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

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

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

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

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

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

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

<i>Mtb</i>:ΔRv2745c 60 min post-diamide treatment.

<p>Genes induced and repressed by functional category at 60 minutes post-diamide treatment in the isogenic mutant. n = 3. MT # and Rv # denote the CDC1551 and the H37Rv gene IDs, respectively.</p

Diamide Susceptibility.

<p>a.) Disc diffusion assay was performed using discs containing 20 μmol diamide. The zone of inhibition of <i>Mtb</i>:ΔRv2745c (red bar) is significantly larger when compared to both <i>Mtb</i> (blue bar) and <i>Mtb</i>:ΔRv2745c (comp) (green bar) indicating that the isogenic mutant is more susceptible to redox stress. n = 3. *p<0.001 b.) OD graph of diamide treated cultures. In the initial stages of treatment, there is no significant difference in growth between the different groups.</p