PPARγ mediated enhanced lipid biogenesis fuels Mycobacterium tuberculosis growth in a drug-tolerant hepatocyte environment
- Immunometabolism Laboratory, National Institute of Immunology, India
- Cellular Immunology Group, International Centre for Genetic Engineering and Biotechnology, India
- CSIR-Institute of Microbial Technology, India
- Indian Institute of Science Education and Research, India
- Council of Scientific and Industrial Research–Institute of Genomics and Integrative Biology (CSIR-IGIB), India
- Academy of Scientific and Innovative Research (AcSIR), India
Figures
Figure 1 with 1 supplement
Presence of Mycobacterium tuberculosis (Mtb) in the liver of human miliary tuberculosis patients.
(A) Hematoxylin and eosin (H and E) staining of the human autopsied liver tissue sections showing the presence of granuloma-like structure in the Mtb-infected liver. (B) Acid-fast staining shows the presence of Mtb in the liver section of miliary tuberculosis (TB) patients (arrows point to the presence of Mtb in the enlarged image). (C–D) Auramine O-Rhodamine B (A–O) staining and fluorescence in situ hybridization (FISH) with Mtb-specific 16 s rRNA primer further confirms the presence of Mtb in human liver tissue sections. (E) Dual staining of β-actin (green) and Ag85B (red) using respective antibodies shows the presence of Mtb in hepatocytes of human biopsied liver sections. (F) Dual staining of β-actin (green) and Ag85B (red) using respective antibodies shows no distinct signal of Ag85B in the uninfected liver biopsied sample (control). The data is representative from six human patient samples.
Figure 1—figure supplement 1
Histopathological changes in the liver biopsy samples of miliary tuberculosis (TB) patients: (A and B) A-O staining and fluorescence in situ hybridization (FISH) in the liver biopsy of the uninfected (control) group shows no Mycobacterium tuberculosis (Mtb)-specific signals.
(C) Hematoxylin and eosin (H&E) staining shows enhanced immune cell infiltration in the liver biopsy of the pulmonary TB patients compared to the uninfected control. (D) Dual staining of β-actin (green) and Ag85B (red) using respective antibodies shows the presence of Mtb in hepatocytes and other cells in liver biopsy sections (indicated by yellow arrows) (E) H&E staining shows distinct granuloma in the lung section of pulmonary TB patient and (F) Acid-fast staining in the same lung section shows a high bacterial load (G) A-O staining in the same lung section further validates the presence of high Mtb load. Scale bars in A, B, and C are 50 μm and in D is 20 μm. and E and F are 150 μm. Data shown in this figure is representative of 5 patients with miliary TB.
Figure 2 with 2 supplements
Involvement of the liver and hepatocytes in a mouse aerosol model of Mycobacterium tuberculosis (Mtb) infection.
(A) Schematic denoting the flow of experimental set up of studying the liver at tissue level and cellular level (generated using Biorender.com). (B) C57BL/6 mice were infected with 200 colony-forming units (CFU) of H37Rv through the aerosol route and the bacterial burden of the lung and liver was enumerated at different time points post-infection in lung and spleen. n=5 mice/group and the data is representative from three independent biological experiments (C) Alexa Fluor 488 Phalloidin and DAPI staining of uninfected and infected lungs at 8 weeks post-infection. Images were taken in 40 X magnification as mentioned (scale bar is 20 μm). (D) Immunofluorescence staining of Alexa Fluor 488 Phalloidin, DAPI, and Ag85B in the infected mice liver at 8 weeks post-infection shows the presence of Ag85B signals within hepatocytes (magnified image) (scale bar is 20 μm). (E) Isolated primary hepatocytes stained with phalloidin green and DAPI show the typical polygonal shape with binucleated morphology. (F) Anti-asialoglycoprotein receptor (ASGR1) antibody staining specifically stains primary hepatocytes isolated from infected mice, as validated by the contour plot in flow cytometry. (G) Antiasialoglycoprotein receptor (ASGR1) antibody staining specifically stains primary hepatocytes isolated from mice, as visualized through confocal microscopy. (H) Primary hepatocytes were isolated from the infected mice, lysed and CFU enumeration was done at the mentioned time points. (I) Ag85B staining of cultured primary hepatocytes, isolated from mice at 8 weeks post-Mtb infection. (Scale bar is 20 μm). (J) Dual immunostaining of Ag85B (red) and albumin (green) shows distinct signals of Mtb within the isolated hepatocytes from infected mice at 8 weeks post-infection. The figures show representative data from four independent biological experiments.
