Multiple transcription factors co-regulate the Mycobacterium tuberculosis adaptation response to vitamin C

Abstract

Background: Latent tuberculosis infection is attributed in part to the existence of Mycobacterium tuberculosis in a persistent non-replicating dormant state that is associated with tolerance to host defence mechanisms and antibiotics. We have recently reported that vitamin C treatment of M. tuberculosis triggers the rapid development of bacterial dormancy. Temporal genome-wide transcriptome analysis has revealed that vitamin C-induced dormancy is associated with a large-scale modulation of gene expression in M. tuberculosis.

Results: An updated transcriptional regulatory network of M.tuberculosis (Mtb-TRN) consisting of 178 regulators and 3432 target genes was constructed. The temporal transcriptome data generated in response to vitamin C was overlaid on the Mtb-TRN (vitamin C Mtb-TRN) to derive insights into the transcriptional regulatory features in vitamin C-adapted bacteria. Statistical analysis using Fisher's exact test predicted that 56 regulators play a central role in modulating genes which are involved in growth, respiration, metabolism and repair functions. Rv0348, DevR, MprA and RegX3 participate in a core temporal regulatory response during 0.25 h to 8 h of vitamin C treatment. Temporal network analysis further revealed Rv0348 to be the most prominent hub regulator with maximum interactions in the vitamin C Mtb-TRN. Experimental analysis revealed that Rv0348 and DevR proteins interact with each other, and this interaction results in an enhanced binding of DevR to its target promoter. These findings, together with the enhanced expression of devR and Rv0348 transcriptional regulators, indicate a second-level regulation of target genes through transcription factor- transcription factor interactions.

Conclusions: Temporal regulatory analysis of the vitamin C Mtb-TRN revealed that there is involvement of multiple regulators during bacterial adaptation to dormancy. Our findings suggest that Rv0348 is a prominent hub regulator in the vitamin C model and large-scale modulation of gene expression is achieved through interactions of Rv0348 with other transcriptional regulators.

Keywords: Dormancy; Mtb-TRN (Mtb-transcriptional regulatory network); Mycobacterium tuberculosis; Transcriptome; Vitamin C.

Fig. 1

a A systematic flow of the protocol used for the regulator-target analysis of Mtb in response to vit C. b Venn diagram showing the total number of regulator-target interactions in the Mtb-TRN, where the inner grey circle shows 10,061 interactions from previous available literature and the outer yellow circle shows additional 5979 interactions from the new updated Mtb-TRN. c Network view of updated Mtb-TRN, wherein nodes are coloured in pink and edges (links) are coloured in blue

Fig. 2

a Temporal enrichment of regulators. Regulators enriched at each time point (FDR corrected pvalue ≤ 0.05) are coloured in blue. The Early (0.25 to 1 h), Intermediate (2 to 8 h) and Late (24 h) time points are marked in pink. b COG functional enrichment of targets of the enriched regulator. COG functional categories enriched at each time point are shown. **, FDR corrected pvalue ≤ 0.05; *, pvalue ≤ 0.05 (without FDR correction)

Fig. 3

Temporal network analysis of enriched regulators and their DRG targets in vit C Mtb-TRN. Representative time points from the temporal response are shown. The regulator nodes are indicated in pink, the up-regulated target gene nodes are indicated in red, while the down-regulated target gene nodes are indicated in green. The size of the node depends upon its connectivity (links) in the network

Fig. 4

Temporal expression patterns of Rv0348 target genes. Major DRG targets of Rv0348 arranged in 4 functions with their temporal expressions is shown. Genes belonging to the same operon are arranged together and the horizontal arrows indicate the directions in which the genes are transcribed in the genome

Fig. 5

DevR and Rv0348 co-regulate DevR-dependent genes. a Rv3134c promoter region. Rv3134c-devR-devS operon genes are depicted by black arrows and the promoter region consists of primary (P) and secondary (S) Dev boxes where DevR binds to the promoter. The DNA probe for EMSA depicted by the black box was generated from the promoter region by PCR amplification. b EMSA of the Rv3134c promoter fragment in the presence of Rv0348 and phosphorylated DevR (DevR~P). Formation of the complex of DevR alone with DNA and a higher molecular weight complex of Rv0348, DevR and DNA are depicted on the left. c EMSA of the Rv3134c promoter fragment in the presence of Rv0348 and unphosphorylated DevR (DevR-unP). d. Interaction of DevR-unP (3.8 to 19 pmol) and Rv0348 (15 pmol) proteins demonstrated by ELISA. BSA was used as a negative control in the assay

Fig. 6

Summary of vit C-Mtb TRN involving multiple regulators and target genes. Regulators and their differentially regulated target genes arranged in 4 functions are shown. The regulators are written in pink box, up-regulated gene targets are written in red coloured font and the down-regulated gene targets are shown in green coloured font