Identifying a novel mechanism of L-leucine uptake in Mycobacterium tuberculosis using a chemical genomic approach
- Translational Health Science and Technology Institute, NCR Biotech Science Cluster, India
- National Institute of Animal Biotechnology (NIAB), India
Figures
Figure 1 with 1 supplement
Screening of the FDA-approved library of molecules against Mtb mc2 6206 reveals the growth-inhibitory effect of semapimod, an anti-inflammatory small molecule.
(a) Status of inhibitors showing activity against Mtb mc2 6206. The bar graph depicts Mtb mc2 6206 inhibitors acting on different host metabolic pathways. (b) MIC90 status of Mtb inhibitors. The bar graph shows inhibitors with different MIC90 against Mtb mc2 6206, in vitro. Molecules with <0.025 µM MIC90 (encircled) are tabulated in the inset. (c) Determination of the minimum dose of semapimod for complete inhibition of Mtb mc2 6206 growth by visual inspection. Isoniazid (INH) was used as a control in the 96-well plate-based assay. The drug concentration beyond which no visual growth is observed is shown in bold. (d) Effect of semapimod on the in vitro growth of Mtb mc2 6206. Shown is the CFU enumeration of drug-treated and untreated (control) bacteria at the indicated time points. (e) Effect of semapimod on the intracellular proliferation of Mtb mc2 6206. Intracellular growth of Mtb mc2 6206 in the THP1-derived macrophages was examined after 6 days of infection in the absence (UT) or presence of 0.10–1.0 µM semapimod. Treatment with 2.5 µM INH was used as control. A significant reduction in CFU counts of Mtb mc2 6206 is observed in the presence of semapimod under in vitro culture conditions (d) as well as during intracellular (P<0.005) growth (e). Data represent mean ± SD of at least n=2 replicates in d and n=4 replicates in (e). Data in (c) are representative of n=2 independent experiments. p Values in (e) were obtained after comparison of CFUs between the UT and semapimod-treated samples, as described in Materials and methods.
Figure 1—figure supplement 1
Characteristics of the small molecules’ library.
(a) The bar graph shows the status of molecules in the library acting on different metabolic pathways. (b) The pie chart depicts various diseases that are targeted by inhibitors. (c) Shown is the ranking of FDA-approved molecules as per the different clinical trial phases.
Figure 2 with 2 supplements
Effect of semapimod treatment on the expression profile of Mtb mc2 6206 transcripts.
(a) Volcano plot of differentially expressed genes in semapimod-treated Mtb mc2 6206. The plot shows the distribution of genes that are differentially expressed via log2 fold-change and the –log p values. Broken vertical lines represent the cutoff of >1.0 log2 fold-change, and the horizontal line represents the cutoff of >2.583 –log p values. Genes below the -log p cutoff are represented by grey dots. Downregulated genes are represented by red dots, and those showing upregulation in response to semapimod treatment are shown with green dots. (b) Status of differentially accumulated transcripts. Heatmap representation of transcripts showing accumulation (left) or suppression (right) upon exposure to semapimod across three biological replicates. (c) Functional categorization of differentially regulated genes. The butterfly chart shows the distribution pattern of differentially regulated genes according to their function, as classified in the Mycobrowser database (https://mycobrowser.epfl.ch/genes/). Mean fold-change values from n=3 biological replicates are shown in (a).
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Figure 2—source data 1
List of differentially expressed genes in semapimod-treated Mtb mc2 6206.
Shown are genes exhibiting >2.0-fold change in expression. p Values were determined by using the two-stage setup method of Benjamini, Krieger, and Yekutieli, keeping FDR (Q) of 1% with the help of GraphPad Prism v7.0e software.
- https://cdn.elifesciences.org/articles/107025/elife-107025-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Validation of RNASeq data by qRT-PCR.
The bar graph shows the expression levels of representative genes in semapimod-treated bacteria, estimated by qRT-PCR. Fold-change was calculated with respect to untreated samples after normalization with rrs, which remained consistent across the two groups. Mean ± SD values of n=2 biological replicates are shown.
Figure 2—figure supplement 2
Semapimod treatment causes a reduction in intracellular ATP level.
Intracellular ATP was estimated in UT and treated Mtb mc2 6206 exposed to 50 nM semapimod (Sem), using the Bac TiterGlo assay kit, as suggested by the manufacturer (Promega). The bar graph shows the percentage change in the intracellular ATP levels following drug exposure. Mean ± SD values of n=2 biological replicates are shown.
Figure 3 with 2 supplements
Semapimod treatment perturbs L-leucine uptake in Mtb mc2 6206.
