Shishodia Sonia K, Shankar Jata
Genomics Laboratory, Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Solan 173234, Himachal Pradesh, India.
Genomics Laboratory, Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Solan 173234, Himachal Pradesh, India.
J Proteomics. 2020 Jul 30;224:103849. doi: 10.1016/j.jprot.2020.103849. Epub 2020 May 30.
Aspergillus terreus is an emerging fungal pathogen in immunocompromised patients. Due to the intrinsic resistance of AmB against A. terreus and acquiring resistance to azoles, an alternative antifungal strategy needs investigation. Thus, we explored the activity of phytochemicals such as shikonin, gallic acid, coumaric acid and quercetin against A. terreus. Among these, shikonin showed significant inhibition at MIC; 2 μg/ml. SEM analyses revealed delayed swelling and distorted cell wall organization in shikonin treated A. terreus conidia. Further, we performed differential proteome profiling using nLC-ESI-MS/MS, qRT-PCR and catalase assay with and without shikonin treated A. terreus. Protein data generated using Proteome Discoverer showed 882 differentially expressed proteins (680 up- and 202 down-regulated). GO analysis showed proteins from signaling pathways, oxidative stress, energy metabolism, and cytoskeleton organization. qRT-PCR of selected genes from ROS detoxification (catalase-peroxidase, superoxide dismutase), respiration (succinate-dehydrogenase, NADH-ubiquinone oxidoreductase), signaling (protein kinase C, Mitogen-activated protein kinase, cAMP-dependent protein kinase, ras-1), and 1, 3-β-glucanosyltransferase, rho-1, β-hexosaminidase showed correlation with expressed proteins. We also observed elevated reactive oxygen species using fluorescence ROS assay correlating with low catalase-peroxidase activity in shikonin treated A. terreus. Modulation of ROS homeostasis and the metabolic shift could be instrumental in shikonin- mediated growth inhibition of A. terreus. SIGNIFICANCE: Aspergillosis caused by A. terreus requires more attention due to the development of drug resistance. In this report, we employed nLC-ESI-MS/MS to identify the differential proteome in A. terreus in response to shikonin. The identified proteins involved in pathways influenced by shikonin could be helpful to understand the molecular mechanism on how shikonin/ phytochemicals or other antifungal agents inhibit fungal growth and may enable the discovery of novel or synergistic drug targets.
土曲霉是免疫功能低下患者中一种新出现的真菌病原体。由于两性霉素B对土曲霉具有内在抗性且对唑类药物产生耐药性，因此需要研究一种替代的抗真菌策略。于是，我们探究了紫草素、没食子酸、香豆酸和槲皮素等植物化学物质对土曲霉的活性。其中，紫草素在最低抑菌浓度（MIC）为2μg/ml时显示出显著抑制作用。扫描电子显微镜（SEM）分析显示，经紫草素处理的土曲霉分生孢子肿胀延迟且细胞壁结构扭曲。此外，我们对经紫草素处理和未处理的土曲霉进行了nLC-ESI-MS/MS差异蛋白质组分析、定量逆转录聚合酶链反应（qRT-PCR）和过氧化氢酶测定。使用蛋白质组发现软件生成的蛋白质数据显示有882种差异表达蛋白（680种上调和202种下调）。基因本体（GO）分析显示这些蛋白涉及信号通路、氧化应激、能量代谢和细胞骨架组织。对来自活性氧解毒（过氧化氢酶-过氧化物酶、超氧化物歧化酶）、呼吸作用（琥珀酸脱氢酶、NADH-泛醌氧化还原酶）、信号传导（蛋白激酶C、丝裂原活化蛋白激酶、环磷酸腺苷依赖性蛋白激酶、ras-1）以及1,3-β-葡聚糖基转移酶、rho-1、β-己糖胺酶等选定基因的qRT-PCR分析显示与表达的蛋白质相关。我们还使用荧光活性氧测定法观察到经紫草素处理的土曲霉中活性氧升高，这与低过氧化氢酶-过氧化物酶活性相关。活性氧稳态的调节和代谢转变可能有助于紫草素介导的对土曲霉生长的抑制。意义：由于耐药性的出现，由土曲霉引起的曲霉病需要更多关注。在本报告中，我们采用nLC-ESI-MS/MS来鉴定土曲霉对紫草素反应中的差异蛋白质组。所鉴定的参与受紫草素影响的通路的蛋白质可能有助于理解紫草素/植物化学物质或其他抗真菌剂如何抑制真菌生长的分子机制，并可能有助于发现新的或协同的药物靶点。
