Department of Chemistry and Bioengineering, Graduate School of Engineering, Osaka Metropolitan University, Osaka, Japan.
Microbiol Spectr. 2022 Aug 31;10(4):e0113822. doi: 10.1128/spectrum.01138-22. Epub 2022 Aug 1.
Toyocamycin (TM) is an adenosine-analog antibiotic isolated from Streptomyces toyocaensis. It inhibits Candida albicans, several plant fungal pathogens, and human cells, but many fungi, including Saccharomyces cerevisiae, are much less susceptible to TM. Aiming to clarify why TM and its analogs tubercidin and 5-iodotubercidin are active against C. albicans but not S. cerevisiae, this study focused on the absence of purine nucleoside transport activity from S. cerevisiae. When the concentrative nucleoside transporter (CNT) of C. albicans was expressed in S. cerevisiae, the recombinant strain became sensitive to TM and its analogs. The expression of C. albicans purine nucleoside permease in S. cerevisiae did not result in sensitivity to TM. Clustered regularly interspaced short palindromic repeat-mediated disruption of CNT was performed in C. albicans. The CNTΔ strain of C. albicans became insensitive to TM and its analogs. These data suggest that the toxicity of TM and its analogs toward C. albicans results from their transport via CNT. Interestingly, S. cerevisiae also became sensitive to TM and its analogs if human CNT3 was introduced into cells. These findings enhance our understanding of the mechanisms of action of adenosine analogs toward pathogens and human cells. We investigated the mechanism of toxicity of TM and its analogs to C. albicans. Inspired by the effect of the copresence of TM and purine nucleosides on cell growth of C. albicans, we investigated the involvement of CNT in the toxicity mechanism by expressing CNT of C. albicans (CaCNT) in S. cerevisiae and deleting CaCNT in C. albicans. Our examinations clearly demonstrated that CaCNT is responsible for the toxicity of TM to C. albicans. S. cerevisiae expressing the human ortholog of CaCNT also became sensitive to TM and its analogs, and the order of effects of the TM analogs was a little different between CaCNT- and hCNT3-expressing S. cerevisiae. These findings are beneficial for an understanding of the mechanisms of action of adenosine analogs toward pathogens and human cells and also the development of new antifungal drugs.
妥布霉素（TM）是一种从链霉菌属（Streptomyces toyocaensis）中分离出来的腺苷类似物抗生素。它能抑制白色念珠菌、几种植物真菌病原体和人类细胞，但许多真菌，包括酿酒酵母（Saccharomyces cerevisiae），对 TM 的敏感性要低得多。本研究旨在阐明为什么 TM 及其类似物放线菌素 D 和 5-碘代放线菌素 D 对白色念珠菌有效，但对酿酒酵母无效，这主要是因为酿酒酵母中不存在嘌呤核苷转运活性。当白色念珠菌的浓缩核苷转运蛋白（CNT）在酿酒酵母中表达时，重组菌株对 TM 及其类似物变得敏感。将白色念珠菌的嘌呤核苷透性酶在酿酒酵母中表达并不会导致对 TM 的敏感性。在白色念珠菌中进行了簇状规则间隔短回文重复（CRISPR）介导的 CNT 敲除。白色念珠菌的 CNTΔ菌株对 TM 和其类似物不敏感。这些数据表明，TM 和其类似物对白色念珠菌的毒性是由于它们通过 CNT 转运。有趣的是，如果将人源 CNT3 导入细胞，酿酒酵母也会对 TM 和其类似物敏感。这些发现增强了我们对腺苷类似物对病原体和人类细胞作用机制的理解。我们研究了 TM 和其类似物对白色念珠菌的毒性机制。受 TM 与嘌呤核苷共同存在对白色念珠菌细胞生长影响的启发，我们通过在酿酒酵母中表达白色念珠菌的 CNT（CaCNT）并在白色念珠菌中敲除 CaCNT 来研究 CNT 在毒性机制中的作用。我们的研究清楚地表明，CaCNT 是 TM 对白色念珠菌毒性的原因。表达 CaCNT 人源同源物的酿酒酵母也对 TM 和其类似物敏感，并且在表达 CaCNT 和 hCNT3 的酿酒酵母中，TM 类似物的作用顺序略有不同。这些发现有助于理解腺苷类似物对病原体和人类细胞的作用机制，也有助于开发新的抗真菌药物。
