THE TUBERCULOSIS PROTEASOME AND PATHOGENESIS

The pathogen and the disease. Tuberculosis is one of the leading causes of death in the world, killing about 2 million people per year. Nearly one-third of the world is infected with Mtb, which is a rod shaped bacterium that persists in phagocytic cells in the lungs of humans. Although healthy individuals usually control Mtb growth, immunosuppression due to a variety of causes can result in increased bacterial replication and the onset of symptoms. Antibiotic therapy is prolonged (6-9 months) and the failure to comply with treatment can lead to the development of multi-drug and extensively drug resistant strains. New drugs to treat tuberculosis are urgently needed, thus researchers are working to identify activities in Mtb that can be targeted.
The task of finding new drugs and drug targets is hindered by the fact that Mtb is dangerous and slow growing, requiring 2-3 weeks to form colonies on solid media. Furthermore, due to the highly infectious nature of the pathogen, all work must be performed in a biosafety level-3 facility. Taken together, Mtb is one of the most significant and challenging organisms to study.

Mycobacterial resistance to host defenses: the bacterial proteasome. Mtb proteasome activity is required for NO-resistance and virulence in mice, linking protein degradation to pathogenesis. Proteolysis by the Mtb proteasome requires the 20S proteasome core protease, Mpa (Mycobacterium proteasomal ATPase), and PafA (proteasome accessory factor A), but other factors are also likely to be needed. The 20S core is a barrel shaped chamber with proteolytic activity. Mpa forms hexamers with ATPase activity similar to eukaryotic proteasomal ATPases, and is believed to cap the base of the 20S core. Importantly, mpa and pafA mutants cannot cause lethal infections in mice. Although it is clear that proteasome activity is required for the pathogenesis of Mtb, we do not fully understand why.

Bacterial ubiquitin-proteasome system. Small protein modifiers like ubiquitin and SUMO are extensively studied in eukaryotes but had not been found in prokaryotes. Using a bacterial 2-hybrid screen, we found that the prokaryotic ubiquitin-like protein Pup interacts with the Mtb proteasomal ATPase Mpa. Further characterization of Pup revealed that it covalently attaches to numerous proteins in Mtb. Pup is the first Ubl to be identified in prokaryotes. Pup is also unique because of its atypical linkage to substrates; ubiquitin and other Ubls attach to substrate lysines by a glycine whereas Pup attaches via a glutamate. Although Pup does not appear to attach to substrates via a glycine, Pup has a penultimate di-glycine (GG) motif that is essential for pupylation (not shown). Another unusual aspect of Pup is that it is predicted to end with glutamine, not glutamate, suggesting Pup is de-amidated before or during conjugation to substrates.
Homologues of the proteasome core and associated ATPases have been easily identified in Mtb, but Ubl activation and conjugation enzymes (E1, E2, E3) have not. This suggests that the mechanism of attachment of Pup to substrates is different from that of eukaryotes, or that the Mtb enzymes involved share little to no similarity with eukaryotic counterparts at the amino acid sequence level.
Taken together these observations have led us to ask:

(a) How is protein degradation linked to tuberculosis pathogenesis?
(b) What are the enzymes required for substrate tagging prior to degradation?
(c) Do other small protein modifiers exist in Mtb or in other bacteria, including those that do not have proteasomes?