Pushparajan, A. R. | Search bioRxiv

bioRxiv2022

Mycobacterium tuberculosis transcriptional regulator Rv1019 is upregulated in hypoxia and negatively regulates Rv3230c-Rv3229c operon encoding enzymes in the oleic acid biosynthetic pathway

The main obstacle in eradicating tuberculosis is the ability of Mycobacterium tuberculosis to remain dormant in the host, and then to get reactivated even years later under immuno-compromised conditions. Transcriptional regulation in intracellular pathogens plays an important role in adapting to the challenging environment inside the host cells. Previously, we demonstrated that Rv1019, a putative transcriptional regulator of M. tuberculosis H37Rv, is an autorepressor. We showed that, Rv1019 is cotranscribed with Rv1020 (mfd) and Rv1021 (mazG) encoding DNA repair proteins and negatively regulates the expression of these genes. In the present study, we show that Rv1019 also regulates the expression of the genes Rv3230c and Rv3229c (desA3) which form a two-gene operon in M. tuberculosis. Constitutive expression of Rv1019 in M. tuberculosis significantly downregulated the expression of these genes. Employing Waynes hypoxia-induced dormancy model of M. tuberculosis, we show that Rv1019 is upregulated (3-fold) under hypoxia. Finally, by reporter assay, using M. smegmatis as a model, we validate that Rv1019 is recruited to the promoter of Rv3230c-Rv3229c during hypoxia and negatively regulates this operon which is involved in the biosynthesis of oleic acid.

molecular biology↗

bioRxiv2020

Putative TetR family transcriptional regulator Rv1019 of Mycobacterium tuberculosis is an auto-repressor and a negative regulator of mfd-mazG operon

Regulation of gene expression at the level of transcription is one of the ways living organisms differentially make proteins in their cells. Intracellular pathogen Mycobacterium tuberculosis persists in the host for years in a latent state in structures called granuloma. Rv1019, a member of an uncharacterized TetR family of transcriptional regulators of Mtb H37Rv, was found to be differentially expressed during dormancy and reactivation in vitro. By GFP reporter assays, electrophoretic mobility shift assays and chromatin immuno-precipitation assays we showed that this protein binds to its own promoter and acts as a negative regulator of its own expression. It forms dimers in vitro which is essential for the DNA binding activity, which is abrogated in the presence of tetracycline. We show that Rv1019 and downstream genes Rv1020 (mfd), Rv1021 (mazG) are co-transcribed. Constitutive expression of Rv1019 in M. smegmatis downregulated MSMEG_5423 (mfd) and MSMEG_5422 (mazG) suggesting that Rv1019 negatively regulates these downstream genes. M. smegmatis expressing Rv1019 was found to be sensitive to UV and H2O2 compared to the control suggesting its role in regulating DNA damage response in mycobacterium. ImportanceOne of the reasons for our inability to eradicate tuberculosis, in spite of the availability of effective drugs and BCG vaccine, is the ingenuity of Mycobacterium tuberculosis to survive, persist and multiply inside the lungs where it employs clever strategies to counter the host defence systems. The number of anti-TB drugs in the current therapeutic regimen is limited, and the long duration of treatment increases the chances for emergence of drug resistant bacteria. Rv1019, a transcriptional regulator differentially expressed in in vitro dormancy-reactivation studies, was found to be an auto-repressor and negatively regulating mfd-mazG operon involved in DNA repair. As Rv1019 is a regulator of critical pathways in bacterial persistence, it could be a prospective target for prophylactic intervention.

microbiology↗