Study On Mechanism Of Azole Drugs’ Cross-resistance Of Candida Albicans Based On Metabonomics Approach

The invasive fungal infection rate and death rate caused by Candida albicans rise with each passing day, and bring a lot of trouble to treatment of fungal infection in clinical practice. Azole drugs, especially fluconazole, which is among the front line therapy because of high safety, good curative potency and little side effect. It only has antibacterial effect which lead to drug resistance during long term use and other antifungal drugs with different structures and targets as well. In recent years, the advent of drug-resistance strain makes a bad situation of fungal control worse. Thus, study of mechanism of azole drugs’cross resistance of Candida albicans become urgent and importantMetabolomics aims to characterize the metabolism network from a whole body perspective, which has a coincidence with the complicated resistance mechanism of Candida albicans. On the basis of preliminary study in our group, the objectives of this study are to analysis metabolic footprinting and phospholipidomics to improve Candida albicans metabolomics research platform. To accomplish the validity, we chose to study on azole drugs’cross resistance mechanism and shikonin antifungal mechanism of Candida albicans.Based on the structural characteristics and characteristic fragment ions of phospholipids, a hydrophilic interaction chromatography-triple quadrupole mass spectrometry system (HILIC-QQQ/MS) was developed. Under the optimization of chromatographic mass spectrometry condition and extraction method, the method is successfully used in the studies of phospholipidomics of Candida albicans. The results show that, phosphatidylcholine and phosphatidylethanolamine are major phospholipids, C16:0, C16:1, C17:1, C18:0, C18:1, C18:2 and C18:3 are the main fatty acid of Candida albicans. The content of PE(32:2) was significantly lower, however unsaturaton indices of phospholipids was significantly higher in amphotericin B group than control group, implying amphotericin B played a role of antifungal by changing the membrane fluidity and destroying cell membrane stability, from another point prove that the target of amphotericin B was located in cell membrane. The established method was offer an assistance for study on mechanism of azole drugs’cross resistance of Candida albicans.In order to maximum obtain the sample effective information from the multi-level and multi-angle, a metabolic fingerprinting and metabolic footprinting method investigation on intracellular and extracellular metabolites of Candida albicans was developed based on combined high throughput analysis technology of ultra-performance liquid chromatography quadrupole time-of-flight/mass spectrometry (UPLC-Q-TOF/MS) and gas chromatography-mass spectrometry (GC-MS).To explore the mechanism of azole drugs’cross resistance of Candida albicans by multivariate statistical methods. With the multivariate statistical analysis,25 potential biomarkers were identified with sensitive strain and resistance strain; 43 potential biomarkers were identified with sensitive strain and drug-sensitive strain; 45 potential biomarkers were identified with resistance strain and drug-resistance strain,20 potential biomarkers were identified with drug-sensitive strain and drug-resistance strain,25 potential biomarkers was found from interaction of strain and drug, primarily involved in amino acid metabolism, citrate cycle, stress metabolism, glycerophospholipid metabolism and sphingolipid metabolism. A phospholipidomics approach based on HILIC-QQQ/MS was used to analyse drug resistance related pathway of Candida albicans and to explore the phospholipids related drug-resistant mechanism. These findings reveal that resistance strain reduce the sensitivity to antifungal agents by enhancing the resistance to external stimuli, decreasing reactive oxygen (ROS), enhancing overexpression of drug efflux transporters, changing cell membrane fluidity, destroying cell membrane stability and affecting mitochondrial function, so as to achieve the effect of antifungal drugs.A metabonomic method based on gas chromatography-mass spectrometry (GC-MS) was employed to explore the mechanism of Candida albicans biofilm treated with shikonin. With the multivariate statistical analysis,19 potential biomarkers have been screened out, primarily involved in amino acid metabolism, citrate cycle, glycolysis, energy metabolism, fatty acid metabolism and stress response, the results showed that shikonin play antifungal role by regulate these metabolism pathways.In conclusion, this is the first time to study Candida albicans based on metabonomics using combined UPLC-Q-TOF/MS、GC-MS and HILIC-QQQ/MS, which maximize obtain the sample effective information from the multi-level and multi-angle, so as to more comprehensive, more accurate understand azole drugs’cross resistance mechanism and shikonin antifungal mechanism of Candida albicans, at the same time provide new ideas for the development of new antifungal drugs.