The genes encoding the Mtb core proteasome, prcB and prcA,
were among the approximately 600 genes of the Mtb genome predicted to be essential or required for optimal growth in vitro6
. This suggested that prcBA
deletion mutants cannot be obtained and we therefore used conditional gene silencing to determine if prcBA
are important for virulence of Mtb. Both Tet- ON and Tet-OFF systems allowed efficient prcBA
silencing and resulted in significant decreases in proteasome transcript and protein levels as well as proteasome activity. The silencing kinetics of the Tet-OFF system were slightly faster than those achieved with the Tet-ON system even though the final proteasome level was lower after silencing with the Tet-ON system. Silencing of prcBA
significantly delayed growth of Mtb on agar plates, which confirmed that the proteasome is required for optimal growth on this medium. However, prcBA
silencing hardly affected growth in liquid culture even though prcBA
transcript levels were reduced more than 10-fold and proteasome activity was reduced approximately 100-fold. This suggests that prcBA
may not be essential for growth in liquid medium.
We further validated the efficiency of prcBA
silencing by demonstrating that depletion of the proteasome rendered Mtb more susceptible to RNI and increased its resistance to H2
These results are consistent with the phenotypes of mutants of the proteasome associated genes mpa
. Increased resistance to organic peroxide has also been reported for S. coelicolor
proteasome mutants and was accompanied by increased levels of haloperoxidase29
. The mechanisms by which the core proteasome protects Mtb against nitrosative damage may include removal of nitrosylated proteins, as demonstrated in mammalian cells30
. However, our and previous data suggest that the role of the Mtb proteasome extends beyond defence against RNI. Infection of IFNγ-deficient mice with proteasome-depleted Mtb extended their life span compared to mice infected with proteasome expressing Mtb. Similarly, an Mtb mpa
mutant was not fully virulent in iNOS-deficient mice19
Silencing of prcBA
transcription during mouse infections demonstrated that the Mtb proteasome is not only required for optimal in vivo
growth but is also essential during the chronic phase of the infection, when the pathogen replicates slowly or not at all31,32
. The eukaryotic proteasome participates in protein turnover, transcription and DNA repair33
. It is unclear by which of these processes Mtb’s proteasome allows the pathogen to persist in its host. Mutants of the proteasomal ATPase Mpa suggested that the Mtb core proteasome is required for growth of Mtb in mice19,20
but did not predict a role for the proteasome during the chronic phase of infection.
Analyses of prcBA
provided proof-of-principle that transcriptional silencing permits inactivation of Mtb genes during different stages of an infection in mice. Reporter gene studies and experiments with other mutants demonstrated that TetR-mediated gene silencing is atc-dose dependent8,10
. This suggests that gene silencing could determine to which extent a gene has to be inactivated before mycobacterial growth or survival are impaired. Both of these features, conditionality and dose-responsiveness, distinguish gene silencing from deletion and transposon mutagenesis and are not only important for functional analyses but also relevant to the development of new drugs against tuberculosis. Chemotherapy must be administered during different phases of an infection, including chronic disease, and is likely most effective if it inhibits processes that are essential during all stages of an infection34
. Conditional gene silencing facilitates the genetic identification of such processes. In addition, the development of drugs that completely inactivate a target is difficult. Experiments that use gene silencing to partially inactivate a gene might therefore help to focus drug development on targets whose inactivation is effective even if it is incomplete.
In addition to the opportunities that gene silencing offers, the approach also has its challenges. The true impact of target inactivation might be masked by compensatory mutations, epigenetic changes or the residual transcription that occurs despite silencing. Careful analysis of bacterial populations after gene silencing, for example with respect to the frequency of suppressor mutations and the residual expression of the target protein, are required to interpret experiments in which gene silencing does not lead to a phenotypic effect. In addition, for some genes it might be necessary to tailor the activity of the regulated promoter to the target gene’s native expression level to achieve promoter replacement. This could be accomplished with a library of well-regulated promoters that differ in their induced activities. The continued improvement of gene silencing tools for mycobacteria should therefore further facilitate the validation of targets for the development of new drugs against tuberculosis.