Figures and data
Non-canonical L-cysteine synthases facilitate mycobacterial survival upon host-like induced stresses in vitro.
(a) 5-way Venn diagram highlighting DEGs belonging to sulfur metabolism pathway under indicated stress conditions (absolute log2 Fold change> 0.5 and Padj value <0.05). (b) Line Diagram illustrating the cysK2 loci in Rv and RvΔcysK2 and the strategy employed for the replacement of cysK2 with hygr. Primer sets used for confirming the generation of RvΔcysK2 are indicated as arrows. The first agarose gel image shows PCR amplicons with a control (sigB) gene-specific primer indicating the presence of nearly equivalent amount of genomic DNA isolated from Rv and RvΔcysK2. The second panel shows PCR amplicons with gene-specific primer set (F1-R1) in Rv and RvΔcysK2 mutant, the third panel shows amplicons (F2-R2) expected only in RvΔcysK2 mutant, and the fourth panel shows amplicons (F2-R3) in Rv and RvΔcysK2 mutant confirming legitimate recombination at native loci. Mr represents 1-kb gene ruler ladder. (c-i) Rv, RvΔcysM, RvΔcysK2, RvΔcysM::cysM, and RvΔcysK2::cysK2 strains were inoculated in Sauton’s (c), PBS (d) or acidic (e), oxidative 50 µM Cumene hydroperoxide (CHP) for 24 h ((f), nitrosative (g), reductive (h) and SDS (i). Bar graphs represent the bacillary survival with data point indicating values CFU log10/mL ± standard deviations (SD) from individual replicate (n=3). Statistical significance was drawn in comparison with Rv using one-way ANOVA followed by a post hoc test (Tukey test; GraphPad prism). **P < 0.005; ***P < 0.0005.
Distinct roles of CysM and CysK2 in attuning cellular processes.
(a) PCA plot demonstrating separation of various bacterial strains under different conditions (b-e) Volcano plots illustrating significantly upregulated (blue) and downregulated (red) genes in indicated strains and conditions with absolute log2 Fold change> 1 and Padj value <0.05. Numbers in the top quadrant highlight the number of significantly upregulated (blue) and downregulated (red) genes in each condition (f) Venn diagram showing the number of significantly downregulated and upregulated DEGs that overlap between indicated strains (g) Heat Maps depicting normalized gene count of differentially expressed genes (DEGs) in independent replicates of RvΔcysM and RvΔcysK2 grown under oxidative conditions with absolute log2 Fold change> 1 and Padj value <0.05. The colour intensity indicates relative upregulated (blue) and downregulated (red) genes compared to the control. (h) Horizontal bar graph depicting the number of DEGs belonging to a particular functional category upon oxidative stress in RvΔcysM compared to RvΔcysK2 under oxidative conditions. (i) Pathway enrichment by DAVID depicting significantly enriched Gene Ontology (GO) biological processes based on DEGs upon oxidative stress in RvΔcysM compared to RvΔcysK2 (absolute log2 Fold change>1 and Padj<0.05).
Key metabolites are differentially affected upon CysM and CysK2 deletion
(a-d) Total abundance (Ion count/Protein concentration) of L-methionine (a), ergothioneine (b), mycothiol (c), and mycothione (d) in Rv with or without 50 μM CHP treatment. (e-g) The percentage of labelled (colored) and unlabeled (black) ergothioneine (e), mycothiol (f), and mycothione (g) in Rv with or without 50 μM CHP treatment at 48 h. Percentage was calculated with respect to 0 h abundance for each replicate. Bars depict the mean of biological replicates (n=3), and error bars represent the standard deviation. The same samples were used for analysis (a-d) and (e-g). Statistical significance was calculated between Rv and RvΔcysM/ Rv ΔcysK2; and between RvΔcysM and RvΔcysK2 using non-parametric t-test. * P < 0.05; ** P < 0.005; *** P < 0.0005.
