Table_3_tpo3 and dur3, Aspergillus fumigatus Plasma Membrane Regulators of Polyamines, Regulate Polyamine Homeostasis and Susceptibility to Itraconazole.XLSX

Aspergillus fumigatus is a well-known opportunistic pathogen that causes invasive aspergillosis (IA) infections, which have high mortality rates in immunosuppressed individuals. Long-term antifungal drug azole use in clinical treatment and agriculture results in loss of efficacy or drug resistance. Drug resistance is related to cellular metabolites and the corresponding gene transcription. In this study, through untargeted metabolomics and transcriptomics under itraconazole (ITC) treatment, we identified two plasma membrane-localized polyamine regulators tpo3 and dur3, which were important for polyamine homeostasis and susceptibility to ITC in A. fumigatus. In the absence of tpo3 and/or dur3, the levels of cytoplasmic polyamines had a moderate increase, which enhanced the tolerance of A. fumigatus to ITC. In comparison, overexpression of tpo3 or dur3 induced a drastic increase in polyamines, which increased the sensitivity of A. fumigatus to ITC. Further analysis revealed that polyamines concentration-dependently affected the susceptibility of A. fumigatus to ITC by scavenging reactive oxygen species (ROS) at a moderate concentration and promoting the production of ROS at a high concentration rather than regulating drug transport. Moreover, inhibition of polyamine biosynthesis reduced the intracellular polyamine content, resulted in accumulation of ROS and enhanced the antifungal activity of ITC. Interestingly, A. fumigatus produces much lower levels of ROS under voriconazole (VOC) treatment than under ITC-treatment. Accordingly, our study established the link among the polyamine regulators tpo3 and dur3, polyamine homeostasis, ROS content, and ITC susceptibility in A. fumigatus.

烟曲霉（Aspergillus fumigatus）是一类公认的机会致病菌，可引发侵袭性曲霉病（invasive aspergillosis, IA），该类感染在免疫抑制人群中具有极高的死亡率。临床治疗与农业生产中长期使用唑类抗真菌药物（azole antifungal drugs），会导致药物疗效衰减或烟曲霉产生耐药性。真菌耐药性与细胞代谢物及对应基因的转录调控息息相关。本研究在伊曲康唑（itraconazole, ITC）处理条件下，通过非靶向代谢组学（untargeted metabolomics）与转录组学（transcriptomics）分析，鉴定得到两个质膜定位的多胺调控因子tpo3与dur3，二者对烟曲霉的多胺稳态（polyamine homeostasis）及伊曲康唑易感性具有关键调控作用。当缺失tpo3和/或dur3时，胞质多胺水平会出现适度升高，这会增强烟曲霉对伊曲康唑的耐受性。与之相对，过表达tpo3或dur3则会导致多胺水平显著升高，进而提升烟曲霉对伊曲康唑的敏感性。进一步分析显示，多胺可通过浓度依赖性方式调控烟曲霉对伊曲康唑的易感性：在适度浓度下清除活性氧（reactive oxygen species, ROS），而在高浓度下则促进活性氧生成，而非通过调控药物转运途径发挥作用。此外，抑制多胺生物合成（polyamine biosynthesis）会降低细胞内多胺含量，引发活性氧积累，并增强伊曲康唑的抗真菌活性。值得注意的是，相较于伊曲康唑处理组，烟曲霉在伏立康唑（voriconazole, VOC）处理下产生的活性氧水平显著更低。综上，本研究明确了烟曲霉中多胺调控因子tpo3与dur3、多胺稳态、活性氧含量及伊曲康唑易感性之间的内在关联。