Banerjee Dithi, Burkard Lauren, Panepinto John C
Department of Microbiology and Immunology, Witebsky Center for Microbial Pathogenesis and Immunology, University at Buffalo, The State University of New York, Buffalo, New York, United States of America.
PLoS One. 2014 Jan 30;9(1):e87246. doi: 10.1371/journal.pone.0087246. eCollection 2014.
The polyene antifungal agent Amphotericin B exhibits potent and broad spectrum fungicidal activity. However, high nephrotoxicity can hinder its administration in resource poor settings. Quantification of early fungicidal activity in studies of HIV patients with cryptococcosis demonstrate that 5-Fluorocytosine therapy in combination with Amphotericin B results in faster clearance than with Amphotericin B alone. In vitro synergy between the two drugs has also been reported but the mechanism by which 5-Fluorocytosine synergizes with Amphotericin B has not been delineated. In this study we set out to investigate the effect of genetic mutation or pharmacologic repression of de novo pyrimidine and purine biosynthesis pathways on the Amphotericin B susceptibility of Cryptococcus neoformans. We demonstrate that a ura- derivative of wild type Cryptococcus neoformans strain H99 is hypersensitive to Amphotericin B. This sensitivity is remediated by re-introduction of a wild type URA5 gene, but not by addition of exogenous uracil to supplement the auxotrophy. Repression of guanine biosynthesis by treatment with the inosine monophosphate dehydrogenase inhibitor, mycophenolic acid, was synergistic with Amphotericin B as determined by checkerboard analysis. As in Cryptococcus neoformans, a ura(-) derivative of Candida albicans was also hypersensitive to Amphotericin B, and treatment of Candida albicans with mycophenolic acid was likewise synergistic with Amphotericin B. In contrast, neither mycophenolic acid nor 5-FC had an effect on the Amphotericin B susceptibility of Aspergillus fumigatus. These studies suggest that pharmacological targeting of nucleotide biosynthesis pathways has potential to lower the effective dose of Amphotericin B for both C. neoformans and C. albicans. Given the requirement of nucleotide and nucleotide sugars for growth and pathogenesis of Cryptococcus neoformans, disrupting nucleotide metabolic pathways might thus be an effective mechanism for the development of novel antifungal drugs.
多烯抗真菌剂两性霉素B具有强大的广谱杀真菌活性。然而，高肾毒性会阻碍其在资源匮乏地区的应用。对感染隐球菌的HIV患者进行的早期杀菌活性定量研究表明，5-氟胞嘧啶与两性霉素B联合治疗比单独使用两性霉素B能更快清除病原体。两种药物在体外也有协同作用，但5-氟胞嘧啶与两性霉素B协同作用的机制尚未明确。在本研究中，我们着手研究从头嘧啶和嘌呤生物合成途径的基因突变或药物抑制对新型隐球菌两性霉素B敏感性的影响。我们证明，野生型新型隐球菌菌株H99的ura-衍生物对两性霉素B高度敏感。通过重新引入野生型URA5基因可恢复这种敏感性，但添加外源尿嘧啶以补充营养缺陷型则不能恢复。通过棋盘分析确定，用肌苷单磷酸脱氢酶抑制剂霉酚酸处理抑制鸟嘌呤生物合成与两性霉素B具有协同作用。与新型隐球菌一样，白色念珠菌的ura(-)衍生物对两性霉素B也高度敏感，用霉酚酸处理白色念珠菌同样与两性霉素B具有协同作用。相比之下，霉酚酸和5-氟胞嘧啶对烟曲霉的两性霉素B敏感性均无影响。这些研究表明，对核苷酸生物合成途径进行药物靶向有可能降低新型隐球菌和白色念珠菌两性霉素B的有效剂量。鉴于新型隐球菌生长和致病需要核苷酸和核苷酸糖，破坏核苷酸代谢途径可能是开发新型抗真菌药物的有效机制。
