黑素介导的monophora着色霉对唑类抗真菌药物耐药机制研究

Chromoblastomycosis is one of the most prevalent and highly mutilating (sub)cutaneous diseases in tropical and subtropical climate zones. In Southern China Fonsecaea monophora is the prevalent etiologic agent. Lesions caused by this fungus are very recalcitrant and are difficult to eradicate. Most of the cases constitute a true therapeutic challenge, relapses are often observed, and therefore patients require long-term therapy with systemic antifungals, which may lead to antifungal resistance. Melanin is considered as an important virulence factor in Fonsecaea, and also conveys interferes with antifungal activity. Mechanisms underlying melanin and antifungal drug interaction are still unclear. In fungi, melanin synthesis is catalyzed by various phenoloxidases, among which laccase is most important one. A natural melanin-deficient mutant, CBS 125149 was obtained by subculturing from a meristematic parent strain of F. monophoraCBS 122845. In the present study, wild type F. monophora and its albino mutant will be used as cell models. Firstly, a time-kill study with CFU determinations will be used to reveal differences in the susceptibilities to azoles between melanized and albino cells. Then, a liquid chromatography tandem mass spectrometry (LC-MS/MS) technique will be used for measurement of cell wall melanin concentrations, and of azole and ergosterin intracellular concentrations. Finally, a DNA microarray will be developed to detect melanin-associated genes conferring resistance to Fonsecaeaand azoles. The development of this project will provide detailed information for impact of melanin on Fonsecaeavirulence and clinical resistance to azoles, and could significantly contribute to investigate the genetic basis of azoles resistance in Fonsecaea isolates. Consequently, our project will improve the treatment of Chromoblastomycosis and provide potential targets for antifungal drug discovery.

由monophora着色霉引起的着色芽生菌病是复发率高、顽固难治性皮下真菌病，其主要原因在于病原菌富含黑素毒力因子。漆酶是黑素合成途径中的关键酶。最近文献及我们初步研究结果提示：真菌黑素野生株较白化株对唑类抗真菌药物的敏感性下降，表明黑素是抗真菌抵抗因子。然而，其确切分子机制有待研究。本项目设计以monphora着色霉黑素野生株（CBS 122845）、白化株(CBS 125149)和siRNA技术构建的漆酶Lac基因干扰株为模型，使用液相色谱-质谱／质谱联用(LC-MS/MS)检测比较细胞壁黑素/胞内唑类药物/胞内麦角固醇含量，探讨黑素抵抗唑类抗真菌药物的机制；基因芯片筛查黑化株与白化株之间的耐药相关差异基因，进一步从分子水平阐明着色霉对唑类药物不敏感/耐药的原因。本项目结果为探明着色芽生菌病系统抗真菌疗效差的原因，以及制定新的治疗策略、开发新的抗真菌药物作用靶点提供新思路和实验依据。

由monophora着色霉引起的着色芽生菌病是复发率高、顽固难治性皮下真菌病，其主要原因在于病原菌富含黑素毒力因子。漆酶是黑素合成途径中的关键酶。最近文献及我们初步研究结果提示：真菌黑素野生株较白化株对唑类抗真菌药物的敏感性下降，表明黑素是抗真菌抵抗因子 。然而，其确切分子机制有待研究。本项目分别通过DOPA黑素合成途径和DHN黑素合成途径抑制剂检测野生株黑素的合成情况，并对其进行定性和定量的测定，发现野生株黑素的含量在叠氮化钠抑制剂和三环唑抑制剂下有明显的下降，单一途径或联合途径抑制剂均未能完全抑制野生株黑素的合成，但显微镜和电镜下显示在叠氮化钠抑制剂和三环唑抑制剂下与白化株的细胞壁黑素颗粒减少。进一步的体外药敏试验结果显示在黑素抑制剂下的不同菌株对氟康唑、伊曲康唑和沃力康唑MIC有差异，但无明显统计学意义。此外，本项目通过对monphora着色霉基因组生物信息学分析、聚类分析和比对野生株及白化株转录组表达结果，初步推断具有4个内含子的漆霉Lac4（AYO21_07092）具有较强的临床联系。本项目结果明确monophora着色霉同时存在DOPA黑素合成途径和DHN黑素合成途径，其黑素存在与否对氟康唑、伊曲康唑和沃力康唑MIC有一定差异，而漆霉Lac4基因可能与monophora着色霉黑素差异有关。

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