Divergent downstream biosynthetic pathways are supported by L-cysteine synthases of Mycobacterium tuberculosis

Mycobacterium tuberculosis's autarkic lifestyle within the host involves rewiring its transcriptional networks to combat host-induced stresses. With the help of RNA-seq, we identified Mtb pathways that are upregulated in response to oxidative, nitrosative, acidic, starvation, and surfactant stresses. Genes belonging to sulfur metabolism were found to be 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 and therapeutic potency of non-canonical L-cysteine synthases, CysM and CysK2. CysM and CysK2 independently facilitate Mtb survival by alleviating host-induced redox stress, suggesting they are not fully redundant within the host. While transcriptome signatures of Rv?cysM and Rv?cysK2 appear similar under normal growth conditions, when subjected to oxidative stress, we identify unique transcriptional signatures. We followed pool size and labelling (34S) of key downstream metabolites viz. mycothione and ergothionine to monitor L-cysteine biosynthesis and utilization, which revealed a significant role of distinct L-cysteine biosynthetic routes on redox stress and homeostasis. 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. Overall design: H37Rv was either left untreated, or treated with 50 micromolar of CHP for 6 h for oxidative stress, resuspended in PBS for starvation, 0.1% SDS for surfactant stress, resuspended in pH 5.5 media for acidic stress or were treated with sodium nitrate in pH 5.5 for nitrosative stress. Comparative gene expression profiling analysis of RNA-seq data for H37Rv, RvDcysM, RvDcysK2 with and without oxidative stress. H37Rv, RvDcysM, , RvDcysK2 were either left untreated or treated with 50 micromolar CHP for 6 hours.