白色念珠菌是一种重要的机会致病菌，具有的形态可塑性使菌株可以以酵母态、真菌丝和假菌丝的形式存在，其形成菌丝的能力被发现与其致病性密切相关。白色念珠菌菌丝形态的发生可被多种条件触发，比如温度，pH，血清，氮饥饿，CO₂等。 ESCRT系统最初被发现是一种涉及多泡体形成的膜萌芽机制。近期，ESCRT系统的功能被大大扩展。早期报道显示，ESCRT系统通过Rim101途径参与在中性-碱性环境下诱导菌丝。本实验前期研究发现，在含血清的环境中，一种ESCRT蛋白Vps4对于菌丝形成期间的极性维持是必需的，因为其缺失导致多菌丝的形成。

本文通过长达8h的观察并统计发现Vps4的缺失不仅导致多个菌丝性状的显着增加，在初期，Vps4的缺失还导致了菌丝萌发数量的显著降低。Vps4是ESCRT-III复合物的解聚蛋白，为了探索ESCRT-III蛋白对菌丝的生长在血清诱导条件下的影响，本文通过显性负突变技术构建了ESCRT-III复合物各亚基的显性负突变菌株，发现Snf7的功能缺失与Vps4的缺失出现了相似的表型。因此本文进一步构建SNF7敲除菌株，发现Snf7的缺失导致了血清诱导下严重的菌丝生长缺陷，包括大部分的菌丝出芽缺陷和形成菌丝的细胞出现收缩。

通过极性必需蛋白荧光定位研究发现，Vps4对于极性蛋白Cdc42和septin蛋白Cdc3在菌丝尖端的定位是必不可少的。通过观察ESCRT对早期和晚期内体标记蛋白Vps21和Ypt72定位的影响，发现ESCRT系统可能是通过参与囊泡运输的方式来完成极性维持的功能。通过RNA水平的检测发现ESCRT蛋白的缺失显著延迟了萌发并导致菌丝特异性基因ECE1、EFG1和HGC1的下调，其中最突出的是HGC1。通过蛋白水平的检测也验证了这一点。由于Hgc1对于菌丝生长的许多方面都是必不可少的，因此其下调可以解释本文观察到萌发延迟的表型。Ras1作为血清诱导途径中的重要调节剂，一般定位于膜上发挥作用，其调控着下游MAPK和cAMP两条途径。本文通过研究ESCRT系统对Ras1在膜上的定位发现，Vps4的缺失会导致Ras1出现在胞内的聚集增多，同时Vps4和Snf7的缺失显著延迟了荧光漂白恢复（FRAP）实验后GFP-Ras1重新定位到膜上的时间。Ras1定位于膜上的活力降低可能破坏信号通路并导致菌丝特异性基因的下调，最终表现出菌丝异常的表型。因此本文发现了独立于Rim101途径的ESCRT系统调节菌丝诱导和极性维持的新机制。

本文通过最新的转录组测序分析方法，对Vps4缺失菌株的转录组与野生型菌株进行基因表达差异性分析、GO数据库和KEGG数据库的功能富集分析，发现Vps4缺失情况下与白色念珠菌生物膜形成和粘附相关基因受到显著影响。因此，本文进一步研究了ESCRT系统缺失菌株对白色念珠菌生物膜形成的影响，结果表明Vps4或Snf7的缺失一定程度上阻碍了生物膜的形成。由于菌丝和生物膜的形成对白色念珠菌的致病性至关重要，本文结果表明ESCRT系统可能在白色念珠菌的致病性中起重要的作用，除了在理论上的重要性之外，可能还可以为研究新型抗念珠菌药物提供新的思路。

Candida albicans is an important opportunistic fungal pathogen and its pathogenicity is closely related to its ability to form hyphae. The morphological plasticity allows C. albicans to exist in yeast, pseudohyphal or hyphal forms. The hyphal form of C.albicans is triggered by a variety of environmental  conditions, including  temperature, pH, serum, nitrogen starvation, CO2, et.al. The ESCRT system was initially discovered as a membrane budding machinery involved in the formation of multivesicular bodies. More recently, the roles of ESCRT are vastly expanded. Early reports show that the ESCRT system is involved in inducing hyphae under the neutral-alkaline environment via the Rim101 pathway. We previously found that under the serum-containing environment, one ESCRT protein, Vps4, is essential for polarity maintenance during hyphal formation, as its deletion caused the formation of multiple hyphae.

We monitored the cells up to 8 hours after being induced by serum  and found that the deletion of VPS4 not only leads to a significant increase in cells that form multiple hyphae, but also causes  a significant decrease in the number of hyphal germination. Vps4 is a depolymerizing protein of ESCRT-III complex. In order to explore the effect of ESCRT-III proteins on the growth of hyphae under the serum-induced condition, we constructed ESCRT-III dominant negative strains,and found that deficiency in Snf7 has a phenotype similar to the deletion of VPS4. We also deleted both copies of SNF7 and found that under serum induction it has a more severe effect on hyphal formation, including the failure of germination in the majority of cells and the formation of constrictions on cells that do form hyphae.

Fluorescence localization studies of proteins essential to polarity revealed that Vps4 is essential for the localization of polarity related protein Cdc42 and the septin subunit Cdc3 to the hyphal tip. By observing early and late endosome localization, we hypothesized that the ESCRT system may perform the function of maintaining the polarity by participating in vesicle trafficking. We also discovered deletions of the ESCRT proteins significantly delay germination and cause down-regulation of hyphal-specific genes (ECE1, EFG1, HGC1), most prominent of which is HGC1. This was also confirmed by the detection of protein levels using Western Blot. Since Hgc1 is essential for many aspects of hyphal growth, its down-regulation could explain our observed phenotypes. Ras1, as an important regulator of hyphal development, is generally localized on the PM, which regulates both MAPK and cAMP pathways. By studying the localization of Ras1, it was found that Vps4 deficiency resulted in increased cytoplasm aggregation of Ras1. Our further studies show that ESCRT proteins are involved in the dynamics of Ras1. Deletions of VPS4 or SNF7 significantly decrease the  fluorescence recovery rate of GFP-Ras1 in the fluorescence recovery after photobleaching (FRAP) experiment. The decreased Ras1 dynamics may disrupt the signaling pathway and lead to down-regulation of hyphal-specific genes. Therefore, in this study we discovered a novel and Rim101 independent mechanism used by the ESCRT system to regulate hyphal induction and polarity maintenance.

The latest RNA-seq method was used to analyze the difference of gene expression between the wild-type and Vps4-deficient strains. The data was further analyzed using the functional enrichment analysis of GO database and KEGG database. It was found that Vps4 was associated with the development of C. albicans biofilm.The expression of adhesion-related genes are significantly affected by the deletion of VPS4. Finally, our data proved that the deletion of the ESCRT system blocks the formation of biofilms. Since the formation of hyphae and biofilms is essential for pathogenicity of C. albicans, our results suggest that the ESCRT system may play an important role in pathogenicity and could provide targets for novel therapeutic agents.