Phosphoproteomics of Aspergillus fumigatus exposed to the antifungal drug caspofungin, Supplementary files

Aspergillus fumigatus is an opportunistic and allergenic pathogenic fungus, responsible for fungal infections in humans. A. fumigatus infections are usually treated with polyenes, azoles, or echinocandins. Echinocandins, such as caspofungin, can inhibit the biosynthesis of the β-1,3-glucan polysaccharide, affecting the cell wall integrity and leading to fungal death. In some A. fumigatus strains, caspofungin treatment at high concentrations induces an increase of fungal growth, a phenomenon called Caspofungin Paradoxical Effect (CPE). Here, we analyze the proteome and phosphoproteome of A. fumigatus wild-type and the mitogen-activated protein kinases mpkA and sakA null mutant strains during CPE (2 µg/ml caspofungin for 1 hour). The wild-type proteome shows 75 proteins and 814 phosphopeptides (corresponding to 520 proteins) altered in abundance in response to caspofungin treatment. The ΔmpkA (ΔmpkA caspofungin/wild-type caspofungin) and ΔsakA (ΔsakA caspofungin/wild-type caspofungin) display 626 proteins and 1236 phosphopeptides (corresponding to 703 proteins) and 101 proteins and 1217 phosphopeptides (corresponding to 645 proteins), respectively, altered in abundance. Functional characterization of the phosphopeptides from the wild-type exposed to caspofungin show enrichment for transcription factors, protein kinases, and cytoskeleton proteins. Proteomic analysis for the ΔmpkA and ΔsakA mutants indicates that the control of proteins involved in metabolism, such as in secondary metabolites production, was highly represented in both mutants. Functional categorization of phosphopeptides from both mutants is very similar, and show a high number of proteins with decreased phosphorylation of proteins involved in transcriptional control, DNA/RNA binding, cell cycle control, and DNA processing. This study reveals novel transcription factors involved in caspofungin tolerance.

烟曲霉（Aspergillus fumigatus）是一种机会性致敏致病真菌，可引发人类真菌感染。该菌感染通常采用多烯类、唑类或棘白菌素类抗真菌药物治疗。棘白菌素类药物如卡泊芬净（caspofungin），可通过抑制β-1,3-葡聚糖多糖的生物合成破坏真菌细胞壁完整性，进而导致真菌死亡。在部分烟曲霉菌株中，高浓度卡泊芬净处理会诱导真菌生长增强，这一现象被称为卡泊芬净悖论效应（Caspofungin Paradoxical Effect, CPE）。本研究针对卡泊芬净悖论效应发生阶段（2 μg/ml卡泊芬净处理1小时）的烟曲霉野生型菌株，以及丝裂原活化蛋白激酶mpkA和sakA缺失突变菌株，开展了蛋白质组与磷酸化蛋白质组分析。野生型菌株的蛋白质组分析显示，经卡泊芬净处理后，共有75种蛋白质与814条磷酸肽（对应520种蛋白质）的丰度发生显著变化。ΔmpkA突变株（ΔmpkA 卡泊芬净处理组/野生型 卡泊芬净处理组）与ΔsakA突变株（ΔsakA 卡泊芬净处理组/野生型 卡泊芬净处理组）的丰度差异蛋白分别为626种蛋白质与1236条磷酸肽（对应703种蛋白质）、101种蛋白质与1217条磷酸肽（对应645种蛋白质）。对野生型菌株经卡泊芬净处理后的磷酸肽进行功能富集分析，结果显示其显著富集于转录因子、蛋白激酶与细胞骨架蛋白相关功能类别。对ΔmpkA与ΔsakA突变株的蛋白质组分析表明，两类突变株中参与代谢过程（如次生代谢产物合成）的蛋白质调控均具有显著代表性。两类突变株的磷酸肽功能分类高度相似，均存在大量参与转录调控、DNA/RNA结合、细胞周期调控与DNA加工过程的蛋白质，其磷酸化水平出现下调。本研究揭示了一批参与卡泊芬净耐受性调控的新型转录因子。