Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.
Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.
Antimicrob Agents Chemother. 2021 Mar 18;65(4). doi: 10.1128/AAC.01967-20.
Protein turnover via the Pup-proteasome system (PPS) is essential for nitric oxide resistance and virulence of , the causative agent of tuberculosis. Our study revealed components of PPS as novel determinants of intrinsic antifolate resistance in both and nonpathogenic 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 Cross-species expression of homologs restored wild-type resistance to proteasomal mutants. Targeted deletion of and , 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 , targeted mutagenesis followed by genetic complementation of , 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.
通过 Pup-蛋白酶体系统（PPS）进行的蛋白质周转对于耐一氧化氮和结核分枝杆菌的毒力至关重要。我们的研究揭示了 PPS 的成分是分枝杆菌和非致病性分枝杆菌固有抗叶酸耐药性的新型决定因素。缺乏原核泛素样蛋白（Pup）或负责将 Pup 连接到其蛋白质靶标的连接酶 PafA 的表达，增强了分枝杆菌对叶酸拮抗剂的敏感性。种间表达的同源物恢复了野生型对蛋白酶体突变体的耐药性。靶向缺失编码 PPS 蛋白水解核心结构成分的和，同样导致抗叶酸耐药性降低。此外，磺胺类药物与酸化的亚硝酸盐具有协同作用，并且在缺乏蛋白酶体活性的情况下，对分枝杆菌的协同作用增强。在分枝杆菌中，通过遗传互补靶向突变，编码负责将 pupylated 蛋白转运到蛋白水解核心的调节亚基，表明 PPS 在抗叶酸耐药性中具有类似的功能。二氢叶酸还原酶的过表达，负责将二氢叶酸还原为四氢叶酸，或 Lonely Guy 基因的破坏，负责 PPS 控制细胞分裂素的产生，消除了 PPS 介导的抗叶酸敏感性。总之，我们的研究结果表明，PPS 通过调节叶酸还原和细胞分裂素的产生来保护分枝杆菌免受抗菌抗叶酸药物的侵害。
