State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen, Fujian Province, China.
Department of Infectious Diseases, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui Province, China.
Antimicrob Agents Chemother. 2021 Feb 17;65(3). doi: 10.1128/AAC.01595-20.
Widespread antimicrobial resistance encourages repurposing/refining of nonantimicrobial drugs for antimicrobial indications. Gallium nitrate (GaNt), an FDA-approved medication for cancer-related hypercalcemia, recently showed good activity against several clinically significant bacteria. However, the mechanism of GaNt antibacterial action is still poorly understood. In the present work, resistant and tolerant mutants of were sought via multiple rounds of killing by GaNt. Multiround-enrichment yielded no resistant mutant; whole-genome sequencing of one representative GaNt-tolerant mutant uncovered mutations in three genes (, , and ) potentially linked to protection from GaNt-mediated killing. Subsequent genetic analysis ruled out a role for and , but two gain-of-function mutations in conferred tolerance. The mutation-mediated GaNt tolerance depended on EvgS-to-EvgA phosphotransfer; EvgA-mediated upregulation of GadE. YdeO, and SarfA also contributed to tolerance, the latter two likely through their regulation of GadE. GaNt-mediated killing of wild-type cells correlated with increased intracellular reactive oxygen species (ROS) accumulation that was abolished by the -tolerant mutation. Moreover, GaNt-mediated killing was mitigated by dimethyl sulfoxide, and the -tolerant mutation upregulated genes encoding enzymes involved in ROS detoxification and in the glyoxylate shunt of the tricarboxylic acid (TCA) cycle. Collectively, these findings indicate that GaNt kills bacteria through elevation of ROS; gain-of-function mutations in confer tolerance by constitutively activating the EvgA-YdeO/GadE cascade of acid resistance pathways and by preventing GaNt-stimulated ROS accumulation by upregulating ROS detoxification and shifting TCA cycle carbon flux. The striking lethal activity of GaNt suggests that clinical use of the agent may not quickly lead to resistance.
广泛存在的抗菌药物耐药性鼓励重新利用/改进非抗菌药物用于抗菌适应症。硝酸镓（GaNt）是一种获得美国食品和药物管理局批准的用于治疗癌症相关高钙血症的药物，最近显示出对几种具有临床意义的细菌具有良好的活性。然而，GaNt 抗菌作用的机制仍知之甚少。在本工作中，通过 GaNt 多次杀伤寻找 的耐药和耐受突变体。多轮富集未产生耐药突变体；对一个具有代表性的 GaNt 耐受突变体的全基因组测序发现了三个基因（、和）中的突变，这些突变可能与免受 GaNt 介导的杀伤有关。随后的遗传分析排除了和的作用，但中的两个功能获得性突变赋予了耐受性。突变介导的 GaNt 耐受性依赖于 EvgS 到 EvgA 的磷酸转移；EvgA 介导 GadE、YdeO 和 SarfA 的上调也有助于耐受，后两者可能通过调节 GadE 起作用。野生型细胞中 GaNt 介导的杀伤与细胞内活性氧（ROS）积累的增加相关，而突变则消除了这种相关性。此外，二甲基亚砜减轻了 GaNt 介导的杀伤，而 -耐受突变上调了编码参与 ROS 解毒和三羧酸（TCA）循环乙醛酸支路的酶的基因。总的来说，这些发现表明 GaNt 通过升高 ROS 杀死细菌；中的功能获得性突变通过组成型激活酸抗性途径的 EvgA-YdeO/GadE 级联反应以及通过上调 ROS 解毒和改变 TCA 循环碳通量来防止 GaNt 刺激的 ROS 积累赋予了耐受性。GaNt 的惊人致死活性表明，该药物的临床应用可能不会迅速导致耐药性。