(a) Effect of semapimod on the in vitro growth of different mycobacterial species. The growth inhibitory effect of semapimod was assessed against slow-growing Mtb H37Rv and M. bovis BCG, and fast-growing M. abscessus and M. smegmatis, respectively, by visual inspection using a 96-well plate-based assay. Rifampicin (Rif) and INH were used as control drugs against Mtb H37Rv. (b) Estimation of intracellular leucine in Mtb mc2 6206. Intracellular leucine was estimated in untreated (UT) and semapimod-treated (Sem) bacteria after 24 hr of drug treatment by mass spectrometry as described in Materials and methods. (c–e) Effect of leuC-leuD and panC-panD expression in Mtb mc2 6206 on bacterial killing by semapimod. Time- (c) and dose- (d–e) dependent growth kinetics reveal loss of bactericidal effect of semapimod against Mtb mc2 6206 by leuC-leuD, and not by panC-panD expression. Percent viability in e was calculated with respect to untreated (UT) cultures after 2 weeks of exposure to different drug concentrations, using a 96-well plate-based method as described in Materials and methods. Data in (a) and (c–d) are representative of n=2 independent experiments. Mean ± SD values from n=4 replicates are shown in (b).
Figure 3—figure supplement 1
Effect of withdrawal of L-leucine on expression of semapimod-regulated genes in Mtb mc2 6206.
The bar graph shows the expression level of various genes in the presence or absence of metabolites, as estimated by qRT-PCR. PL+, 7H9-PLO; PL-, 7H9-O; P-, 7H9-LO; L-, 7H9-PO. Fold-change in expression was calculated in all the samples with respect to PL + after normalization with sigA, which remained consistent in all the samples. Mean ± SD values of n=2 biological replicates are shown. p Values were calculated using GraphPad Prism version 10.
Figure 3—figure supplement 2
Effect of semapimod treatment on the intracellular levels of valine and L-proline in the Mtb mc2 6206.
Intracellular valine and L-proline were estimated in untreated (Control) and semapimod-treated (Treated) bacteria, after 24 hr of drug treatment, by mass spectrometry as described in Materials and methods. Mean + SD values from n=2 replicates are shown.
Figure 4 with 3 supplements
Generation and characterization of semapimod-resistant strain of Mtb mc2 6206.
(a) Propagation of the putative semapimod-resistant (SemR) strain, but not the wild-type (WT) Mtb mc2 6206 in the presence of 50 nM semapimod confirms drug-resistance in SemR. In contrast, both the strains exhibit substantial growth on 7H11-PLO agar plate in the absence of drug. Shown is the growth pattern of two different colonies, C1 and C2, of the respective strains. (b) In vitro growth analysis of the WT and the SemR strains of Mtb mc2 6206. Comparative analysis of OD600 at different time points reveals substantially increased growth of SemR strain compared to WT under in vitro culture conditions. (c) In vitro susceptibility of the WT and the SemR strains of Mtb mc2 6206 to vancomycin. Viability of WT and SemR Mtb strains was determined in the presence of vancomycin by a 96-well plate-based assay, as described in Materials and methods. Percent viability was calculated with respect to the untreated (UT) cultures after 2 weeks of exposure to different concentrations of the antibiotic. Results show a substantial increase in the susceptibility of SemR to vancomycin compared to WT. (d) Whole-genome map analysis of SemR. Shown is the genome map of SemR highlighting the positions of genes undergoing substitutions, when compared with WT Mtb mc2 6206. Mutations leading to corresponding changes at the amino acid level in the respective proteins are indicated alongside. Suspect genes presumably involved in providing semapimod resistance are marked in red fonts. Data are representative of at least n=2 independent experiments in (a) and (b). Mean ± SD values from n=3 biological replicates are shown in (c).
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Figure 4—source data 1
Analysis of MIC90 of Rif and Inh against SemapimodR strain of Mtb mc2 6206.
MIC90 of standard TB drugs, rifampicin, and INH was determined against WT and SemR strains using a 96- well plate- based assay, as described in the Materials and methods. As can be seen, resistance to semapimod does not alter bacterial susceptibility to either of the frontline TB drugs.
- https://cdn.elifesciences.org/articles/107025/elife-107025-fig4-data1-v1.docx
Figure 4—figure supplement 1
List of M. smegmatis genes potentially involved in L-leucine transport.
(a) Organization of the prospective genes involved in L-leucine transport at different loci in M. smegmatis. (b) Description of the function of M. smegmatis genes associated with L-leucine transport.
Figure 4—figure supplement 2
Estimation of intracellular L-leucine and valine in the Mtb mc2 6206.
