Determinants Outside the DevR C-Terminal Domain Are Essential for Cooperativity and Robust Activation of Dormancy Genes in Mycobacterium tuberculosis

Background: DevR (also called as DosR) is a two-domain response regulator of the NarL subfamily that controls dormancy adaptation of Mycobacterium tuberculosis (M. tb). In response to inducing signals such as hypoxia and ascorbic acid, the N-terminal receiver domain of DevR (DevRN) is phosphorylated at Asp54. This results in DevR binding to DNA via its C-terminal domain (DevRC) and subsequent induction of the DevR regulon. The mechanism of phosphorylation-mediated activation is not known. The present study was designed to understand the role of the N- and C-terminal domains of DevR in DevR regulon genes activation. Methodology/Principal Findings: Towards deciphering the activation mechanism of DevR, we compared the DNA binding properties of DevRC and DevR and correlated the findings with their ability to activate gene expression. We show that isolated DevRC can interact with DNA, but only with the high affinity site of a representative target promoter. Therefore, one role of DevR N is to mask the intrinsic DNA binding function of DevR C. However, unlike phosphorylated DevR, isolated DevR C does not interact with the adjacent low affinity binding site suggesting that a second role of DevRN is in cooperative binding to the secondary site. Transcriptional analysis shows that consistent with unmasking of its DNA binding property, DevRC supports the aerobic induction, albeit feebly, of DevR regulon genes but is unable to sustain gene activation during hypoxia

Validation of <i>Mtb</i> gene expression in mouse lungs by quantitative RT-PCR.

<p>The expression of indicated genes in intracellular bacteria was compared to that of bacteria growing exponentially in 7H9 broth by RT-PCR. The expression of each gene was normalized to <i>sigA</i> and fold change were calculated from three biological replicates.</p

Functional categories with significant changes in gene expression in DNA microarray and <i>Mtb</i> growth.

<p>A. The graph shows the total number of genes (left) changed in DNA microarray and mycobacterial colony-forming units (CFU) in mouse lungs during <i>Mtb</i>, <i>Mtb</i>:Δ<i>dosR</i>, <i>Mtb</i>:Δ<i>dosS</i> and <i>Mtb</i>:Δ<i>dosT</i> infection. B. Functional categories with significant changes in gene expression and number of genes either up or down (cut off 1.5 fold, P<0.05) are shown in each data set. <b>C</b>. Percentage of genes (obtained from panels A and B) is shown for each functional category.</p

Hierarchical clustering of bacterial genes expressed in C3HeB/FeJ mouse lungs.

<p>A snapshot of few bacterial genes induced in C3HeB/FeJ mouse lungs upon infection with <i>Mtb</i> or <i>Mtb</i>:Δ<i>dosR</i> or <i>Mtb</i>:Δ<i>dosS</i> or <i>Mtb</i>:Δ<i>dosT</i> and their comparison with genes expressed during NRP [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135208#pone.0135208.ref024" target="_blank">24</a>] is shown. A gradual decrease or increase in color intensity indicates low (blue) or high (orange) expression. For example, a gradual increase in gene expression over 80 days of hypoxia indicates their requirement during both hypoxia <i>in-vitro</i> and chronic phase of infection in C3HeB/FeJ mouse lungs.</p

