The temporal expression profile of Mycobacterium tuberculosis infection in mice

Abstract

Infection with Mycobacterium tuberculosis causes the illness tuberculosis with an annual mortality of approximately 2 million. Understanding the nature of the host-pathogen interactions at different stages of tuberculosis is central to new strategies for developing chemotherapies and vaccines. Toward this end, we adapted microarray technology to analyze the change in gene expression profiles of M. tuberculosis during infection in mice. This protocol provides the transcription profile of genes expressed during the course of early tuberculosis in immune-competent (BALB/c) and severe combined immune-deficient (SCID) hosts in comparison with growth in medium. The microarray analysis revealed clusters of genes that changed their transcription levels exclusively in the lungs of BALB/c, SCID mice, or medium over time. We identified a set of genes (n = 67) activated only in BALB/c and not in SCID mice at 21 days after infection, a key point in the progression of tuberculosis. A subset of the lung-activated genes was previously identified as induced during mycobacterial survival in a macrophage cell line. Another group of in vivo-expressed genes may also define a previously unreported genomic island. In addition, our analysis suggests the similarity between mycobacterial transcriptional machinery during growth in SCID and in broth, which questions the validity of using the SCID model for assessing mycobacterial virulence. The in vivo expression-profiling technology presented should be applicable to any microbial model of infection.

Fig. 1.

Microarray analysis of Mtb growing in vivo. Histograms show the number of genes with measurable expression levels during Mtb growth in either BALB/c (A) or SCID (B) mice. The left scale represents the total number of mycobacterial colony-forming units (cfu/lung) cultured before RNA extraction, and the right scale represents the percentage of the expressed genes.

Fig. 2.

Expression profiles for Mtb in different environments. (A) Venn diagram showing the number of overlapping and unique set of growth-dependent genes. (B) Self-organizing maps of gene expression profiles of Mtb under different conditions. Genes with Z scores ≥ ±2 in at least one time point from BALB/c mice, SCID mice, or medium samples were included in the analysis. The means of each cluster of genes (15 clusters) are represented with red, blue, or black boxes indicating up-regulation, down-regulation, or no change of gene expression levels, respectively.

Fig. 3.

Identification of immune-responsive genes by using DNA microarrays. (A) The fold change in genes with significant change in expression levels when Mtb bacilli were grown in BALB/c (red) vs. SCID (blue) mice at 7, 14, 21, and 28 days after infection (n = 122, 67 up- and 55 down-regulated in BALB/c relative to 7H9 samples). (B) The dendogram tree of hierarchical clustering analysis of genes expressed in BALB/c or SCID mice and broth over the first 28 days after infection. List of genes is provided in the supporting information.

Fig. 4.

Genomic organization of the iVEGI where arrowheads indicate the direction of transcription as predicted from the genome sequence of Mtb strain H37Rv. Filled arrows represent mycobacterial genes with higher expression levels in mice samples compared with in vitro samples.