Transcriptional characterization of the antioxidant response of Mycobacterium tuberculosis in vivo and during adaptation to hypoxia in vitro

Abstract

Transcriptional profiling of antioxidant genes of Mycobacterium tuberculosis was performed by real-time RT-PCR during mouse lung infection and during adaptation to gradual oxygen depletion in vitro. M. tuberculosis genes involved in major detoxification pathways of oxidative stress were not up-regulated during chronic mouse lung infection, which is established in response to expression of host adaptive immunity. This result suggests that a major function of bacterial antioxidant enzymes is to protect from oxidants generated during the early, acute phase of infection. In vivo transcription profiles of bacterial antioxidant enzymes differed from those seen under adaptation to low oxygen in vitro, indicating differences between growth arrest in vivo and that induced by hypoxia in vitro.

Figure 1. M. tuberculosis growth and normalized copy number of bacterial transcripts encoding antioxidant enzymes in infected mouse lung

Panel A: M. tuberculosis growth and copy numbers of cytochrome bd oxidase (cydA) during infection. In lungs of C57BL/6 mice infected with M. tuberculosis H₃₇Rv via the respiratory route, M. tuberculosis grew exponentially for about 20 days followed by chronic phase characterized with stabilization of bacterial counts (filled circles). Copy numbers of cydA transcripts were determined by real time RT-PCR and normalized against 16S rRNA (filled triangles, data from 27). Panels B–D: Copy numbers of bacterial transcripts encoding antioxidant enzymes. Normalized copy numbers of M. tuberculosis mRNAs against 16S rRNA were expressed as mean ± SD of data obtained from lungs of 3 or 4 mice per time point.

Figure 2. M. tuberculosis growth and changes of bacterial transcripts encoding antioxidant enzymes in liquid cultures during adaptation to gradual oxygen depletion

M. tuberculosis was cultured under conditions of gradual O₂ depletion established by Wayne and Hayes . Bacterial RNA was extracted, and transcripts were enumerated by real-time RT-PCR with molecular beacons. Panel A: M. tuberculosis growth curve and 16S rRNA levels. Panel B: Changes of M. tuberculosis transcripts encoding antioxidant enzymes during gradual oxygen depletion. Levels of transcripts per cell were obtained by dividing mRNA copy number by the corresponding 16S rRNA copy number. Shown are ratios of normalized mRNA copy numbers determined at hour 78, 102, 126, 168, 240, 342, 460 and 578 relative to aerated mid-log cultures (hour 0). Replicate experiments produced similar results.