Figure 6.

Live but not dead mycobacteria downregulate carboxypeptidase-M (CPM) transcript and protein in macrophages (MAs). (a) mRNA levels of CPM during in vitro differentiation of monocytes into MAs are shown in the presence of interferon-γ (IFNγ) or Mycobacterium bovis-bacillus Calmette–Guérin (BCG) tuberculosis bacilli (TB) at various time points. As shown, control samples at 12 h vs 0 time point and TB-treated vs control (non-infected) samples at all the indicated time points were found significant. (b and c) The immunofluorescence (IF)-stained macrophages revealed striking downregulation in CPM-protein expression by the 48th hour of culture when infected with TB (c) compared to control (non-infected) macrophages expressing CPM (b, red fluorescence). (d) In separate experiments (n=3), cells were harvested at 24 h after treatment with live (L) or heat-killed (HK) TB bacilli followed by a reverse transcription-polymerase chain reaction (RT-PCR) analysis for CPM-RNA expression. As shown in this figure, significant CPM-mRNA downregulation occurred only in MAs that were infected with live TB, but no remarkable decrease in transcript was found when HK bacilli were used, suggesting that CPM downregulation is likely dependent on the viability of mycobacterium. All PCR data shown in panel a and panel d are expressed as a ratio of the CPM transcript relative to cyclophilin expression. Error bars indicate the s.d. of the relative expression. ^*Significant (P<0.05) compared with the respective control value.