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

Abstract

Despite considerable progress in understanding the pathogenesis of Mycobacterium tuberculosis (Mtb), 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 in-vivo Mtb 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 Mtb and thus make available, a faithful and highly tractable system to study the physiology of the pathogen in-vivo. We compared the transcriptomics of Mtb and various mutants in the DosR (DevR) regulon derived from Kramnik mouse granulomas to those cultured in-vitro. We recently showed that mutant ΔdosS 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 Mtb:ΔdosR; 'Lipid Metabolism' in Mtb:ΔdosT; 'Virulence, Detoxification, Adaptation' in both Mtb:ΔdosR and Mtb:ΔdosT; and 'PE/PPE' family in all mutant strains compare to wild-type Mtb H37Rv, suggesting that the inability to induce DosR functions to different levels can modulate the interaction of the pathogen with the host. The Mtb 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 glnB, Rv0744c, Rv3281, sdhD/B, mce4A, dctA etc. downregulated in mutant ΔdosS indicate their requirement for bacterial growth and flow of carbon/energy source from host cells. We conclude that genes expressed in Mtb during in-vivo chronic phase of infection in Kramnik mice mainly contribute to growth, cell wall processes, lipid metabolism, and virulence.

Fig 1. In-Situ hybridization.

In-Situ hybridization detected the presence of Mtb specific sigA transcripts in mice lung samples (derived at chronic phase of infection) infected with Mtb, Mtb:ΔdosR, Mtb:ΔdosS and Mtb:ΔdosT strains. Representative images with low (left) and high (right) magnification for each Mtb strain is shown.

Fig 2. Functional categories with significant changes in gene expression in DNA microarray and Mtb growth.

A. The graph shows the total number of genes (left) changed in DNA microarray and mycobacterial colony-forming units (CFU) in mouse lungs during Mtb, Mtb:ΔdosR, Mtb:ΔdosS and Mtb:ΔdosT 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. C. Percentage of genes (obtained from panels A and B) is shown for each functional category.

Fig 3. Gene expression in C3HeB/FeJ mouse lungs.

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

Fig 4. Hierarchical clustering of Mtb genes expressed in C3HeB/FeJ mouse lungs.

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 Mtb genes described in the ‘Tuberculist’ database.

Fig 5. Scatter plot diagram showing similarity and dissimilarity in gene expression from various datasets.

A). Comparison of gene expression in C3HeB/FeJ mouse lungs infected with Mtb strains (red- Mtb; black- Mtb:ΔdosR; blue- Mtb:ΔdosS; green- Mtb:ΔdosT) versus gene expression profile in BALB/c mice [7] 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 [23].

Fig 6. Validation of Mtb gene expression in mouse lungs by quantitative RT-PCR.

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 sigA and fold change were calculated from three biological replicates.

Fig 7. Hierarchical clustering of bacterial genes expressed in C3HeB/FeJ mouse lungs.

A snapshot of few bacterial genes induced in C3HeB/FeJ mouse lungs upon infection with Mtb or Mtb:ΔdosR or Mtb:ΔdosS or Mtb:ΔdosT and their comparison with genes expressed during NRP [24] 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 in-vitro and chronic phase of infection in C3HeB/FeJ mouse lungs.