阿萨希毛孢子菌HSP90基因调控生物膜形及耐药的机制研究

Recent years，with the increasing number of immunodeficient population, the incidence of invasive trichosporonosis increased significantly year by year. The genus Trichosporon now ranks as the second most common cause of disseminated yeast infections, which remain lack of optimal antifungal therapy.Previous study found that Trichosporon asahii was not sensitive to commonly used antifungal agents, and compared to planktonic cells, the MIC values of Trichosporon asahii biofilm rised a hundred times over. Trichosporon biofilm formation and related antifungal drug resistance are the main reasons，which contribute the treatment failure. Based on our previous works of Trichosporon asahii biofilm,we aim at Trichosporon asahii heat shock protein 90 gene. Based on the establishment of HSP90-deleted strain and HSP90-revertent strain, we plan to study the function of heat shock protein 90 (HSP90) gene in the biofilm formation and non-specific drug resistance of Trichosporon asahii, using in vitro and in vivo biofilm models,the confocal scanning electron microscopy, the M27-A3 antifungal drug susceptibility broth microdilution method, XTT-based biofilm antifungal drug susceptibility method, quantitative real-time PCR and Southern Blot methods. We want to reveal the stress protein system play a key role in Trichosporon asahii biofilm infection and adaptive response to antifungal agents. The findings is of great importance for understanding that the Trichosporon asahii resist external stress pressure, regulate biofilm formation and non-specific antifungal drug resistance, through the activation of its stress response signaling pathway system. The results of this research may provide a scientific basis to commence new HSP90-targeted antifungal strategy for treatment of Trichosporon asahii biofilm related infections and non-specific drug resistance.

近年来，由于免疫缺陷人群的不断增加，侵袭性毛孢子菌感染的发病率逐年升高，已成为导致人类真菌血源播散性感染第二位的病原酵母菌。研究发现，阿萨希毛孢子菌对常用抗真菌药物不敏感，且相比游离细胞其生物膜的MIC值可上升百倍以上；而阿萨希毛孢子菌体内生物膜的形成及相关耐药是血源播散性毛孢子菌感染抗真菌治疗失败的重要原因。基于前期对阿萨希毛孢子菌生物膜的研究基础，本研究拟针对阿萨希毛孢子菌热休克蛋白90，通过构建HSP90基因缺失及回复菌株，运用生物膜体内外模型、共聚焦扫描电镜、M27-A3真菌药敏检测法、XTT真菌生物膜药敏检测法、实时定量PCR及Southern Blot等分子生物学方法，研究热休克蛋白90在阿萨希毛孢子菌生物膜形成及耐药中的作用，揭示其在阿萨希毛孢子菌所致生物膜感染及对抗真菌药的适应性反应中所发挥的关键功能，为阿萨希毛孢子菌生物膜相关感染和非特异性耐药的治疗提供新策略及科学依据。

近40年来，由于免疫缺陷人群的不断增加，侵袭性毛孢子菌感染的发病率逐年升高。阿萨希毛孢子菌对常用抗真菌药物不敏感，且相比游离细胞其生物膜的MIC值可上升百倍以上；而阿萨希毛孢子菌体内生物膜的形成及相关耐药是血源播散性毛孢子菌感染抗真菌治疗失败的重要原因。基于前期对阿萨希毛孢子菌生物膜的研究基础，本研究针对阿萨希毛孢子菌热休克蛋白90，通过化学抑制的方法研究，运用生物膜体内外模型、M27-A3真菌药敏检测法、XTT真菌生物膜药敏检测法方法，研究发现热休克蛋白90在阿萨希毛孢子菌生物膜形成及耐药中的发挥作用。同时，通过体外模型研究常用抗真菌药联合、非甾体抗炎药及舍曲林与联合常规抗真菌药联合研究，乙醇抑制阿萨希毛孢子菌临床株生物膜研究，文献计量学研究毛孢子菌菌血症临床特征及预后影响因素，环介导等温扩增检测阿萨希毛孢子菌的临床应用研究。与抗真菌药物联合并出现协同作用时，HSP90抑制剂、非甾体抗炎药及舍曲林部分情况下其MIC值在安全的血药浓度范围内，特别是对AMB具有明显增敏效果，提示我们可以有新的治疗策略用于对抗毛孢子菌感染，能够提高抗真菌药的疗效并避免因为长时间应用而产生耐药性。乙醇可有效抑制阿萨希毛孢子菌生物膜形成并清除成熟生物膜，说明其对导管相关感染是具有预防及治疗的临床应用价值。环介导等温核酸扩增技术的敏感度显著优于是传统PCR方法。LAMP检测阿可有效检测萨希毛孢子菌实验动物模型体液标本和临床样本。伏立康唑可作为推荐的一线抗真菌药来治疗该疾病；宿主的免疫状态是影响预后的重要因素；在临床允许的条件下，建议尽快拔出患者体内留置的中心静脉导管。上述研究结果，为阿萨希毛孢子菌生物膜相关感染的诊断及治疗提供新策略及科学依据。