Intracellular L-leucine and valine were estimated in the wild-type (WT) and SemR bacteria, after 7 days of culturing, by mass spectrometry as described in Materials and methods. Mean + SD values from n=3 replicates are shown. The p values were calculated using GraphPad Prism version 10.
Figure 4—figure supplement 3
Comparative sequence analysis of Ppe60 in the wild-type and SemR strains of Mtb 6206.
Sequence alignment of Ppe60 from WT and SemR strains was performed by Clustal Omega (https://www.ebi.ac.uk/jdispatcher/msa/clustalo) using default parameters. ‘*’ represents identical residues; ‘.’ represents weakly similar residues; ‘:’ signifies conserved residues with strongly similar properties, whereas completely dissimilar changes are shown by gaps.
Figure 5 with 2 supplements
Overexpression of ppsB alters susceptibility of SemR strain of Mtb mc2 6206 to semapimod and vancomycin.
(a–b) In vitro susceptibility of different strains of Mtb mc2 6206 to vancomycin (a) and semapimod (b). Viability of WT and SemR Mtb strains in the presence of drugs was compared with those constitutively expressing ppsB by a 96-well plate-based assay, as described in Materials and methods. Percent viability was calculated with respect to UT cultures after 2 weeks of exposure to different concentrations of inhibitors. Remarkably, the response of SemR to both semapimod and vancomycin is reversed upon overexpression of ppsB. Contrarily, ppsB expression in WT does not affect bacterial sensitivity to either of these drugs, thus indicating a specific effect of the PDIM biosynthesis gene in SemR. Mean values from n=3 biological replicates are shown in (a) and (b).
Figure 5—figure supplement 1
Overexpression of mce1D does not affect susceptibility of Mtb mc2 6206 to semapimod.
Effect of mce1D overexpression in the WT and SemR strains on bacterial viability in the presence of semapimod. Viability of WT and SemR strains constitutively expressing mce1D was assessed by a 96-well plate-based assay, as described in Materials and methods. Percent viability was calculated with respect to UT cultures after 2 weeks of exposure to different concentrations of inhibitors. Mean ± SD values of n=2 biological and n=2 technical replicates are shown.
Figure 5—figure supplement 2
Overexpression of ppe60 does not affect susceptibility of Mtb mc2 6206 to semapimod.
(a–b) Effect of ppe60 overexpression in the WT and SemR strains on bacterial viability was determined in the presence of semapimod by a 96-well plate-based assay, as described in Materials and methods. Viability was assessed by visual inspection of the plate (a) as well as by determining percent viability (b). Percent viability was calculated with respect to UT cultures after 2 weeks of exposure to different concentrations of semapimod. Mean ± SD values of n=2 biological replicates are shown.
Figure 6 with 2 supplements
Semapimod targets the PDIM biosynthesis protein, PpsB.
(a) Analysis of semapimod-PpsB interaction by BLI-Octet. Interaction of semapimod with Mtb PpsB was examined by optical interference-based biolayer interferometry from the Octet system (ForteBIO). Briefly, dialyzed 6xHis-PpsB was immobilized onto an AR2G sensor to a level of 2.1 nm. Binding was observed at the indicated concentrations of the ligand (semapimod) to acquire a differential graded response. The binding constant was calculated as per the standard steps described in Materials and methods. (b) Analysis of cell-wall apolar lipids in the WT and SemR strains of Mtb mc2 6206. Cell-wall apolar lipids were extracted and analyzed by two-dimensional TLC as described in Materials and methods, revealing a significant reduction in PDIMs in SemR compared with the WT strain. Contrary to PDIMs, no change was observed in the triacylglycerol (TAG) levels between the two strains. The position of PDIMs is marked by broken circles in both images for clarity.
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Figure 6—source data 1
PDF file containing original TLC images for Figure 6B, indicating the positions of TAG and PDIM in SemR and WT strains.
- https://cdn.elifesciences.org/articles/107025/elife-107025-fig6-data1-v1.zip
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Figure 6—source data 2
Original file for TLC images displayed in Figure 6B.
- https://cdn.elifesciences.org/articles/107025/elife-107025-fig6-data2-v1.zip
Figure 6—figure supplement 1
SDS-PAGE analysis of purified PpsB of Mtb.
Shown is the Coomassie Brilliant Blue-stained denaturing polyacrylamide gel with 10 µg of purified PpsB with 6 x His tags at the N-terminus. The molecular mass of the purified protein was determined by using the prestained molecular mass marker.
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Figure 6—figure supplement 1—source data 1
PDF file containing original Coomassie-stained polyacrylamide gel for Figure 6—figure supplement 1 showing the elution fractions of purified PpsB.
