Whole genome CRISPRi screening identifies druggable vulnerabilities in an isoniazid resistant strain of Mycobacterium tuberculosis

Abstract

Drug-resistant strains of Mycobacterium tuberculosis are a major global health problem. Resistance to the front-line antibiotic isoniazid is often associated with mutations in the katG-encoded bifunctional catalase-peroxidase. We hypothesise that perturbed KatG activity would generate collateral vulnerabilities in isoniazid-resistant katG mutants, providing potential pathway targets to combat isoniazid resistance. Whole genome CRISPRi screens, transcriptomics, and metabolomics were used to generate a genome-wide map of cellular vulnerabilities in an isoniazid-resistant katG mutant strain of M. tuberculosis. Here, we show that metabolic and transcriptional remodelling compensates for the loss of KatG but in doing so generates vulnerabilities in respiration, ribosome biogenesis, and nucleotide and amino acid metabolism. Importantly, these vulnerabilities are more sensitive to inhibition in an isoniazid-resistant katG mutant and translated to clinical isolates. This work highlights how changes in the physiology of drug-resistant strains generates druggable vulnerabilities that can be exploited to improve clinical outcomes.

Fig. 1. WG-CRISPRi screening in M. tuberculosis strain mc26206.

a Experimental design for WG-CRISPRi essentiality screening. The pooled CRISPRi plasmid library is transformed into appropriate M. tuberculosis strains. WG-CRISPRi screens were performed for 14 days, with varying concentrations of ATc and at day 5 and 10 back diluted (1/20) into fresh media + ATc to maintain log phase growth. gDNA was extracted, gRNA amplified and sequenced from samples collected on days 5, 10 and 14. The proportion of each gRNA at each ATc concentration within the pooled population was quantified relative to ATc-0 for each sampled time point and plotted on a log2-fold scale as a reduction in gRNA abundance. Increased depletion of gRNA was used to identify genes that are either synthetic lethal or have increased vulnerability in INH^R-katG. Created in BioRender. Wang, X. (2023) BioRender.com/q45z115. b–g Summary of gRNA abundance in the M. tuberculosis strain (b) mc²6206 DS-parent, (d) INH^R-katG, and (f) the depletion difference between the DS-parent and INH^R-katG. The gRNA abundance is relative to the ATc-0 control at each time point calculated by the exact test and p-values adjusted by the Benjamini–Hochberg method. Unchanged gRNAs are coloured grey, whilst gRNA with significant > 2-fold depletion (Benjamini–Hochberg adjusted p < 0.01) are coloured green. The red dot within each violin plot denotes the mean gRNA depletion. A total of 3991 unique protein-encoding genes were screened. Of these, the number of essential genes identified in the (c) DS-parent and (e) INH^R-katG were illustrated under different time points and ATc concentrations. g Genes identified as being more vulnerable to inhibition in INH^R-katG are defined as when (i) they were called essential in INH^R-katG and (ii) had no less than 2 gRNAs that were depleted by >1-log2 fold more than observed in the DS-parent. Of these, genes were identified as “synthetic lethal” when had no less than 2 gRNAs that were (1) not depleted in the DS-parent and (2) were depleted by >1-log2 fold in INH^R-katG more than observed in the DS-parent. Source data are provided as a Source Data file.

Fig. 2. Pathway analysis identifies diverse functions that are more vulnerable to inhibition in INH^R-katG.

a Pathway analysis identifies functional classifications of genes with increased vulnerability in INH^R-katG (i.e., Fig. 1g). The bubble plot represents data from day 14. The x-axis quantifies the proportion of genes called “more vulnerable” over the total number of M. tuberculosis genes per functional pathway, and the y-axis shows the name of each functional pathway with the total number of genes in each class labelled in brackets. Within each functional class, the dot size indicates the average ratio of gRNAs targeting each gene that is more depleted in INH^R-katG. The dot colour denotes the ATc concentration from which the amplicon sequencing was performed. b Scatter plot illustrating experimental validation of each of the 30 gRNAs. Data is plotted as the difference in gRNA abundance between INH^R-katG and the DS-parent from WG-CRISPRi screens (Day14+ATc-300) (x-axis) against the fold change in ATc MIC between INH^R-katG and the DS-parent from ATc dose response assays (y-axis). The vertical dotted line indicates the cut-off for a gRNA to be significantly depleted, whilst the horizontal dotted indicates a two-fold change in ATc MIC. c–e Growth of M. tuberculosis DS-parent and INH^R-katG expressing for gRNA targeting (c) gyrB, (d) atpD and atpF and (e) clpC1 in ATc dose response assays (mean ± extrema of two biological replicates, n=3 independent experiments). The (gx) after each gRNA denotes the specific gRNA targeting each gene. f Viability plots of M. tuberculosis DS-parent and INH^R-katG expressing for gRNA targeting atpD (mean ± extrema of two biological replicates, n=3 independent experiments). g M. tuberculosis survival in macrophages. THP-1 macrophage cells were infected with M. tuberculosis DS-parent and INH^R-katG cells expressing the stated gRNA (mean ± SD of three biological replicates, n = 2 independent experiments). h, i The susceptibility of M. tuberculosis DS-parent and INH^R-katG to increasing concentrations of (h) levofloxacin (mean ± extrema of two biological replicates, n=3 independent experiments) and (i) SQ109 (mean ± extrema of two biological replicates, n = 3 independent experiments). f, h, i Inoc denotes the starting CFU/ml and no-cpd denotes the detected CFU/ml in the absence of compound Dashed line represents the lower detection limit. Source data are provided as a Source Data file.

