Divergent downstream biosynthetic pathways are supported by <sc>L</sc>-cysteine synthases of Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis's (Mtb) autarkic lifestyle within the host involves rewiring its transcriptional networks to combat host-induced stresses. With the help of RNA sequencing performed under various stress conditions, we identified that genes belonging to Mtb sulfur metabolism pathways are significantly upregulated during oxidative stress. Using an integrated approach of microbial genetics, transcriptomics, metabolomics, animal experiments, chemical inhibition, and rescue studies, we investigated the biological role of non-canonical L-cysteine synthases, CysM and CysK2. While transcriptome signatures of RvΔcysM and RvΔcysK2 appear similar under regular growth conditions, we observed unique transcriptional signatures when subjected to oxidative stress. We followed pool size and labelling (³⁴S) of key downstream metabolites, viz. mycothiol and ergothioneine, to monitor L-cysteine biosynthesis and utilization. This revealed the significant role of distinct L-cysteine biosynthetic routes on redox stress and homeostasis. CysM and CysK2 independently facilitate Mtb survival by alleviating host-induced redox stress, suggesting they are not fully redundant during infection. With the help of genetic mutants and chemical inhibitors, we show that CysM and CysK2 serve as unique, attractive targets for adjunct therapy to combat mycobacterial infection.

Keywords: Mycobacterium; cysteine; infectious disease; microbiology; synthases; tuberculosis.

Figure 1.. Non-canonical L-cysteine synthases facilitate mycobacterial survival upon host-like induced stresses in vitro.

(a) Five-way Venn diagram highlighting differentially expressed genes (DEGs) belonging to sulfur metabolism pathway under indicated stress conditions (absolute log₂ Fold change >0.5 and p_adj value <0.05). (b) Line diagram illustrating the cysK2 loci in Rv and RvΔcysK2 and the strategy employed for the replacement of cysK2 with hyg^r. Primer sets used for confirming the generation of RvΔcysK2 are indicated as arrows. The first agarose gel image shows PCR (Polymerase chain reaction) 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. Rv, RvΔcysM, RvΔcysK2, RvΔcysM::cysM, and RvΔcysK2::cysK2 strains were inoculated in Sauton’s (c), PBS (Phosphate-Buffered Saline) (d), or acidic (e), oxidative 50 µM cumene hydroperoxide (CHP) for 24 hr (f), nitrosative (g), reductive (h), and sodium dodecyl sulfate (SDS) (i). Bar graphs represent the bacillary survival with data point indicating values CFU log₁₀/ml ± standard deviations (SDs) from individual replicate (n = 3). Statistical significance was drawn in comparison with Rv using one-way analysis of variance (ANOVA) followed by a post hoc test (Tukey test; GraphPad prism). **p < 0.005; ***p < 0.0005.

Figure 1—figure supplement 1.. 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.

Figure 1—figure supplement 2.. 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 p_adj 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 p_adj value <0.05. The colour intensity indicates relative upregulated (blue) and downregulated (red) genes compared to the control.

Figure 1—figure supplement 3.. 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 heat map 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.

Figure 1—figure supplement 4.. Genes belonging to the sulfur metabolism pathway are upregulated upon oxidative stress.

(a) Horizontal bar graph demonstrating the number of differentially expressed genes (DEGs) belonging to a particular functional category under indicated stress condition. Pathway enrichment by DAVID depicting significantly enriched Gene Ontology (GO) biological processes based on DEGs in starvation (b), sodium dodecyl sulfate (SDS) (c), nitrosative (d), and pH 5.5 (e). (f) Pathway enrichment by DAVID depicting significantly affected GO biological processes based on DEGs (log₂ Fold change >0.5 and p_adj value <0.05).

Figure 1—figure supplement 5.. L-Cysteine synthases facilitate mycobacterial survival upon oxidative stresses in vitro.

