Investigation of molecular factors involved in mycobacterial stress responses and non-replicating persistence

Mycobacterium tuberculosis is a remarkably successful human pathogen due to its ability to switch into dormancy or non-replicating persistence (NRP) phase driven by the host stress microenvironments. Identifying the panoply of genes or pathways involved in dormancy will progress our understanding on latent tuberculosis infection. Rv2660c and Rv2661c (conserved hypothetical proteins) and Rv1675c (transcriptional regulator) were implicated in mycobacterial stress response and transition to dormancy, hence their biological importance in M. tuberculosis biology was further explored. Rv2660c and rv2661c were highly upregulated in vitro starvation and in vivo infection model, however, recent high upregulation of a noncoding RNA, ncRv12659 in these models challenged the importance of these genes for NRP. A panel of single and double in-frame deletion mutants and over-expressing strains of rv2660c and rv2661c in M. tuberculosis were generated. A deletion of rv2660c and rv2661c also resulted in partial inactivation of ncRv12659 and rv2662 respectively. The deletion mutants exhibited normal growth in vitro and in mice. Furthermore, the strains showed unimpaired survival under nutrient starvation, hypoxia, oxidative and nitrosative stresses. Quantitative RT-PCR analysis revealed that neither target gene was highly expressed throughout starvation, oxidative and acidic pH stresses. Rv1675c (Cmr) is a redox sensor that regulates the DosR signalling pathway. Cmr binding to DNA was severely reduced by nitrosation of the two conserved cysteine residues. The cmr mutant displayed survival advantage during exposure to nitrosative stress. The over-expression of cmr or cmrC2A form (mutated cysteines) had a mild inhibitory effect on growth of M. tuberculosis. The over-expressing strain of cmrC2A was more resistant to hydrogen peroxide, suggesting that Cmr may also control the response to oxidative stress. Our study clarified the role of Rv2660c and Rv2661c in growth, NRP and infection, and further highlighted a novel Cmr-mediated regulatory network involved during nitrosative stress and transition to dormancy.