Comprehensive analysis of amino acid alterations during Candida albicans biofilm inhibition by Shikonin using non-targeted and stable isotope-labeled targeted metabolomics

Shikonin exerts a strong inhibitory effect on Candida albicans biofilm formation. This study integrated nontargeted metabolomics and stable isotope-labeled targeted metabolomics to investigate the alterations in amino acids during the inhibition of Candida albicans biofilm formation by shikonin, as well as its potential antibacterial mechanism. The non-targeted metabolomics studies identified a total of 35 potential biomarkers between the shikonin-treated and control groups. The enrichment analysis of all metabolites and their associated pathways indicated that alterations in amino acid pathways were the main characteristic. The developed 4, 6Dimethoxy-2-isothiocyanate pyrimidine derivatization based on ultra performance liquid chromatographytandem mass spectrometry was applied to precisely quantify 20 amino acids present during Candida albicans biofilm formation under shikonin intervention. Significant variations were found in the content of these amino acids in Candida albicans, ranging from 0.53 mu g/mg to 91.44 mu g/mg. The majority of the amino acids exhibited a notable increasing trend during the initial to intermediate phases of biofilm formation, followed by a gradual decline, consistent with the non-targeted results. Shikonin impeded the continuation of this trend to a certain extent. In our study, we integrated the results from non-targeted metabolomics to conduct the first comprehensive analysis of amino acids during the Candida albicans biofilm formation process and further explored the underlying intervention mechanism of shikonin. Additionally, the convenient quantitative method we established for analyzing amino compounds using stable isotope labeling significantly enhanced the response of the compounds in mass spectrometry, making it a valuable approach worthy of wider application.