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Figure 2—source data 1
Data used for plotting the graph in Figure 2B.
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Figure 2—source data 2
Data used for plotting the graph in Figure 2H.
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Figure 2—figure supplement 1
Histopathological changes in the murine liver post Mycobacterium tuberculosis (Mtb) infection.
(A) 105 number of H37Rv were administered intraperitoneally (IP) and the bacterial burden was enumerated in primary hepatocytes that were isolated from the infected mice at the mentioned time points post-infection. (B) Analysis of the serum parameters of albumin, alanine transaminase (ALT), aspartate transaminase (AST), and gamma-glutamyl transpeptidase (GGT) in the uninfected and infected mice at the mentioned time points, mice were infected through the aerosol route with 200 colony-forming units (CFU). (C) Representative hematoxylin and eosin (H&E)-stained liver sections show enhanced immune cell infiltration in Mtb-infected mice. (D) Multiplex immunostaining of infected mice liver with CD 45.2 and Ag85B indicates the presence of Ag85B-positive signals in CD 45.2 cells. Sample size (E) Bacterial load in the primary hepatocytes of mice infected with 50 CFU of H37Rv at 6- and 8 weeks post-infection. The figures show representative data from three independent biological experiments.
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Figure 2—figure supplement 1—source data 1
Data used for generating the graph in Figure 2—figure supplement 1A.
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Figure 2—figure supplement 1—source data 2
Data used for generating the graph in Figure 2—figure supplement 1B.
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Figure 2—figure supplement 1—source data 3
Data used for generating the graph in Figure 2—figure supplement 1E.
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Figure 2—figure supplement 2
Involvement of the liver in the guinea pig aerosol infection model.
(A) Guinea pigs were infected with 200 colony-forming units (CFU) of H37Rv through the aerosol route and the bacterial burden of the lungs, liver, and spleen was enumerated at different time points post-infection (B) Hematoxylin and eosin (H&E) images of the lung and the liver at 4- and 8 weeks post-infection showing distinct granulomas in both the organs. Sample size (n)=3 guinea pigs in each group.
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Figure 2—figure supplement 2—source data 1
Data used for generating the plot in Figure 2—figure supplement 2A.
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Figure 3 with 1 supplement
Mycobacterium tuberculosis (Mtb) uses hepatocytes as a replicative niche.
(A and B) Representative microscopic images showing the infection of primary hepatocytes (PHCs), AML-12, HepG2, and Huh-7 with labeled Mtb-H37Rv strains and subsequent quantification of percentage infectivity in the respective cell types. RAW 264.7, THP-1, and murine bone marrow-derived macrophages (BMDMs) were used as macrophage controls. The scale bar in all images is 10 µm except in HepG2, which is 5 µm (C) colony-forming unit (CFU) enumeration of Mtb-H37Rv in different hepatic cell lines. (D and E) Representative confocal microscopy images of Mtb-GFP-H37Rv infected PHCs at the respective time points post-infection and bar blot depicting mean fluorescent intensity (MFI)/cell at the respective time points. (F and G) Representative confocal microscopy images of MtbH37Rv-GFP infected HepG2 at the respective time points post-infection and bar blot depicting MFI/cell at the respective time points, n=4 independent experiments, with each dot representing five fields of 30–60 cells. Scale bar 10 µm. (H) Fold change of growth rate of Mtb within HepG2, PHCs, RAW 264.7, THP-1, and BMDMs at the mentioned time points post-infection. All the figures show representative data from four independent biological experiments.
