Transcription factors Rv0081 and Rv3334 connect the early and the enduring hypoxic response of Mycobacterium tuberculosis

Abstract

The ability of Mycobacterium tuberculosis (M. tb) to survive and persist in the host for decades in an asymptomatic state is an important aspect of tuberculosis pathogenesis. Although adaptation to hypoxia is thought to play a prominent role underlying M. tb persistence, how the bacteria achieve this goal is largely unknown. Rv0081, a member of the DosR regulon, is induced at the early stage of hypoxia while Rv3334 is one of the enduring hypoxic response genes. In this study, we uncovered genetic interactions between these two transcription factors. RNA-seq analysis of ΔRv0081 and ΔRv3334 revealed that the gene expression profiles of these two mutants were highly similar. We also found that under hypoxia, Rv0081 positively regulated the expression of Rv3334 while Rv3334 repressed transcription of Rv0081. In addition, we demonstrated that Rv0081 formed dimer and bound to the promoter region of Rv3334. Taken together, these data suggest that Rv0081 and Rv3334 work in the same regulatory pathway and that Rv3334 functions immediately downstream of Rv0081. We also found that Rv3334 is a bona fide regulator of the enduring hypoxic response genes. Our study has uncovered a regulatory pathway that connects the early and the enduring hypoxic response, revealing a transcriptional cascade that coordinates the temporal response of M. tb to hypoxia.

Structural basis of hypoxic gene regulation by the Rv0081 transcription factor of Mycobacterium tuberculosis

“…One cell length being very long could be explained by the presence of two dimers in the crystal asymmetric unit arranged in a side‐to‐side manner. Earlier it has been reported that the molecule is likely to be a monomer; however, recently it has been reported that Rv0081 exist in a dimeric form and also forms higher oligomers at higher concentration . Therefore, we tested the presence of dimeric association through multi‐angle light scattering coupled with size‐exclusion chromatography (SEC‐MALS).…”

Intricate genetic programs controlling dormancy inMycobacterium tuberculosis

“…The number in parentheses indicates the number of genes evaluated in each group. (C) The log2 fold-change distribution of Late hypoxia genes with significant differential expression (P-value < 0.05, absolute log2 fold-change >1) from the ΔRv0081 strain in hypoxia (Sun et al, 2018). (D) Boxplots representing log2 fold-change of various gene groups related to Late hypoxia state from Rv0081 transcription factor overexpression (TFOE) (Rustad et al, 2014).…”

“…Numbers on top of the dendrogram branches indicate the unbiased P-value (au; black) and bootstrap support (bp; purple) for each branch. T0 T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 T17 T18 T19 T20 T21 T22 T23 T24 T25 T0 T1 T2 T3 T4 T5 T6 T7 T10 T12 T13 T14 T15 T16 T17 T18 T22 T23 T24 T25 T21 T19 T20 T9 T8 T11 T18 T16 T17 T0 T1 T25 T22 T23 T24 T2 T3 T4 T7 T5 T6 T14 T15 T10 T12 T13 0 20 40 60 80 Height 100 100 100 100 100 58 100 62 100 100 100 100 100 100 100 100 100 100 100 100 99 99 99 100 au 100 100 100 100 99 57 100 56 100 100 100 100 100 100 100 100 100 100 Plot is based on the comparison between the normoxic TFOE data and hypoxic ΔRv0081 data (Rustad et al, 2014;Sun et al, 2018). Only genes with differential expression in the Rv0081deletion s...…”

“…Another frequent pair combines Rv0023 and Rv0324 to control 70 genes enriched in Mid hypoxia genes (P-value = 3x10 -5 ). We explored the directionality of these statespecific FFL target genes using gene expression data from MTB TF overexpression (TFOE) strains in normoxia (Rustad et al, 2014) and a MTB Rv0081 gene deletion (ΔRv0081) strain in hypoxia (Sun et al, 2018). For example, the majority of Depletion genes with differential expression in the ΔRv0081 strain were up-regulated, suggesting a negative relationship with Rv0081 in hypoxia (Fig 4A).…”

“…The first hypothesis is that Rv0081 plays a pivotal role in the adaptation of MTB to hypoxia (Galagan et al, 2013) and its regulatory activity may be oxygen-dependent. The bifunctional activity of Rv0081 is based on comparison between the normoxic TFOE data (Rustad et al, 2014) and hypoxic ΔRv0081 data (Sun et al, 2018) (Fig S5). The regulatory targets of Rv0081 had very little concordance of fold-change expression in these different conditions.…”