Fig. 3. INH^R-katG utilizes alternative redox detoxification pathways to compensate for the loss of katG.

Susceptibility of M. tuberculosis DS-parent and INH^R-katG to growth inhibition by (a) H₂O₂ and (b) ascorbic acid (mean ± extrema of two biological replicates, n = 3 independent experiments). c CellRox based detection of reactive oxygen stress in M. tuberculosis DS-parent and INH^R-katG when exposed to H₂O₂ (mean ± SD of three biological replicates, n = 2 independent experiments). d Changes in gene expression INH^R-katG relative to the DS-parent and increased depletion of gRNA abundance in INH^R-katG relative to DS-parent. Genes are named using gene name or rv number. gRNAs that are more depleted in INH^R-katG are presented as white dots, with the positioning of each dot denoting the level of increased depletion on a log2FC scale. Only gRNAs that show a statistically significant depletion on day 14+ ATc-300 are presented. e–h INH^R-katG is more sensitive to iron dysregulation: (e) Abundance of gRNAs targeting ideR throughout the WG-CRISPRi screen as detected by amplicon sequencing. Data is presented for cultures exposed to ATc-300. f Growth of M. tuberculosis DS-parent and INH^R-katG expressing gRNA targeting ideR (mean ± SD of two biological replicates, n ≥ 3). The (gx) after each gRNA denotes the specific gRNA targeting ideR. g Intracellular survival of M. tuberculosis DS-parent and INH^R-katG expressing gRNA targeting ideR (mean ± SD of three biological replicates, n = 2 independent experiments). h Susceptibility of M. tuberculosis DS-parent and INH^R-katG to 2.5 mM FeCl as determined by CFU/ml (mean ± SD of three biological replicates, n = 2 independent experiments). i Growth kinetics of M. tuberculosis DS-parent and INH^R-katG expressing gRNA targeting ideR, Bacterial growth was measured by OD600. All strains were grown in 7H9 media with ATc-300 and back diluted 1/20 with fresh media on days 5 and 10. Data is mean ± SD of three biological replicates, n = 2 independent experiments. j The ability of M. tuberculosis DS-parent and INH^R-katG to deplete oxygen during the transition to hypoxia was detected using PreSens oxygen sensing spots. Data represents the individual oxygen consumption curves of two biological replicates from a representative experiment. k The viability of M. tuberculosis DS-parent and INH^R-katG once the concentration of dissolved oxygen was less than 1% was detected by plating for viable colonies. Data is shown as the mean ± SD of three biological replicates, n = 2 independent experiments. Source data are provided as a Source Data file.

Fig. 4. Amino acid metabolism is altered in a INH^R-katG mutant.

