Proteomic analysis revealed ROS-mediated growth inhibition of Aspergillus terreus by shikonin

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 MIC50; 2 mu g/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-beta-glucanosyltransferase, rho-1, beta-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.