data are mean ± SD of n=7, dTTP not detected in two cultures; *p<0.05; **p<0.01; for WT vs. ATF4 KO by unpaired t-test. (F) Total folate species (%) in uninf and inf ES-2 cells in minimal essential media, normalized to cell number. DHF: dihydrofolate; CH2-THF: 5,10-methylene-tetrahydrofolate; CH3-THF: 5-methyl-tetrahydrofolate; CHO-THF: 10-formyl-tetrahydrofolate. Data are mean ± SD of n=4; **p<0.01; ***p<0.001 for uninf vs. inf by multiple unpaired t-test. (G) Inf WT ES-2 cells treated ± MTX (200 nM) and either 68 μM folic acid; 2mM formate, or 25μM CH3-THF, and analyzed by flow cytometry for Toxoplasma mCherry median FI. Data are mean ± SEM of n=3; **p < 0.001 for untreated vs. folate(s), ###p < 0.001, ####p<0.0001 for DMSO vs. MTX by two-way ANOVA. (H) ES-2 cells treated ± MTX (200 nM) and analyzed as in (G). Data are mean ± SEM of n=3; ***p < 0.001 for DMSO vs. MTX; ##p< 0.01 for WT vs. ATF4 KO by two-way ANOVA analysis. (I) ES-2 cells as indicated analyzed as in (G). (B-I) Harvested at 24 hpi. Data are mean ± SEM of n=3; *p<0.05; ***p< 0.001 by one-way ANOVA. (J) Inducible DHFR-TS system. (K) IF images of HFFs inf with Di-Cre parasites expressing killer-red (-rapa) or DHFR-TS (+rapa). (L-M) Plaque assays and quantification of Di-Cre parasites ± rapa (50nM) in HFFs at 7 dpi, n=15; **p<0.01 by unpaired t-test. (N) Model of ATF4-drived folate-based immunity.

Fig. 4.. ATF4 protects against Toxoplasma infection in vivo.

(A) Schematic of infection and ISRIB administration. Mice (n=5) where injected with ISRIB one day before infection with 50 tachyzoites or 1x PBS injection and every other day after (B-D) Peritoneal exudate cells (PECs) were isolated from mice treated as in (A) at 5 days post injection (dpi) and analyzed for the indicated ATF4 target genes by qPCR. Transcripts were normalized to Hprt and relative to uninfected PECs mice (n=5). ***p<0.001; ****p<0.0001 for vehicle versus ISRIB and #p < 0.05; ###p < 0.001; and ###p < 0.0001 for PBS versus Toxoplasma by means of two-way ANOVA analysis. (E) Peritoneal exudate was isolated 3 dpi from mice treated as in (A) and analyzed by means of flow cytometry for (E) % infected PECS; (F) Toxoplasma parasite burden in infected PECs (mCherry median FI) and (G) % of extracellular parasites. Data are mean ± SD of n=5 mice, *p<0.05 by unpaired t-test analysis (vehicle vs. ISRIB). (H) Representative scatter plots of peritoneal exudate isolated from mice treated as in (A). (I-L) Same as E-H except at 5 dpi.