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Figure 3—source data 1
Data used for generating the graph in Figure 3B.
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Figure 3—source data 2
Data used for generating the graph in Figure 3C.
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Figure 3—source data 3
Data used for generating the graph in Figure 3E.
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Figure 3—source data 4
Data used for generating the graph in Figure 3G.
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Figure 3—source data 5
Data used for generating the graph in Figure 3H.
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Figure 3—figure supplement 1
Standardization of multiplicity of infection (MOI) in primary hepatocytes (PHCs) and HepG2.
(A) Confocal microscopic images of Phalloidin Alexa Fluor 555-stained PHCs infected with different multiplicity of infection (MOI) of Mtb-H37Rv-GFP. (B) Quantification of % infectivity at different MOIs in primary hepatocytes (PHCs) (C) Quantification of % infectivity at different MOIs in HepG2. Relative CFU of Mtb in (D) HepG2, (E) PHCs, (F) RAW 264.7, (G) THP-1, (H) murine BMDMs at the respective time points post-infection.
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Figure 3—figure supplement 1—source data 1
Data used for generating Figure 3—figure supplement 1B.
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Figure 3—figure supplement 1—source data 2
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Figure 3—figure supplement 1—source data 3
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Figure 3—figure supplement 1—source data 6
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Figure 3—figure supplement 1—source data 7
Data used for generating Figure 3—figure supplement 1H.
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Figure 4 with 1 supplement
RNA sequence analysis of infected and sorted hepatocytes at 0 hr and 48 hr post-infection.
(A) Experimental setup for infection of HepG2 with Mb-H37Rv-mCherry at 10 multiplicity of infection (MOI) with histogram of mCherry signals at 0- and 48 hr post-infection (schematic depicting experimental setup generated using Biorender.com) (B) Principal component analysis (PCA) plot illustrating the HepG2 transcriptome, identified through global transcriptomic analysis of 0 hr uninfected and infected and 48 hr uninfected and infected. (C) Gene ontology (GO) pathway enrichment analysis was done for differentially expressed genes (DEGs) with adjusted o-value <0.05 at 0- and 48 hr post-infection. The bubble plot depicts the enrichment of pathways on the mentioned time points post-infection, where the coordinate on the x-axis represents the gene ratio, the bubble size represents the gene count, and color represents the p-value. (D and E) Volcano plot depicting the fold change of different genes in 0 hr and 48 hr post-infection, the red dots represent the significantly upregulated and downregulated genes.
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Figure 4—source data 1
Data used to generate the volcano plot in Figure 4D.
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Figure 4—source data 2
Data used to generate the volcano plot in Figure 4E.
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Figure 4—figure supplement 1
Comparison of significant pathways between Mycobacterium tuberculosis (Mtb)-infected THP-1 and HepG2, 48 HPI.
(A) Gene ontology (GO) pathway enrichment analysis was done for differentially expressed genes (DEGs) with adjusted p-value <0.05 at 48 hr post-infection for Mtb-infected THP-1. (B) GO pathway enrichment analysis was done for DEGs with adjusted p-value <0.05 at 48 hr post-infection for Mtb-infected HepG2. The bubble plot depicts the enrichment of pathways on the mentioned time points post-infection, where the coordinate on the x-axis represents the gene ratio, the bubble size represents the gene count and color represents the p-value The bubble plot depicts the enrichment of pathways on the mentioned time points post-infection, where the coordinate on the x-axis represents the gene ratio, the bubble size represents the gene count and color represents the p-value.
Figure 5 with 3 supplements
Increased fatty acid biogenesis drives Mycobacterium tuberculosis (Mtb) growth in hepatocytes.