- https://cdn.elifesciences.org/articles/107025/elife-107025-fig6-figsupp1-data1-v1.zip
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Figure 6—figure supplement 1—source data 2
Original file for Coomassie-stained polyacrylamide gel displayed in Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/107025/elife-107025-fig6-figsupp1-data2-v1.zip
Figure 6—figure supplement 2
Comparative analysis of cell-wall mycolic acid profile of WT and SemR strains of Mtb mc2 6206.
Cell-wall mycolic acid methyl esters (MAMES) were extracted from both strains and analyzed by one-dimensional TLC, as described in Materials and methods. As can be seen, no change was observed in the levels of different MAMES between the two strains.
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Figure 6—figure supplement 2—source data 1
PDF file containing original TLC image for Figure 6—figure supplement 2 showing MAMES in SemR and WT strains.
- https://cdn.elifesciences.org/articles/107025/elife-107025-fig6-figsupp2-data1-v1.zip
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Figure 6—figure supplement 2—source data 2
Original file for TLC image displayed in Figure 6—figure supplement 2.
- https://cdn.elifesciences.org/articles/107025/elife-107025-fig6-figsupp2-data2-v1.zip
Figure 7 with 1 supplement
Effect of semapimod treatment on the survival of Mtb H37Rv in the organs of infected BALB/c mice.
(a) Schematic of mouse infection. Infection was performed by the aerosol route with the virulent Mtb H37Rv strain. After 21 days of infection, mice were divided into two groups: one receiving only 5% sucrose (sham) and the other receiving 5 mg/kg semapimod prepared in 5% sucrose. Intracellular bacterial load was determined by CFU plating of lung homogenates at days 1, 21, and 49, and of spleen homogenates prepared on day 49. (b) Gross pathology of lungs. Images of lungs obtained from both the sham- and semapimod-treated groups of mice after 28 days of treatment (i.e. day 49 post-infection) are presented. Scale bar is shown for size reference. (c–d) Effect of semapimod treatment on intracellular survival of Mtb H37Rv. Intracellular survival was determined by estimating the bacterial burden in the lungs at the respective time points (c), and in the spleen at day 49 post-infection (d), by CFU enumeration. Data represent mean ± SD values from n=3 animals in (c) and (d). p Values in (c) and (d) were obtained after comparison of CFUs between the two groups, as described in Materials and methods. The illustration was created in BioRender.
Figure 7—figure supplement 1
Susceptibility of Mtb H37Rv isolated from mice lungs post-semapimod treatment to vancomycin.
Susceptibility of Mtb H37Rv obtained from the lungs of untreated (UT) and semapimod-treated (Sem) infected mice to vancomycin was assessed by a 96-well plate-based assay, as described in Materials and methods. Percent viability was calculated with respect to UT cultures after 2 weeks of exposure to different concentrations of vancomycin. Mean ± SD values of n=2 biological replicates are shown.
Figure 8
Proposed model describing the effect of semapimod on L-leucine uptake in Mtb mc2 6206.
(a) Uptake of L-leucine is controlled by cell-wall PDIM. (b) Semapimod targets the PDIM biosynthesis protein (PpsB) causing an altered PDIM profile, which restricts L-leucine uptake in the auxotroph by an unknown mechanism (denoted by ‘?’), leading to cell death. (c) L-leucine is freely accessible to the SemR strain, owing to perturbation of cell-wall PDIM. As a consequence, semapimod is ineffective against this strain. However, overexpression of wild-type ppsB in SemR reverts its susceptibility to semapimod. The illustration was created in BioRender.
Author response image 1
Tables
Table 1
Analysis of MIC90 of Rif and Inh against SemapimodR strain of Mtb mc2 6206.
MIC90 of standard TB drugs, rifampicin, and INH was determined against WT and SemR strains using a 96-well plate-based assay, as described in the Materials and methods. As can be seen, resistance to semapimod does not alter bacterial susceptibility to either of the frontline TB drugs.
| Rifampicin | INH | |
|---|---|---|
| WT | 0.11 µM | 0.40 µM |
| SemR | 0.11 µM | 0.40 µM |
Additional files
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Supplementary file 1
List of oligonucleotides.
Shown are the oligonucleotides used in this study.
- https://cdn.elifesciences.org/articles/107025/elife-107025-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/107025/elife-107025-mdarchecklist1-v1.docx
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Identifying a novel mechanism of L-leucine uptake in Mycobacterium tuberculosis using a chemical genomic approach
eLife 14:RP107025.
https://doi.org/10.7554/eLife.107025.3
