白色念珠菌是一种与人类共生的条件致病真菌，具有酵母态，菌丝态和假菌丝态；菌 丝态是白色念珠菌致病的主要形态。课题组前期研究发现，ESCRT  系统成员如 Snf（ESCRT-III 中的核心亚基）、Vps4 缺失使白色念珠菌的菌丝萌发和生长异常；同时 Snf7 还参与了抗真菌药物两性霉素 B 造成的白色念珠菌液泡膜损伤修复。表明 ESCRT 系统在 白色念珠菌的致病性和对抗真菌药物的抗药性中发挥一定作用。Did2 为 ESCRT-III 中的调 节蛋白，发挥刺激 Vps4 活性以解聚 Snf7 的作用，Vps4 则利用 ATP 中的能量促进 ESCRT- III 的解聚，两者功能的正常行使保证 ESCRT-III 可被细胞循环利用。因此，在前期对于 ESCRT 系统蛋白亚基的研究基础之上，我们继续探究了 Did2 在菌丝萌发和生长中的作用 以及 Did2 和 Vps4 在白色念珠菌耐药性中的作用。

结果发现，Did2 缺失并未影响白色念珠菌的菌丝萌发和形态，对两性霉素 B 的敏感性 无明显变化；荧光聚集实验表明 Did2 可能没有参与液泡膜修复过程。但 Vps4 在抗药性中 发挥作用。通过 Quinacrine 染色实验发现，AMB 处理 1 小时，vps4Δ/Δ，vps4Δ/VPS4 和野 生型菌株中荧光在液泡中的细胞比例依次上升，并且 vps4Δ/Δ 和野生型菌株之间存在显著 差异（P<0.01）,表明 Vps4 在维持液泡膜完整性中发挥一定作用。接着通过对 vps4Δ/Δ， vps4Δ/VPS4 和野生型菌株中 Snf7 聚集点观察发现，在 AMB 处理 2 小时和 3 小时，vps4Δ/Δ， vps4Δ/VPS4 和野生型菌株中含 2 个以上 Snf7 聚集点的细胞数量逐渐下降，并且三种菌株 两两之间的细胞比例均存在显著性差异（P<0.01）。综合以上内容，在两性霉素 B 引发的白 色念珠菌液泡膜损伤过程中，Snf7 会被招募至液泡膜进行修复，y 与 MVB 途径相似，Vps4 在此过程中发挥解聚作用，间接参与 ESCRT 系统的膜修复功能。最后，通过 GFP-Atg8 荧 光定位实验，我们初步发现两性霉素 B 可促进白色念珠菌自噬。

本研究进一步完善了 ESCRT 系统在菌丝生长和膜修复方面的发挥的作用，对两性霉 素 B 对白色念珠菌自噬的影响做了初步探究，为白色念珠菌致病性和抗药性的研究提供了 一定帮助。

Candida albicans is an opportunistic pathogenic fungus that can grow in three forms: the yeast, hyphal or pseudohyphal forms. The hyphal form is infectious and a major cause of candidiasis. In previous studies, we found that the deletion of ESCRT system members such as Snf7 (a core subunit in ESCRT-III) and Vps4 causes abnormal mycelial germination and slows the growth of Candida albicans; meanwhile, Snf7 is also involved in the repair of vesicle membrane damage caused by the antifungal drug amphotericin B. These results indicate ESCRTs influence hyphal growth and drug resistance of C. Albicans. Did2 is a regulatory protein in ESCRT-III and plays a role in stimulating the ATPase activity of Vps4 to promote depolymerization of Snf7 and other ESCRT-III members. Based on previous studies, we investigated the role of Did2 in mycelial germination and growth and the role of Did2 and Vps4 in drug resistance in Candida albicans.

The results suggest that Did2 deficiency does not affect the hyphal germination and morphology of Candida albicans, as well as the sensitivity to amphotericin B. Fluorescence aggregation experiments proves that Did2 may not be involved in the membrane repair process. But Vps4 played a role in drug resistance. We carried out the Quinacrine staining experiment to study the vacuole membrane integrity after the treatment of amphotericin B. Our data suggest that, compared to that in wild type cells, the vacuole membrane in vps4Δ/Δ cells shows significant leakage, which could be partially rescued in vps4Δ/ VPS4 cells. We also observed the Snf7’s puncta in vps4Δ/Δ, vps4Δ/VPS4, and the wild type cells with and without amphotericin B incubation. The results show vps4Δ/Δ cells are more likely to contain more than two Snf7 foci, followed by vps4Δ/VPS4 cells. The wild type cells contains the least number of Snf7 foci.

Combined with the results of previous research we proposed that Vps4 has an indirect impact on the vacuolar membrane repair via promoting Snf7 depolymerization. In the last part of this thesis, we described our findings indicating the treatment of amphotericin B could trigger autophagy in C. albicans.

To summarize, this study provides an insight on the mechanisms of hyphal growth and membrane repair in C. albicans and suggests ESCRTs play an important role in both of these processes.