Composition Analysis On Extracellular Matrix Of Candida Albicans Biofilms And Interaction Of Sodium Houttuyfonate With Candida Albicans Cell Wall

Objective:This study aimed to improve the extraction methods of Candida albicans biofilm matrix,clarify the content of carbohydrate,protein and lipid in the matrix,and study interaction of sodium houttuyfonate with Candida albicans cell wall.Methods:1.Firstly,C.albicans biofilm was formed under static and dynamic conditions.The efficiency of six methods for extracting C.albicans biofilm matrix?EM?was compared,i.e.vortexing and ultrasonication?VU?,formaldehyde plus vortexing and ultrasonication?Formaldehyde-VU?,EDTA plus vortexing and ultrasonication?EDTA-VU?,NaOH plus vortexing and ultrasonication?NaOH-VU?,ethanol plus vortexing and ultrasonication?Ethanol-VU?,ion exchange resin?including cation/anion exchange resin?plus vortexing and ultrasonication?CER/AER-VU?.Subsequently,the stability of the methods of extracting biofilm matrix was monitored by XTT and plate counting;the weight of biofilm and martix was measured;the contents of carbohydrate,protein and triglyceride in the EM of Candida albicans,Candida glabrata,Candida krusei and Candida tropicalis were detected.2.The susceptibility of C.albicans to sodium Houttuynia?SH?was analyzed by targeting enzymes of seven EM chemical components?including Protease K,protease XIV,?-N-acetyl-glucosamindase,DNase I,RNase A and lyticase?.After incubation of SH with EM,the cell wall,Laminarin?commercially available?-1,3-glucan?and chitin?shrimp shell?,the adsorption of SH to C.albicans was analyzed by UPLC.The standard strain SC5314 and the mutant of C.albicans phr 1-/-were used.In homology domain containing,family B?evectins?member 1?phr1?mutant phr1-/-was co-incubated with?-1,3-glucan antibody and wheat germ Agglutinin?WGA?,respectively,and then exposed to observe the physical interaction between SH and glucan,chitin.Results:1.Among the methods of extracting biofilm matrix of Candida,VU method had high efficiency,the recovery rate was 58.65%,and cell lysis was less.The ion exchange resin method,especially CER-VU pH 7.8 was obviously better than VU method,and the recovery rate was 90.30%.XTT,plate counting and dry weight measurement showed that the cell viability damage caused by ion exchange resin extraction was negligible and had no significant effect on cell integrity.The yield of three substances extracted from C.albicans membrane matrix showed that:carbohydrate content was the highest in C.albicans EM,protein content followed,and triglyceride content was the lowest;carbohydrate content of Candida glabrata was the highest,triglyceride followed;carbohydrate content of Candida krusei was the same as triglyceride content and protein content was the lowest.The three major substances in C.tropicalis had little difference,and the carbohydrate content was the highest,triglycerides and proteins followed.2.The high efficiency of CER-VU was further confirmed by detecting differentially expressed metabolites by UPLC-Q-TOF-MS using non-target metabonomics.A large number of metabolites?CER-VU/VU=217/198?were identified,including a large number of matched lipids and derivatives?CER-VU/VU=43/45?,carbohydrates and derivatives?CER-VU/VU=20/18?,amino acids and derivatives?CER-VU/VU=54/48?,nucleic acids and derivatives?CER-VU/VU=19/15?.Compared with the VU,the CER-VU showed a high degree of involvement in signaling pathways.3.C.albicans SC5314 and phr1-/-were used to study the antifungal mechanism of SH related to fungal cell wall by drug sensitivity test,enzyme digestion,UPLC detection and fluorescence exposure test.Compared with SC5314?512?g/mL?,the SMIC of SH was decreased 8 times in phr1-/-?64?g/mL?.After enzymatic digestion,only lysozyme could significantly increase the antifungal activity of SH in SC5314?64?g/mL?,but not in phr1-/-.Although the MIC of SH to SC5314 and phr1-/-is not distinct,the MIC of SH to SC5314 was 64?g/mL,and to phr1-/-was 32?g/mL,the SMIC80 of the two strains was very different.Compared with SC5314(SMIC₈₀ was 512?g/mL),phr1-/-biofilm was more sensitive to SH?SMIC80 is 64?g/mL?.When SH was incubated with laminarin,EM of two strains and cell wall for a short time,UPLC showed that the peak area of SH+laminarin decreased 71.6%,the peak area of SH+SC5314 EM decreased38.2%,the peak area of SH+SC5314 cell wall was decreased by 62.6%,the peak area of SH+phr1-/-EM was decreased by 70%,and the peak area of SH+phr1-/-cell wall was decreased by 53.2%at the same concentration.Fluorescence measurements showed that SH induced cell wall remodeling in SC5314 and phr1-/-by exposing?-1,3-glucan and chitin.Conclusion:We introduced a series of commonly used methods to extract EM from static and dynamic C.albicans biofilm,and show that CER-VU was a more effective method to extract EM.Compared with VU method,CER-VU could produce more carbohydrate,protein and lipid;SH might be hindered by?-1,3-glucan in cell wall and matrix,and ultimately through cell wall remodeling?including?-1,3-glucan and chitin exposure?caused fungi to respond to SH attack.Exposing?-1,3-glucan could provide a target for new antifungal agents.This study is helpful to understand the composition and function of biofilm matrix and provide experimental basis for the potential antifungal mechanism of SH.