The Pup-Proteasome System Protects Mycobacteria from Antimicrobial Antifolates

Protein turnover via the Pup-proteasome system (PPS) is essential for nitric oxide resistance and virulence of Mycobacterium tuberculosis, the causative agent of tuberculosis. Our study revealed components of PPS as novel determinants of intrinsic antifolate resistance in both M. tuberculosis and nonpathogenic M. smegmatis. The lack of expression of the prokaryotic ubiquitin-like protein (Pup) or the ligase, PafA, responsible for ligating Pup to its protein targets, enhanced antifolate susceptibility in M. smegmatis. Cross-species expression of M. tuberculosis homologs restored wild-type resistance to M. smegmatis proteasomal mutants. Targeted deletion of prcA and prcB, encoding the structural components of the PPS proteolytic core, similarly resulted in reduced antifolate resistance. Furthermore, sulfonamides were synergistic with acidified nitrite, and the synergy against mycobacteria was enhanced in the absence of proteasomal activity. In M. tuberculosis, targeted mutagenesis followed by genetic complementation of mpa, encoding the regulatory subunit responsible for translocating pupylated proteins to the proteolytic core, demonstrated a similar function of PPS in antifolate resistance. The overexpression of dihydrofolate reductase, responsible for the reduction of dihydrofolate to tetrahydrofolate, or disruption of the Lonely Guy gene, responsible for PPS-controlled production of cytokinins, abolished PPS-mediated antifolate sensitivity. Together, our results show that PPS protects mycobacteria from antimicrobial antifolates via regulating both folate reduction and cytokinin production.

Automated summary

The study reveals the importance of the Pup-proteasome system in mediating nitric oxide resistance and virulence of Mycobacterium tuberculosis, as well as intrinsic antifolate resistance in both M. tuberculosis and nonpathogenic M. smegmatis. Disruption of Pup or PafA expression enhances susceptibility to antifolates in M. smegmatis, while cross-species expression of M. tuberculosis homologs restores antifolate resistance in M. smegmatis mutants. Targeted deletion of structural components of PPS proteolytic core also reduces antifolate resistance, highlighting the role of PPS in protecting mycobacteria from antimicrobial antifolates.