Rv, RvΔcysM, RvΔcysK2, RvΔcysM::cysM, and RvΔcysK2::cysK2 strains were inoculated in 7H9-ADC (a), 7H9-ADS (b), 7H9-ADS plus 10 mM diamide (c) medium. Bar graphs represent the bacillary survival with data point indicating values CFU log₁₀/ml ± standard deviation (SD) from individual replicate (n = 3). Statistical significance was drawn in comparison with Rv using one-way analysis of variance (ANOVA) followed by a post hoc test (Tukey test; GraphPad prism). **p < 0.005; ***p < 0.0005.

Figure 2.. 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 p_adj 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 down- and upregulated differentially expressed genes (DEGs) that overlap between indicated strains. (g) Heat maps depicting normalized gene count of DEGs in independent replicates of RvΔcysM and RvΔcysK2 grown under oxidative conditions with absolute log₂ Fold change >1 and p_adj 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 p_adj <0.05).

Figure 2—figure supplement 1.. 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 p_adj 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 p_adj 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 down- and upregulated DEGs that overlap between indicated strains (log₂ Fold change >1 and p_adj value <0.05). 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 p_adj value <0.05).

Figure 3.. Key metabolites are differentially affected upon CysM and CysK2 deletion.

Total abundance (ion count/protein concentration) of L-methionine (a), ergothioneine (b), mycothiol (c), and mycothione (d) in Rv strains with or without 50 μM cumene hydroperoxide (CHP) treatment. The percentage of labelled (coloured) and unlabeled (black) ergothioneine (e), mycothiol (f), and mycothione (g) in Rv strains with or without 50 μM CHP treatment at 48 hr. Since mycothione (g) has two ionizable sulfur, the two-coloured stacked bar graphs represent percentage of labelled M+2 and M+4 ions. Percentage was calculated with respect to 0 hr 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/ RvΔcysK2; and between RvΔcysM and RvΔcysK2 using non-parametric t-test. *p < 0.05; **p < 0.005; ***p < 0.0005.

Figure 3—figure supplement 1.. Overview of metabolites tracked through ³⁵S sulfur incorporation.

Figure 4.. 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 (Interferon gamma) overnight and lipopolysaccharide (LPS) 2 hr prior to the infection. In specific cases, iNOS inhibitor 1400 W was added along with IFNγ/LPS. The intracellular bacillary survival was calculated 96 hr post-infection (p.i.). (b) Peritoneal macrophages from C57BL/6 mice were infected, and intracellular bacillary survival was enumerated 96 hr p.i. (c) Peritoneal macrophages from C57BL/6 mice were infected, left untreated (control) or treated with 0.2 mM 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, 1400 W. Intracellular bacillary survival was enumerated 96 h p.i. (b–f) Data points are presented as CFU log₁₀/ml ± standard deviation (SD) of each replicate (n = 3). Statistical significance was drawn in comparison with Rv using one-way analysis of variance (ANOVA) followed by a post hoc test (Tukey test; GraphPad prism). **p < 0.005; ***p < 0.0005.

Figure 5.. 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. Colony-forming unit (CFU) was enumerated at day 1 (n = 2), week 4 (n = 5), and week 8 (n = 5). 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). 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), and 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). 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 analysis of variance (ANOVA) followed by a post hoc test (Tukey test; GraphPad prism). *p<0.05, **p<0.005, ***p < 0.0005.

Figure 6.. L-Cysteine synthases inhibitors can effectively kill Mtb within the host.

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. Four hours 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 ± standard deviation (SD), and values from independent replicates are represented by individual data points (n = 3). Alamar blue assay is used to determine MIC values of C1 (d), C2 (e), 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 twofold 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 twofold dilution. The concentration of INH + C1 or C2 in 3 A was 0.015 μg/ml + 0.15 mg/ml, and 3B was 0.0075 μg/ml + 0.075 mg/ml. and each subsequent column twofold 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 twofold dilution. ND – no drug and NC – no culture. ***p<0.0005.

Author response image 1.