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.

Methylfolate Trap Promotes Bacterial Thymineless Death by Sulfa Drugs.

The methylfolate trap, a metabolic blockage associated with anemia, neural tube defects, Alzheimer's dementia, cardiovascular diseases, and cancer, was discovered in the 1960s, linking the metabolism of folate, vitamin B12, methionine and homocysteine. However, the existence or physiological significance of this phenomenon has been unknown in bacteria, which synthesize folate de novo. Here we identify the methylfolate trap as a novel determinant of the bacterial intrinsic death by sulfonamides, antibiotics that block de novo folate synthesis. Genetic mutagenesis, chemical complementation, and metabolomic profiling revealed trap-mediated metabolic imbalances, which induced thymineless death, a phenomenon in which rapidly growing cells succumb to thymine starvation. Restriction of B12 bioavailability, required for preventing trap formation, using an "antivitamin B12" molecule, sensitized intracellular bacteria to sulfonamides. Since boosting the bactericidal activity of sulfonamides through methylfolate trap induction can be achieved in Gram-negative bacteria and mycobacteria, it represents a novel strategy to render these pathogens more susceptible to existing sulfonamides.