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In vivo gene silencing identifies the Mycobacterium tuberculosis proteasome as essential for the bacteria to persist in mice
Sheetal Gandotra1,2, Dirk Schnappinger1,3, Mercedes Monteleone1, Wolfgang Hillen4 & Sabine Ehrt1,2
Abstract
The success of Mycobacterium tuberculosis (Mtb) as a human pathogen relies on its ability to resist eradication by the immune system. The identification of mechanisms that enable Mtb to persist is key for finding ways to limit latent tuberculosis, which affects one-third of the world's population. Here we show that conditional gene silencing can be used to determine whether an Mtb gene required for optimal growth in vitro is also important for virulence and, if so, during which phase of an infection it is required. Application of this approach to the prcBA genes, which encode the core of the mycobacterial proteasome, revealed an unpredicted requirement of the core proteasome for the persistence of Mtb during the chronic phase of infection in mice. Proteasome depletion also attenuated Mtb in interferon-
–deficient mice, pointing to a function of the proteasome beyond defense against the adaptive immune response. Genes that are essential for growth in vitro, in vivo or both account for approximately 20% of Mtb's genome. Conditional gene silencing could therefore facilitate the validation of up to 800 potential Mtb drug targets and improve our understanding of host-pathogen dynamics.
Figure 1 - Tet-ON and Tet-OFF systems allow efficient and rapid silencing of proteasome expression.
(a) Map of the prcBA genomic region in H37Rv wild-type (top) and H37Rv Pmyc1 tetO:prcBA Mtb (bottom). Probe location and EcoRV (E) restriction sites are indicated. (b) Southern blot of EcoRV-digested genomic DNA from wild-type H37Rv Mtb and from three transductants of H37Rv Pmyc1 tetO:prcBA (T1, T2, T3) probed with the DNA fragment indicated in a. (c) PrcB levels analyzed by immunoblotting in H37Rv Pmyc1 tetO:prcBA Mtb without TetR and transformed with wtTetR (PrcBATet-ON, left) or revTetR (PrcBATet-OFF, right) after growth for 7 d in the presence or absence of anhydrotetracycline (Atc). DlaT was used as loading control. (d) PrcBA transcript levels after transformation of H37Rv Pmyc1 tetO:prcBA Mtb with wtTetR (PrcBATet-ON, left) or revTetR (PrcBATet-OFF, right) and growth for 7 d in the presence or absence of anhydrotetracycline. H37Rv Pmyc1 tetO:prcBA Mtb not transformed with TetR (no TetR) were included as control; normalization of prcBA transcript to rpoB and 16S rRNA instead of sigA gave similar results (data not shown); data are means 
s.e.m. of triplicate samples. (e) Proteasome activities in PrcBATet-ON (left) and PrcBATet-OFF (right) mutants in the presence or absence of anhydrotetracycline. ND, not detected. Vmax represents the cleavage velocity of the fluorogenic peptide substrate and reports proteasome activity. (f) PrcB levels in PrcBATet-ON (left) and PrcBATet-OFF (right) mutants in the presence and absence of anhydrotetracycline at the indicated time points. DlaT was used as loading control.