a Genes within the amino-acid class with increased vulnerability in INH^R-katG. Bubble plot represents day 14 data. The x-axis quantifies the proportion of genes with increased vulnerability per subclass. The y-axis shows the names and total number of genes per subclass. Dot size indicates the mean ratio of gRNAs (per gene) more depleted in INH^R-katG. Dot colour denotes ATc concentrations used. Pro: Proline and 4-hydroxyproline. Lys-Thr-Met-Cys: Lysine, threonine, methionine, and cysteine. Branched-chain: Branched-chain amino acids. Glu-Asp-NH₄: Glutamine, glutamate, aspartate, asparagine, ammonia assimilation. Aromatics: Aromatic amino acid metabolism. His: histidine metabolism. b Significantly depleted metabolites are labelled with asterisks (p < 0.05 determined by two-sided t-test, n = 5 biological replicates). Schematics of (c) pathways linking aspartate metabolism with the TCA cycle and nucleotide metabolism and (d) aromatic amino acid metabolism. Bolded metabolites highlight those that were detected. Blue, red and black denotes metabolites that are increased, decreased or no-change in INH^R-katG. Bolded genes highlight those that are more vulnerable. e–g DS-parent and INH^R-katG expressing gRNAs targeting (e) metC, (f) lysA and (g) aroK in ATc dose-response assays (mean ± extrema of two biological replicates, n = 3 independent experiments). The (gx) denotes the gRNA name. h Viability plots of DS-parent and INH^R-katG expressing gRNA targeting aroK (mean ± extrema of two biological replicates, n ≥ 3). Inoc denotes the starting CFU/ml and no-cpd denotes the detected CFU/ml in the absence of ATc. Dashed line represents the minimum detection limit. i Growth kinetics of M. tuberculosis DS-parent and INH^R-katG expressing a gRNA targeting aroK (mean ± SD of three biological replicates, n = 2 independent experiments). j Intracellular survival of DS-parent and INH^R-katG cells expressing an aroK gRNA within THP-1 macrophages (mean ± SD of three biological replicates, n = 2 independent experiments). k DS-parent pre-depleted for metA, aroK and lysA for 5 days was exposed to ascorbic acid. No-cpd is the absence of compound but with 300 ng/ml of ATc. Data is the reduction in viable colonies on day 10, relative to the starting inoculum. Non-targeting gRNA is a negative control. l The DS-parent or INH^R-katG were grown in 7H12 media with 1 mM ascorbic acid and the stated amino acids. Data is expressed as the reduction in viable colonies at each time point relative to the starting inoculum. Source data are provided as a Source Data file.

Fig. 5. Ribosome biogenesis is more vulnerable to inhibition in an INH^R-katG mutant.

a Pathway analysis of more vulnerable genes in INH^R-katG within the protein synthesis class. Subclasses are described using classifications from the curated PATRIC database. The bubble plot represents data from day 14. The x-axis quantifies the proportion of genes called “more vulnerable” over the total number of M. tuberculosis genes per functional subclass of the protein synthesis pathway, and the y-axis shows the name of each functional subclass and the total number of genes in each subclass. Within each functional subclass, the dot size indicates the average ratio of gRNAs targeting each gene that is more depleted in INH^R-katG. The dot colour denotes the ATc concentration from which the amplicon sequencing was performed. Ribo-bio: Ribosome biogenesis. Aa-tRNA: Aminoacyl-tRNA-synthetases. b Growth of M. tuberculosis DS-parent and INH^R-katG expressing gRNAs targeting rplC in ATc dose response assays (mean ± extrema of two biological replicates, n=3 independent experiments). The (gx) after each gRNA denotes the specific gRNA targeting each gene. c, d M. tuberculosis DS-parent and INH^R-katG expressing a gRNA targeting rplC and assessed (c) in continuous log phase growth in 7H9 media with ATc-300, (d) for viability in ATc dose response assays and (e) for intracellular survival within THP-1 macrophage cells (mean ± SD of three biological replicates, n = 2 independent experiments). Data presentation is consistent with Fig. 4 and experiments were performed as described in material and methods. f–h Susceptibility of M. tuberculosis DS-parent and INH^R-katG to increasing concentrations of LZD as assessed (f) in 96 well plates, (g) in time kill assays and (h) against intracellular M. tuberculosis within THP-1 infected macrophages. For h, the MIC of LZD was 1 µM. For (f) Inoc denotes the starting CFU/ml and no-cpd denotes the detected CFU/ml in the absence of compound (mean ± SD of three biological replicates, n = 2 independent experiments). Dashed line represents the lower limit of detection. i M. tuberculosis DS-parent grown with INH (9 µM) with or without increasing concentrations of LZD (mean ± SD of three biological replicates, n = 2). Source data are provided as a Source Data file.

Fig. 6. Collateral vulnerabilities translate to clinically relevant INH^R genotypes.

a, b Susceptibility of M. tuberculosis H37RV, INH^R-KatG^M225R and INH^R-KatG^W351G to inhibition by BDQ and LZD. Growth of M. tuberculosis was determined using REMA assay. c–e Susceptibility of M. tuberculosis DS-parent, INH^R-katG, INH^R-KatG^S315N and a MDR- KatG^S315N strain to inhibition and killing by (c) isoniazid and (d, e) bedaquiline. MIC and MBC assays are mean ± extrema of two biological replicates, n = 2 independent experiments. For MBC assays Inoc denotes the starting CFU/ml and no-cpd denotes the detected CFU/ml in the absence of ATc. The dashed line represents the lower limit of detection. For c–e all strains contain the chromosomally integrated plasmid pKM427 that is used as part of mycobacterial recombineering. The BDQ MIC is 0.2 µM as determined in d). f Susceptibility of INH sensitive and resistant clinical isolates to LZD The INH^S isolate is TDR-TB 77, whilst INH^R isolate is TDR-TB 42. Both strains are from the TDR-TB strain bank. Source data are provided as a Source Data file.