(A) Increased number of LDs/cells in HepG2 and primary hepatocytes (PHCs) post-Mtb infection as compared to the uninfected cells (B) Quantification of the number of lipid droplets in the infected HepG2 and PHCs with their uninfected control, 50–70 cells were analyzed from 3 independent experiments. (C) Increase in the BODIPY intensity at different days post-infection in infected hepatocytes (D). A high degree of colocalization of Mtb-H37Rv-GFP (green) with lipid droplets (red) within PHCs (E) Mass spectrometric quantification of the different species of diacylglycerols (DAGs), triacylglycerols (TAGs), and cholesterol esters in the Mtb-infected hepatocytes. (F) Confocal images showing puncta of fluorescently labeled fatty acid in Mtb derived from metabolically labeled (with BODIPY 558/568 C12) HepG2. Relative colony-forming unit (CFU) of Mtb under the administration of different inhibitors of the lipid metabolic pathway in (G). PHCs (H). HepG2 and (I). THP-1. Representative data from three independent biological experiments. Data were analyzed using the two-tailed unpaired Student’s t-test in B and one-way ANOVA in C, F, G, and H.*p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001. ns = non-significant. The figures represent data from three independent biological experiments.
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Figure 5—source data 1
Data used for generating the graph in Figure 5B.
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Figure 5—source data 2
Data used for generating the graph in Figure 5C.
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Figure 5—source data 3
Data used for generating the graphs in Figure 5E.
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Figure 5—source data 4
Data used for generating the graph in Figure 5G.
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Figure 5—source data 5
Data used for generating the graph in Figure 5H.
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Figure 5—source data 6
Data used for generating the graph in Figure 5I.
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Figure 5—figure supplement 1
Increased lipid biogenesis provides a growth advantage to Mycobacterium tuberculosis (Mtb) within hepatocytes.
(A) Confocal microscopy showing the LDs in both primary hepatocytes (PHCs) and HepG2 under inhibitors of fatty acid biosynthesis and triacylglycerols (TAG) biosynthesis. (B) Mtb RNA isolated from DMEM grown Mtb and HepG2-derived Mtb,48 HPI, with the RNA expression pattern of key lipid biogenesis genes and TAG biosynthesis genes in Mtb as determined by qRT PCR, the fold change has been calculated by considering the expression in the DMEM grown Mtb to be 1 (C) Experimental plan for metabolic labeling of HepG2 and subsequent infection by Mtb (schematic of experimental procedure generated using Biorender.com) (D) Fluorescently labeled fatty acid (BODIPY 558/568 C12) staining in DMSO-treated, C75-treated, and T863-treated HepG2. Data were analyzed by using the two-tailed unpaired Student’s t-test in B. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001, ns = non-significant. Representative data from three biologically independent experiments.
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Figure 5—figure supplement 1—source data 1
Data used for generating Figure 5—figure supplement 1B.
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Figure 5—figure supplement 2
Confocal microscopy images showing the puncta of fluorescently labeled fatty acid (BODIPY 558/568 C12) in Mycobacterium tuberculosis (Mtb) isolated from metabolically labeled HepG2.
(A) Confocal images showing puncta of labeled fatty acid in Mtb derived from DMSO, C75, and T863 treated metabolically labeled HepG2.
Figure 5—figure supplement 3
Enhanced lipid accumulation in the liver of Mycobacterium tuberculosis (Mtb)-infected mice.
(A) BODIPY staining in the liver of the uninfected and 8 weeks post-Mtb-infected murine liver (B) Dual staining of BODIPY (green) and Ag85B (red) shows Ag85B colocalization with BODIPY (highlighted in the zoomed image). (C) Representative images showing the mean lipidTOX Neutral Red staining in infected liver, 2-, 4-, and 8 weeks post-infection (D) Plot depicting the mean lipidTOX intensity at the mentioned time points post-infection normalized to the area. Data were analyzed using one-way ANOVA. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001. ns = non-significant in I. Representative data from experiment having n=4–6 mice/group.
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Figure 5—figure supplement 3—source data 1
Data used for generating the plot Figure 5—figure supplement 3D.
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Figure 6 with 1 supplement
Peroxisome proliferator-activated receptor-gamma (PPARγ) driven lipid biogenesis drives Mycobacterium tuberculosis (Mtb) growth in hepatocytes.