L-cysteine synthases ameliorate mycobacterial survival in response to host-induced oxidative and nitrosative stress
(a) Pictorial representation of peritoneal macrophage infection experiments. Thioglycolate was injected into the peritoneum cavity of C57BL/6, phox−/−, IFNγ−/− mice. Four days post-injection, peritoneal macrophages were extracted and activated by treatment with IFNγ overnight and LPS - 2 h prior to the infection. In specific cases, iNOS inhibitor 1400 W was added along with IFNγ/LPS. The intracellular bacillary survival was calculated 96 h p.i. (b) Peritoneal macrophages from C57BL/6 mice were infected, and intracellular bacillary survival was enumerated 96 h p.i. (c) Peritoneal macrophages from C57BL/6 mice were infected, left untreated (control) or treated with 0.2mM L-cysteine at the time of infection and intracellular bacillary survival was assessed 96 h p.i. (d-f) Peritoneal macrophages isolated from mice of indicated genotypes were either left untreated or treated with iNOS inhibitor, 1400W. Intracellular bacillary survival was enumerated 96 h p.i. (b-f) Data points are presented as CFU log10/ml ± SD of each replicate (n=3). Statistical significance was drawn in comparison with Rv using one-way ANOVA followed by a post hoc test (Tukey test; GraphPad prism). **P < 0.005; ***P < 0.0005.
Deletion of non-canonical L-cysteine synthases attenuates mycobacterial survival in murine lungs and spleen
(a) Schematic outline of murine infection experiment. C57BL/6 (n = 12 per group) were infected with Rv, RvΔcysM, or RvΔcysM::cysM strains via an aerosol route. CFU was enumerated at day 1 (n=2), week 4 (n=5) and week 8 (n=5). (b-c) Each data point represents Log10 CFU in lung (b) and spleen (c) of an infected animal, and the error bar indicates the median with interquartile range for each group. (d) Schematic outline of murine infection experiment. C57BL/6 (n = 12 per group) were infected with Rv, RvΔcysK2, or RvΔcysK2::cysK2 strains via an aerosol route. CFU was enumerated at day 1 (n=2), week 4 (n=5) and week 8 (n=5). (e-f) Each data point represents Log10 CFU in the lung (e) and spleen (f) of an infected animal, and the error bar indicates the median with interquartile range for each group. (g) Schematic outline of murine infection experiment. C57BL/6 (n = 7 per group), phox−/− (n = 7 per group), IFNγ−/− (n = 7 per group) were infected with Rv, RvΔcysM, or RvΔcysM::cysM strains via an aerosol route. CFU was enumerated at day 1 (n=2) and week 4 (n=5). (h-i) Each data point represents Log10 CFU in the lung (h) and spleen (i) of an infected animal, and the error bar indicates the median with interquartile range for each group. (a-i) Statistical significance was drawn in comparison with Rv using one-way ANOVA followed by a post hoc test (Tukey test; GraphPad prism). ***P < 0.0005.
L-cysteine synthases inhibitors can effectively kill Mtb within the host.
(a) Chemical structure and formula of lead compounds. (b). Venn diagram illustrating the specificity of compounds on mycobacterial L-cysteine synthases. (c). Peritoneal macrophages were infected with Rv. 4 h post-infection, cells were either left untreated (control) or 5 mg/ml C1 (a), C2 (b) or C3 (c) was added to the infected cells. Bar graphs represent mean Log10 CFU/ml ± SD, and values from independent replicates are represented by individual data points (n=3). (d-f). Alamar blue assay is used to determine MIC values of C1 (d), C2, and C3 (f) with and without Isoniazid in Rv. Starting concentration of INH in 1A is 0.96 μg/ml, 1B-0.43 μg/ml and each subsequent column 2 fold dilution. Concentration of C1, C2 and C3 were 2.4 mg/ml in 2A and 1.2 mg/ml in 2B and in each subsequent column 2 fold dilution. The concentration of INH + C1 or C2 in 3A was 0.015 μg/ml + 0.15 mg/ml, and 3B was 0.0075 μg/ml + 0.075 mg/ml. and each subsequent column 2-fold dilution. The concentration of INH + C3 in 3A was.015 μg/ml + 37 μg/ml, and 3B was 0.0075 μg/ml + 18.5 and each subsequent column 2-fold dilution. ND-no drug and NC-no culture.
