Regulation of macrophage gene expression following invasion by Mycobacterium tuberculosis

Introduction: Mycobacteria are intracellular pathogens which survive and grow in host macrophages. M. tuberculosis bacilli enter the macrophage via binding to several distinct cell surface molecules. Following phagocytosis, sustained intracellular bacterial growth depends on the ability to avoid destruction by macrophage-mediated host defences such as lysosomal enzymes, reactive oxygen and the reactive nitrogen intermediates. We used differential display reverse transcription polymerase chain reaction (DD RT-PCR) to identify host genes which are regulated during infection and hence which might be involved in the host-parasite cross talk. Results: Live M. tuberculosis (strain H₃₇Rv) was used to infect Balb/c peritoneal murine macrophages. mRNA from infected and uninfected macrophages was isolated at different time intervals after phagocytosis and subjected to DD RT-PCR. Oligo dT₁₂NV and random 10mer primers were used for PCR amplification of cDNA. Macrophage genes which appeared to be differently regulated during infection were subjected to further reamplification by PCR in order to clone and sequence them. The differential expression of the selected bands was further analysed by an RNA protection assay and a Northern blot. Results: Several differentially regulated bands were identified. One band, of 158 bp, was down regulated after infection. Sequencing of this band revealed a high level of homology (95% identity) to mouse cytochrome c oxidase subunit VIIc. The downregulation was specific for live virulent Mtb, while live BCG, heat killed Mtb and latex beads-mediated phagocytosis did not affect the transcriptional level of this enzyme. Conclusions: The cytochrome oxidase enzyme complex of the inner mytochondrial membrane catalyzes the reaction between ferrocytochrome c and oxygen. The reaction is the terminal event in the electron transport scheme. Downregulation of cytochrome c oxidase subunit VIIc could interfere with: (1) the host apoptotic programme; or (2) the host respiratory burst.