Exploring the Role of Sulfur Metabolism Machinery in Host-pathogen Interaction During Mycobacterial Infections

Abstract

Hydrogen sulfide (H₂S) allows bacteria to persist in hostile environments and is involved in drug resistance mechanisms of various infectious pathogens, including Mycobacteria tuberculosis (Mtb). It is produced by the activity of CBS, CSE and 3-MPST in humans and homologues of these enzymes are present in various pathogenic bacteria. In Mtb, MtbCbs (Rv1077) and MtbMetB (Rv1079) are the known homologues of human CBS and CSE, respectively. However, the mechanism of H2S production in Mtb requires better understanding and characterisation. In this study, MtbSseB is hypothesised to be the homologue of human 3-MPST, this has been validated using multiple sequence alignment and molecular docking analysis. Next, the homologous proteins were cloned, expressed and purified. Furthermore, the substrate and cofactor requirements of MtbCbs (20mM of L-Cysteine and 500µM of SAM) and MtbMetB (4mM L-Cysteine) were standardised using the agarose gel-based lead acetate assay for optimal activity. This study further explored the potential role of Mtb H2S-producing enzymes as potential drug targets. We studied the effect of known inhibitors of the human enzymes, Aminooxyacetic acid (AOAA) and Propargylglycine (PAG), on the MtbCbs and MtbMetB enzymes. Interestingly, AOAA inhibited MtbCbs and MtbMetB activity to produce H2S with IC₅₀ values of 72 ± 52 µM and 3.34 ± 0.66 µM, respectively. On the other hand, PAG inhibited only MtbMetB activity with an IC₅₀ of 140.6 µM ± 76 µM and could not inhibit MtbCbs. This establishes that AOAA preferentially inhibits MtbMetB over MtbCbs, whereas PAG selectively inhibits MtbMetB over MtbCbs. Thus, our study provides an understanding of pharmacological research on H2S-producing enzymes of Mtb, which can be used in future to target MtbCbs and MtbMetB enzymes as potential drug targets to combat TB.