Antifungal Mechanism Of Glyceryl Monolaurate(GML) On Pathogenic Candida

Candida spp.is a zoonotic conditional pathogenic fungus.The drug resistance is getting worse and candida-induced infections and mortality rates continue to rise due to the widespread use of antifungal drugs,broad-spectrum antibiotics,immunosuppressants,and glucocorticoid drugs.The combined use of antifungal drugs with decreasing efficacy every year improved the efficacy reducing toxicity and the lower of acquired resistance.It was found that Monolaurin(GML)used as a food additive has good antibacterial and antiviral effects,but GML interacted fungi is still unknown.In this study,the antimicrobial activity of GML against Candida spp.were tested by drug sensitivity test and the effect of drug combination was evaluated;Using the wind photometer,the cell wall special staining,GC/MS,flow cytometry and DAPI stainiing,the integrity of Candida spp.cell wall or cell membrane and the synthesis of cell membrane after GML,the leakage of macromolecular protein in the cytoplasm and the influence of nuclear structure after GML were observed and detected;the changes of total protein expression and intracellular ROS level after GMLdetected were using SDS-PAGE and fluorescence microplate reader;Then,the effects of GML on the formation of Candida spp.biofilm and the activity and structure of mature biofilm were observed,and the effects of virulence factors such as hydrolase secretion,adhesion ability and hypha formation were detected by XTT,Sport assay,CLSM and other methods.The expression levels of related genes ALS3,HWP1,ECE1,and ERG11 during biofilm formation was tested using qRT-PCR.The results show that the MIC of GML acting on Candida spp.was 15.6 ?g/mL-250 ?g/mL and the MFC is between 31.3?g/mL-500 ?g/mL.The study found that The FICI of GML combined with fluconazole,ketoconazole,itraconazole,amphotericin B,5-fluorocytosine,and tea saponin for Candida albicans is between 0.14-0.37,showed a synergistic effect;The FICI of GML combined with fluconazole,itraconazole and tea saponin for Candida krusei is between 0.28-0.49,showed a synergistic effect.GML prolonged effectively the lag period of Candida spp.growth and inhibited its growth in the logarithmic growth period.The effect of GML on Candida damaged its cell wall and cell membrane integrity,and inhibited the synthesis of cell membranes,resulting in increased permeability of large amount of intracellular macromolecular material leakageCompared with the control group,the extracelular content increased significantly(P<0.05).Reduced cell volume,damaged the nucleus,and hindered the normal expression of bacterial proteins,which affected cell metabolism and various physiological activities;Compared with the control group,the level of ROS produced by mitochondria in the bacteria after GML was significantly increased(P<0.01),which ultimately causes cell death.GML also significantly down-regulate the expression(P<0.01)of C.candida albicans ALS3?HWP1?ECE1?ERG11 and Candida krusei ERG11 genes by reducing the surface hydrophobicity of candida cells,inhibiting adhesion and mycelium formation,inhibiting the biofilm formation and destroying the maintenance,effectively reducing the thickness and viability of the biofilm.