Transcriptional signatures of Mycobacterium tuberculosis in mouse model of intraocular tuberculosis

Abstract

Background: Studies on human intraocular tuberculosis (IOTB) are extremely challenging. For understanding the pathogenesis of IOTB, it is important to investigate the mycobacterial transcriptional changes in ocular environment.

Methods: Mice were challenged intravenously with Mycobacterium tuberculosis H37Rv and at 45 days post-infection, experimental IOTB was confirmed based on bacteriological and molecular assays. M. tuberculosis transcriptome was analyzed in the infected eyes using microarray technology. The identified M. tuberculosis signature genes were further validated and investigated in human IOTB samples using real-time polymerase chain reaction.

Results: Following intravenous challenge with M. tuberculosis, 45% (5/12) mice showed bacilli in the eyes with positivity for M. tuberculosis ribonucleic acid in 100% (12/12), thus confirming the paucibacillary nature of IOTB similar to human IOTB. M. tuberculosis transcriptome in these infected eyes showed significant upregulation of 12 M. tuberculosis genes and five of these transcripts (Rv0962c, Rv0984, Rv2612c, Rv0974c and Rv0971c) were also identified in human clinically confirmed cases of IOTB.

Conclusions: Differentially expressed mycobacterial genes identified in an intravenously challenged paucibacillary mouse IOTB model and presence of these transcripts in human IOTB samples highlight the possible role of these genes for survival of M. tuberculosis in the ocular environment, thus contributing to pathogenesis of IOTB.

Keywords: Mycobacterium tuberculosis H37Rv; human vitreous sample; intraocular tuberculosis; microarray; mouse model; transcriptome.

Figure 1.

M. tuberculosis transcriptional signatures in the mouse model of intraocular tuberculosis. (A) Heatmap view of upregulated and downregulated M. tuberculosis genes. Each row represents a specific gene, whereas each column represents each sample. The last column depicts the average. Red signifies upregulated and green signifies downregulated genes (≥1.5-fold change). Data represents fold change from eight hybridization slides (four biological replicates and four technical replicates). (B) Histogram shows the fold change (average + S.D.) of statistically differentially expressed genes depicted in (A) in comparison to reference RNA (in vitro logarithmically grown inoculum)

Figure 2.

Validation of M. tuberculosis transcriptional signatures. RT-qRT-PCR analysis of the upregulated genes on microarray analysis of M. tuberculosis in the eyes of i.v. challenged mouse model of IOTB, 45^th dpi. Expression is compared to the in vitro logarithmically grown inoculum and transcripts were normalized against 16s transcripts. Shown is the log₂ fold change in gene expression ± S.D. P-values ≤0.05 are statistically significant. NS, not significant; M. tb, Mycobacterium tuberculosis.