In-Vivo Gene Signatures of <i>Mycobacterium tuberculosis</i> in C3HeB/FeJ Mice

<div><p>Despite considerable progress in understanding the pathogenesis of <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>), development of new therapeutics and vaccines against it has proven difficult. This is at least in part due to the use of less than optimal models of <i>in-vivo Mtb</i> infection, which has precluded a study of the physiology of the pathogen in niches where it actually persists. C3HeB/FeJ (Kramnik) mice develop human-like lesions when experimentally infected with <i>Mtb</i> and thus make available, a faithful and highly tractable system to study the physiology of the pathogen <i>in-vivo</i>. We compared the transcriptomics of <i>Mtb</i> and various mutants in the DosR (DevR) regulon derived from Kramnik mouse granulomas to those cultured <i>in-vitro</i>. We recently showed that mutant Δ<i>dosS</i> is attenuated in C3HeB/FeJ mice. Aerosol exposure of mice with the mutant mycobacteria resulted in a substantially different and a relatively weaker transcriptional response (< = 20 genes were induced) for the functional category ‘Information Pathways’ in <i>Mtb</i>:Δ<i>dosR</i>; ‘Lipid Metabolism’ in <i>Mtb</i>:Δ<i>dosT</i>; ‘Virulence, Detoxification, Adaptation’ in both <i>Mtb</i>:Δ<i>dosR</i> and <i>Mtb</i>:Δ<i>dosT</i>; and ‘PE/PPE’ family in all mutant strains compare to wild-type <i>Mtb</i> H37Rv, suggesting that the inability to induce DosR functions to different levels can modulate the interaction of the pathogen with the host. The <i>Mtb</i> genes expressed during growth in C3HeB/FeJ mice appear to reflect adaptation to differential nutrient utilization for survival in mouse lungs. The genes such as <i>glnB</i>, Rv0744c, Rv3281, <i>sdhD/B</i>, <i>mce4A</i>, <i>dctA</i> etc. downregulated in mutant Δ<i>dosS</i> indicate their requirement for bacterial growth and flow of carbon/energy source from host cells. We conclude that genes expressed in <i>Mtb</i> during <i>in-vivo</i> chronic phase of infection in Kramnik mice mainly contribute to growth, cell wall processes, lipid metabolism, and virulence.</p></div

Hierarchical clustering of <i>Mtb</i> genes expressed in C3HeB/FeJ mouse lungs.

<p>Hierarchical clustering demonstrates the expression of common genes (low, blue to high, orange) in two or more datasets in C3HeB/FeJ mice. The data was compared to functional categories of <i>Mtb</i> genes described in the ‘Tuberculist’ database.</p

In-Situ hybridization.

<p><i>In-Situ</i> hybridization detected the presence of <i>Mtb</i> specific <i>sigA</i> transcripts in mice lung samples (derived at chronic phase of infection) infected with <i>Mtb</i>, <i>Mtb</i>:Δ<i>dosR</i>, <i>Mtb</i>:Δ<i>dosS</i> and <i>Mtb</i>:Δ<i>dosT</i> strains. Representative images with low (left) and high (right) magnification for each <i>Mtb</i> strain is shown.</p

Scatter plot diagram showing similarity and dissimilarity in gene expression from various datasets.

<p><b>A)</b>. Comparison of gene expression in C3HeB/FeJ mouse lungs infected with <i>Mtb</i> strains (red- <i>Mtb</i>; black- <i>Mtb</i>:Δ<i>dosR</i>; blue- <i>Mtb</i>:Δ<i>dosS</i>; green- <i>Mtb</i>:Δ<i>dosT</i>) versus gene expression profile in BALB/c mice [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135208#pone.0135208.ref007" target="_blank">7</a>] <b>B)</b>. Graph shows the bacterial genes and their expression levels in C3HeB/FeJ mouse lungs (this study) compared to infected macrophages at 4- and 24-hr post infection [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135208#pone.0135208.ref023" target="_blank">23</a>].</p

Gene expression in C3HeB/FeJ mouse lungs.

<p>The unique genes (on X-axis) expressed (gene expression obtained in microarray using <i>sigA</i> normalized RNA of <i>Mtb</i> and Dos mutants derived from mouse lungs compared to <i>in-vitro</i> grown culture are shown on Y-axis) in C3HeB/FeJ mouse lungs infected with <i>Mtb</i> (red circle) or <i>Mtb</i>:Δ<i>dosR</i> (black diamond) or <i>Mtb</i>:Δ<i>dosS</i> (blue, upright triangle) or <i>Mtb</i>:Δ<i>dosT</i> (green, inverted triangle) are shown. Various functional categories are indicated according to the information available in the ‘Tuberculist’ database.</p

Strains used in this study.

<p>Strains used in this study.</p