BLAST search result using MFCYP51A2 as a query.

Background Mycetoma is a deep fungal infection caused by several microorganisms, with Madurella mycetomatis being the most common causative agent. Another related species, Madurella fahalii, is also known to cause eumycetoma. However, unlike M. mycetomatis, M. fahalii exhibits resistance to itraconazole, the standard treatment for eumycetoma, and the underlying cause of this resistance remains unknown. Therefore, understanding the mechanism of this resistance is critical for developing more effective therapies. Principal Findings Using the high-quality draft genome sequence of Madurella fahalii IFM 68171, we identified two copies of the gene encoding cytochrome P450 14-α sterol demethylase (CYP51), the target enzyme of itraconazole. These include a gene conserved among Madurella species (Mfcyp51A1) and a M. fahalii-specific gene (Mfcyp51A2). Both genes are actively transcribed in M. fahalii and are upregulated in response to itraconazole. Furthermore, heterologous expression in Saccharomyces cerevisiae revealed that transformants carrying the Mfcyp51A2 gene exhibited reduced susceptibility to itraconazole compared to those with Mfcyp51A1. Conclusion We demonstrated that itraconazole resistance in M. fahalii may be attributed to the presence of an additional CYP51 gene. This study represents the first report on the physiological characteristics of Madurella species using genetic engineering techniques.背景 足菌肿（Mycetoma）是一类由多种微生物引发的深部真菌感染，最常见的致病菌为足菌肿马杜拉菌（Madurella mycetomatis）。另有近缘物种法哈马杜拉菌（Madurella fahalii），同样可引发真菌性足菌肿（eumycetoma）。与足菌肿马杜拉菌不同，法哈马杜拉菌对伊曲康唑——真菌性足菌肿的标准治疗药物——具有耐药性，而该耐药性的潜在分子机制至今仍未阐明。因此，解析该耐药机制对于开发更高效的治疗方案至关重要。主要研究结果 借助法哈马杜拉菌IFM 68171的高质量草图基因组序列，我们成功鉴定出两个编码细胞色素P450 14-α甾醇去甲基酶（cytochrome P450 14-α sterol demethylase，CYP51，伊曲康唑的作用靶标酶）的基因拷贝。其中一个为马杜拉菌属物种间保守的基因（Mfcyp51A1），另一个为法哈马杜拉菌特异性基因（Mfcyp51A2）。两个基因在法哈马杜拉菌中均呈现活跃转录状态，且在伊曲康唑诱导下表达上调。进一步的酿酒酵母（Saccharomyces cerevisiae）异源表达实验显示，携带Mfcyp51A2基因的转化子相较于携带Mfcyp51A1基因的菌株，对伊曲康唑的敏感性显著降低。结论 本研究证实，法哈马杜拉菌对伊曲康唑的耐药性可能源于其额外携带的CYP51基因拷贝。本研究亦是首次通过基因工程技术解析马杜拉菌属物种的生理特性。