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Figure 6

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ZDB-FIG-221029-14

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Pagán et al., 2022 - mTOR-regulated mitochondrial metabolism limits mycobacterium-induced cytotoxicity

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Figure 6

Deficiencies in mTOR, glycolysis, and OXPHOS sensitize macrophages to mycobacterial ESAT-6-dependent cytotoxicity

(A) Cytochrome c release 7 hpi in THP-1 macrophages infected with BFP2-expressing WT or ΔESX-1 Mm at MOI = 3.

(B) Percentage of dying cells (Sytox Green⁺) during 4-h time-lapse at 1 dpi with tdTomato-expressing WT or ΔESX-1 Mm at MOI = 1. Values from uninfected (U) and infected (I) cells from the same fields are shown. See Video S4.

(C–J) Zebrafish were infected with dose-matched inocula of tdTomato-expressing Mm of the indicated strains via the caudal vein.

(C) Intramacrophage Mm burdens at the beginning of 6-h time-lapse confocal microscopy of mtor^fh178/fh178 and mtor-sufficient siblings expressing Tg(mpeg1:YFP) 2 dpi. See Video S5.

(D) Percentage of dying infected macrophages in same experiment shown in (C). See Video S5.

(E) Cording in mtor^fh178/fh178 animals and mtor-sufficient siblings 4 dpi.

(F and G) Cording in WT zebrafish treated with 2DG, UK5099, or DMSO 5 dpi.

(H) Cording in ndufaf1 G0 crispants and WT siblings 5 dpi.

(I) Cording in mtor^{sa16755/sa16755} animals and mtor-sufficient siblings 4 dpi. See also Figure S3.

(J) Cording in mtor^fh178/fh178 animals and mtor-sufficient siblings 4 dpi with ΔesxA Mm complemented with WT or point mutant Mtb esxA.

Symbols represent values from individual (A) wells, (B) imaging fields, or (C) animals. (A and B) Bars and (C and D) horizontal lines indicate mean values. (E–J) Numbers within columns indicate animals per group. Statistical analyses, (A–D) one-way ANOVA with Sidak’s post-test or (E–J) Fisher’s exact test. (B, E, and H) Data are representative of two experiments. Zebrafish time-lapse data were pooled from four experiments.

See also Figure S4.

Expression Data

Expression Detail

Antibody Labeling

Phenotype Data

Fish:	w200Tg/w200Tg w200Tg/w200Tg + CRISPR1-ndufaf1 + CRISPR2-ndufaf1 + CRISPR3-ndufaf1 + CRISPR4-ndufaf1 mtor^{sa16755/sa16755}; w200Tg/w200Tg
Conditions:	chemical treatment by environment: 2-deoxy-D-glucose chemical treatment by environment: UK-5099 biological treatment: Mycobacterium marinum M, chemical treatment by environment: 2-deoxy-D-glucose biological treatment: Mycobacterium marinum M biological treatment: Mycobacterium marinum M, chemical treatment by environment: UK-5099
Knockdown Reagents:	CRISPR1-ndufaf1 CRISPR2-ndufaf1 CRISPR3-ndufaf1 CRISPR4-ndufaf1
Observed In:	caudal vein antibacterial innate immune response caudal vein macrophage apoptotic process
Stage Range:	Long-pec to Day 6

Phenotype Detail

Acknowledgments

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Reprinted from Cell, 185(20), Pagán, A.J., Lee, L.J., Edwards-Hicks, J., Moens, C.B., Tobin, D.M., Busch-Nentwich, E.M., Pearce, E.L., Ramakrishnan, L., mTOR-regulated mitochondrial metabolism limits mycobacterium-induced cytotoxicity, 3720-3738.e13, Copyright (2022) with permission from Elsevier. Full text @ Cell