Overview of Sulfur metabolism pathway in Mycobacterium tuberculosis
Sulfate is imported via the sulfate transporter composed of SufI.CysT.W.A, which is acted upon by CysDNC complex to form Adenosine-5’-phosphosulfate (APS). APS can either form sulfolipids or is reduced by subsequent actions of CysH and SirA to form sulfide. CysK1, the classical L-cysteine synthase, utilizes reduced sulfide and O-acetyl-L-serine as substrates to form L-Cysteine. Alternatively, L-cysteine is produced by CysM, a non-canonical L-cysteine synthase that is unique to actinomycetes and uses O-phospho-L-serine and a small sulfur carrier protein CysO to form L-cysteine. Mtb also encodes for a third L-cysteine synthase, CysK2. However, the major product of this pathway is S-sulfo-L-cysteine, which can get rapidly converted to L-cysteine. The reverse transsulfuration pathway is also depicted.
Sulfur assimilation and L-cysteine biosynthesis pathways are upregulated under stress conditions.
(a-e) Volcano plots illustrating significantly upregulated (blue) and downregulated (red) genes in Rv grown under indicated conditions with Log2 Fold change> 1 and Padj value <0.05 compared to control (untreated) Rv. Numbers in the top quadrant depict the number of significantly upregulated (blue) and downregulated (red) genes in each condition compared to the control. (f-j) Heat Maps depicting normalized gene count of differentially expressed genes (DEGs) in independent replicates of Rv grown under indicated conditions with Log2 Fold change> 1 and Padj value <0.05. The colour intensity indicates relative upregulated (blue) and downregulated (red) genes compared to the control.
Genes that were differentially expressed in at least one of the five stresses.
We compiled a list of genes differentially expressed in at least one of the five stresses against compared untreated Rv. We made a data frame of fold changes for these selected genes in the five comparisons. We then generated a heatmap where the five comparisons were plotted as columns (x-axis), and selected genes were plotted as rows (y-axis). Hierarchical clustering was performed on the y-axis so that genes with similar fold changes across comparisons clustered together.
Genes belonging to the Sulfur metabolism pathway are upregulated upon oxidative stress
(a) Horizontal bar graph demonstrating the number of DEGs belonging to a particular functional category under indicated stress condition. (b-e) Pathway enrichment by DAVID depicting significantly enriched Gene Ontology (GO) biological processes based on DEGs in starvation (b), SDS (c), Nitrosative (d) and pH 5.5 (e). (f) Pathway enrichment by DAVID depicting significantly affected Gene Ontology (GO) biological processes based on DEGs (Log2 Fold change> 0.5 and Padj value <0.05).
L-cysteine synthases facilitate mycobacterial survival upon oxidative stresses in vitro
(a-c) Rv, RvΔcysM, RvΔcysK2, RvΔcysM::cysM, and RvΔcysK2::cysK2 strains were inoculated in 7H9-ADC (a), 7H9-ADS (b), 7H9-ADS plus 10mM diamide (c) medium. Bar graphs represent the bacillary survival with data point indicating values CFU log10/mL ± SD from individual replicate (n=3). Statistical significance was drawn in comparison with Rv using one-way ANOVA followed by a post hoc test (Tukey test; GraphPad prism). **P < 0.005; ***P < 0.0005.
Unique transcriptional signatures of CysM and CysK2.
(a & c) Volcano plots illustrating significantly upregulated (blue) and downregulated (red) genes in indicated strains and conditions with Log2 Fold change> 1 and Padj value <0.05. Numbers in the top quadrant highlight the number of significantly upregulated (blue) and downregulated (red) genes in each condition (b & d) Heat Maps depicting normalized gene count of differentially expressed genes (DEGs) in independent replicates of RvΔcysM and RvΔcysK2 compared to Rv with Log2 Fold change> 1 and Padj value <0.05. The colour intensity indicates relative upregulated (blue) and downregulated (red) genes compared to the control. (e) Venn diagram showing the number of significantly downregulated and upregulated DEGs that overlap between indicated strains (Log2 Fold change> 1 and Padj value <0.05). (f & g) Pathway enrichment by DAVID depicting significantly enriched Gene Ontology (GO) biological processes based on DEGs upon oxidative stress in RvΔcysM (f) and RvΔcysK2 (g) compared to Rv (Log2 Fold change> 1 and Padj value <0.05).
