Global Proteomic Profiling of the Secretome of Candida albicans ecm33 Cell Wall Mutant Reveals the Involvement of Ecm33 in Sap2 Secretion

Candida albicans secretes numerous proteins related to cell wall remodeling, adhesion, nutrient acquisition and host interactions. Also, extracellular vesicles containing cytoplasmic proteins are secreted into the medium. The C. albicans ecm33/ecm33 mutant (RML2U) presents an altered cell wall and is avirulent. The proteomic analysis of proteins secreted by RML2U cells identified a total of 170 proteins: 114 and 154 of which correspond to the vesicle-free secretome and extracellular vesicles, respectively. Notably, 98 proteins were common to both samples, and the groups most represented were metabolic and cell wall-related proteins. The results of this study showed that RML2U had an altered pattern of proteins secreted by the classical secretion pathway as well as the formation of extracellular vesicles, including their size, quantity, and protein composition. Specifically, the secretion of aspartic protease 2 (Sap2) was compromised but not its intracellular expression, with bovine serum albumin (BSA) degradation by RML2U being altered when BSA was used as the sole nitrogen source. Furthermore, as recent research links the expression of Sap2 to the TOR (Target Of Rapamycin) signaling pathway, the sensitivity of RML2U to rapamycin (the inhibitor of TOR kinase) was tested and found to be enhanced, connecting Ecm33 with this pathway.白色念珠菌（Candida albicans）可分泌诸多与细胞壁重塑、黏附、营养获取及宿主互作相关的蛋白质。此外，包含细胞质蛋白的细胞外囊泡亦会被分泌至培养基中。本研究中的白色念珠菌ecm33/ecm33缺失突变株（RML2U）存在细胞壁结构异常，且无致病力。对RML2U菌株分泌的蛋白质进行蛋白质组学分析，共鉴定得到170种蛋白：其中114种对应无囊泡分泌组，154种对应细胞外囊泡组分。值得注意的是，两类样本中共存在98种重叠蛋白，占比最高的蛋白类别为代谢相关蛋白与细胞壁相关蛋白。本研究结果显示，RML2U菌株的经典分泌途径所分泌的蛋白模式以及细胞外囊泡的形成过程均出现异常，具体涉及囊泡的尺寸、数量及蛋白组成。具体而言，天冬氨酸蛋白酶2（aspartic protease 2, Sap2）的分泌过程受到损害，但其胞内表达并未受影响；当以牛血清白蛋白（bovine serum albumin, BSA）作为唯一氮源时，RML2U对BSA的降解能力发生改变。此外，已有研究表明Sap2的表达与雷帕霉素靶蛋白（Target Of Rapamycin, TOR）信号通路相关，本研究检测了RML2U对雷帕霉素（TOR激酶抑制剂）的敏感性，发现其敏感性显著增强，由此将Ecm33与该信号通路建立了关联。