(A) Heat map showing the fold change of the genes involved in the lipid biosynthesis and LD biogenesis in liver across weeks 2, 4, and 8, post-infection. (B) Kinetic increase in the gene expression of Pparγ transcript levels across different weeks post-infection (C) Immunoblot showing increased PPARγ protein levels in primary hepatocytes (PHCs) (MOI: 10) at 5-, 24-, and 48 hr post-infection. (D) Bar plot showing the increased band intensity of PPARγ in the infected PHCs at the mentioned time points. (E) Representative confocal microscopy images showing HepG2 infected with Mtb-mCherry-H37Rv and treated with GW9662 and rosiglitazone (F) mean fluorescent intensity (MFI)/cell of Mtb-mCherry-H37Rv DMSO-treated, GW9662, and rosiglitazone-treated HepG2 cells. (G) Representative confocal images of uninfected, infected, GW9662, and rosiglitazone-treated infected HepG2 showing changes in the number of LDs. (H) Plot depicting the quantification of LDs/cell in the mentioned conditions. Representative data from n=4 biological replicates. Data were analyzed by using the two-tailed unpaired Student’s t-test in D and by one-way ANOVA in B, F, and H. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001, ns = non-significant.
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Figure 6—source data 1
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Figure 6—source data 2
Data used for generating the graph in Figure 6B.
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Figure 6—source data 3
Labeled and unedited blots shown in Figure 6C.
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Figure 6—source data 4
Data used for generating the graph in Figure 6D.
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Figure 6—source data 5
Data used for generating the plot in Figure 6F.
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Figure 6—source data 6
Data used for generating the plot in Figure 6H.
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Figure 6—figure supplement 1
Mycobacterium tuberculosis (Mtb) induced peroxisome proliferator-activated receptor-gamma (PPARγ) expression in infected hepatocytes.
(A) mRNA levels of PPARG and other lipid biosynthetic genes in infected HepG2, the fold change has been calculated by considering the expression in the uninfected cell to be 1 (B) Relative bacterial burden in DMSO, GW9662, and rosiglitazone-treated HepG2 infected with Mtb H37Rv. (C) Multiplex immunostaining showed increased expression of PPARγ in the liver of infected mice, 8 weeks post-infection. (D) Bar plot showing PPARγ protein intensity (normalized to the area) in the uninfected and infected liver, 8 weeks post-infection. (E) Intensity of PPARγ protein per hepatocyte in uninfected and infected liver, 8 week post-infection. Data were analyzed by using the two-tailed unpaired Student’s t-test in (A, D, and E) and by one-way ANOVA in B. Representative data from n=4 biological replicates *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001. ns = non-significant.
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Figure 6—figure supplement 1—source data 1
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Figure 6—figure supplement 1—source data 2
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Figure 6—figure supplement 1—source data 3
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Figure 7 with 3 supplements
Hepatocytes provide a drug-tolerant niche to Mycobacterium tuberculosis (Mtb).
(A) Experimental scheme of deducting the percentage drug tolerance in hepatocytes (generated using Biorender.com) (B) Percentage tolerance of Mtb-H37Rv against Rifampicin within RAW 264.7, primary hepatocytes (PHCs), and HepG2 at different time points post-infection. (C) Percentage tolerance of Mtb-H37Rv against Isoniazid within RAW 264.7, PHCs, and HepG2 at different time points post-infection. Representative data from three independent experiments. (D) Percentage tolerance of Mtb-H37Rv against rifampicin within bone marrow-derived macrophages (BMDMs), PHCs, and HepG2 as measured microscopically (E) Percentage tolerance of Mtb-H37Rv against isoniazid within BMDMs, PHCs, and HepG2 measured microscopically. Representative data from three independent experiments. Each dot represents a single field having more than four infected cells. 20 such fields were analyzed (F) Transcript levels of the various drug modifying enzymes (DMEs) involved in Rifampicin and Isoniazid metabolism in mice liver, 8 weeks post-infection (n=4 mice), the fold change has been calculated by considering the expression in the uninfected mice to be 1. Data were analyzed by using two-way ANOVA in (B) and (C), one-way ANOVA in (D) and (E) and the two-tailed unpaired Student’s t-test in (F) and Representative data from n=4 biologically independent replicates *p<0.05, **p<0.005, ***p<0.0005, ****p<0. 0001. ns=non-significant.
