Identification of substrates of the Mycobacterium tuberculosis proteasome

Abstract

The putative proteasome‐associated proteins Mpa (Mycobaterium proteasomal ATPase) and PafA (proteasome accessory factor A) of the human pathogen Mycobacterium tuberculosis (Mtb) are essential for virulence and resistance to nitric oxide. However, a direct link between the proteasome protease and Mpa or PafA has never been demonstrated. Furthermore, protein degradation by bacterial proteasomes in vitro has not been accomplished, possibly due to the failure to find natural degradation substrates or other necessary proteasome co‐factors. In this work, we identify the first bacterial proteasome substrates, malonyl Co‐A acyl carrier protein transacylase and ketopantoate hydroxymethyltransferase, enzymes that are required for the biosynthesis of fatty acids and polyketides that are essential for the pathogenesis of Mtb. Maintenance of the physiological levels of these enzymes required Mpa and PafA in addition to proteasome protease activity. Mpa levels were also regulated in a proteasome‐dependent manner. Finally, we found that a conserved tyrosine of Mpa was essential for function. Thus, these results suggest that Mpa, PafA, and the Mtb proteasome degrade bacterial proteins that are important for virulence in mice.

Characterization of the Proteasome Accessory Factor (paf) Operon inMycobacterium tuberculosis

“…We recently determined that Mpa and PafA are required for the apparent degradation of three proteins: FabD (malonyl coenzyme A acyl carrier protein transacylase), PanB (ketopantoate hydroxymethyltransferase), and Mpa itself (24). In this work, we show that there is no PafB or PafC in the pafA mutant; thus, it was possible that PafB and PafC were also important for the stability of these proteins.…”

“…Immunoblot analysis of total M. tuberculosis cell lysates showed that Mpa levels were dramatically increased in the pafA mutant compared to WT M. tuberculosis (Fig. 5A) (24). Complementation of the pafA mutation with pafA or pafABC restored Mpa to WT levels (Fig.…”

“…FLAG-fabD-His 6 and FLAGpanB-His 6 were expressed in M. tuberculosis under the control of a heterologous mycobacterial hsp60 promoter and an E. coli ribosome-binding site (24). In contrast to the mpa and pafA mutants (24), epitope-tagged FabD and PanB did not accumulate in the pafB and pafC mutants ( Fig.…”

“…Mpa is similar to ATPases found in the eukaryotic proteasome base (7), and chemical inhibition of the M. tuberculosis proteasome protease activity sensitizes wild-type (WT) M. tuberculosis to RNI to a degree similar to that of the mpa or pafA mutants (6). The strongest evidence connecting Mpa, PafA, and the proteasome protease is the observation that all three are required for the apparent degradation of three M. tuberculosis proteasome substrates (24).…”

Thiol-Dependent Peroxidases in Mycobacterium tuberculosis Antioxidant Defense

“…Evidence for the first Bcp (Rv2125) expression at a protein level exists, both in the membrane fraction (Gu et al, 2003) and in the cytosol of H37Rv strains (Mawuenyega et al, 2005). The protein has been shown to be target of modification by the small protein Pup, a post-translational modification that targets proteins for degradation by the M. tuberculosis proteosome (Pearce et al, 2006;Festa et al, 2010). To note, pupylation and proteosome function are essential for the virulence of this bacterium, for reasons still unknown (Darwin et al, 2003;Gandotra et al, 2007).…”