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Figure 5

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ZDB-FIG-221029-13

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

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Figure 5

Glycolysis inhibition impairs mitochondrial metabolism and sensitizes infected macrophages to mycobacterium-induced cytotoxicity

(A–C) THP-1 macrophages treated with torin1 (400 nM), 2-deoxy-D-glucose (2DG, 5 mM), or DMSO were infected with Mm expressing (A) BFP2, (B) tdTomato, or (C) Mtb expressing tdTomato (MOI = 1).

(A) TMRE GeoMFI 1 dpi.

(B and C) Percentage of non-viable cells (FVD eFluor 660⁺) 1 dpi.

(D–I) Zebrafish were infected with ∼150 fluorescent Mm via the caudal vein.

(D) 5 dpi macrophage numbers in the body of mock- or Mm-infected Tg(mpeg1:YFP) zebrafish fish treated with 50 mM 2DG or 0.5% DMSO.

(E and F) 6-h time-lapse confocal microscopy of Tg(mpeg1:YFP) 3 dpi. (E) Absolute numbers of infected macrophages per field. (F) Percentage of dying infected macrophages per field. See Video S3.

(G) Cording in wild-type (WT) animals treated with 2DG or DMSO 5 dpi.

(H) Cording in WT animals treated with UK5099 (10 μM) or 0.5% DMSO 5 dpi.

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

Symbols represent values from individual (A–C) and (K) wells or (D–F) animals. (A–C) Bars and (D–F) horizontal lines indicate mean values. (G–I) Numbers within columns indicate animals per group. Statistical analyses, one-way ANOVA with (A–C) Sidak, (D) Tukey post-tests, (E and F) unpaired Student’s t test, or (G–I) Fisher’s exact test. (E and F). Time-lapse data were pooled from two independent experiments. Data are representative of (A), (G), and (H), two independent experiments.

See also Figure S3.

Expression Data

Expression Detail

Antibody Labeling

Phenotype Data

Fish:	w200Tg/w200Tg AB + CRISPR1-ndufaf1 + CRISPR2-ndufaf1 + CRISPR3-ndufaf1 + CRISPR4-ndufaf1
Conditions:	chemical treatment by environment: 2-deoxy-D-glucose 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 caudal vein macrophage migration
Stage Range:	Long-pec to Days 7-13

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