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Figure 7—source data 1
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Figure 7—source data 2
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Figure 7—figure supplement 1
Gene expression analysis of key drug modifying enzymes (DMEs) related to rifampicin and isoniazid metabolism.
(A) Relative expression of CYP3A4, CYP3A43, and NAT2 genes in infected HepG2 post-Mtb infection, the fold change has been calculated by considering the expression in the uninfected cells to be 1. Data were analyzed using the two-tailed unpaired Student’s t-test in C. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001, ns = non-significant. (B) KEGG pathway analysis shows the upregulation of NAT2 gene (N-acetyltransferase 2) in Mycobacterium tuberculosis (Mtb)-infected HepG2.
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Figure 7—figure supplement 1—source data 1
Data used for generating the graph in Figure 7—figure supplement 1A.
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Figure 7—figure supplement 2
Microscopic images of Mycobacterium tuberculosis (Mtb)-infected bone marrow-derived macrophages (BMDMs), primary hepatocytes (PHCs), and HepG2 treated with rifampicin and isoniazid.
(A) Confocal microscopy images of Mtb-H37Rv-GFP infected BMDMs, PHCs, and HepG2. All the cells are treated with DMSO, 0.5 μg/ml rifampicin, and 0.5 μg/ml isoniazid post-infection. The cells were stained with Phalloidin Alexa Fluor-555 to demarcate the cell boundary. Representative data from three biologically independent experiments. Each dot represents a single field having more than four infected cells. 20 such fields were analyzed.
Figure 7—figure supplement 3
Mycobacterium tuberculosis (Mtb) derived from hepatocytes show no changes in drug efflux pumps and antibiotic sensitivity.
(A) Resazurin microtitre assay of the three groups of Mtb in different concentrations of isoniazid and rifampicin (schematic of experimental procedure generated using Biorender.com). (B) Mtb was obtained from DMSO-treated, 0.5 μg/ml rifampicin, and 0.5 μg/ml isoniazid-treated HepG2 by differential lysis and RNA was isolated from those Mtb populations (C and D) qRT-PCR of some of the key efflux pumps genes in these three Mtb groups. Representative data from three biologically independent experiments. Data were analysed using the two-tailed unpaired Student’s t-test in C and D. *p<0.05, **p<112 0.005, ***p<0.0005, ****p<0.0001, ns = non-significant. Representative data from three biological replicates.
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Figure 7—figure supplement 3—source data 1
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Figure 7—figure supplement 3—source data 2
Data used for generating the graph in Figure 7—figure supplement 3D.
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Author response image 1
Ag85B staining in uninfected mice shows no signals.
Author response image 2
Growth kinetics of Mtb in 7H9 medium with DMSO, C75 and T863.
Author response image 3
Colocalisation of Mtb-GFP with various intra-cellular markers within PHCs.
Author response image 4
Percentage Colocalisation of Mtb-GFP with various intra-cellular markers within PHCs, HepG2 and THP-1.
Author response image 5
Gene expression analysis of the mentioned genes in Mtb infected PHCs as compared to the uninfected control.
Additional files
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MDAR checklist
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Supplementary file 1
Details of the human biopsy samples used in the study.
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Supplementary file 2
Differentially expressed genes between uninfected and Mtb-infected HepG2 at 0 hr and 48 hr post-infection.
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PPARγ mediated enhanced lipid biogenesis fuels Mycobacterium tuberculosis growth in a drug-tolerant hepatocyte environment
eLife 14:RP103817.
https://doi.org/10.7554/eLife.103817.3
