Role Of SAP2 And Its Enzyme Inhibitor Ritonavir In Resistance To Azoles Of Candida Albicans

Objective:1.To explore the effect of Saps protease inhibitor ritonavir(RIT)on the activity of Candida albicans virulence factor Saps.2.To explore the drug sensitivity of Candida albicans in vitro to ritonavir combined with azoles fluconazole(FCA)against Candida albicans in free state and biofilm state.3.To explore the relationship between expression and mutation of the virulence factor SAP2 and target enzyme gene ERG11 for azole drug resistance in Candida albicans.Methods:1.Clinical 50 Candida albicans strains identified in the Fungi Laboratory of in Dermatology and Venereology Diseases for the Second Hospital of Shanxi Medical University were tested the activity of virulence factor Saps in BSA medium containing different concentrations of ritonavir and explore whether ritonavir affects the activity of the virulence factor Saps further affected the virulence of Candida albicans.2.The M27-A4 micro-liquid dilution method recommended by the Clinical Laboratory and Standards Institute(CLSI)was used to perform ritonavir combined with FCA in vitro drug sensitivity test on clinical strains to investigate the free state and biofilm state,whether ritonavir combined with FCA can increase the sensitivity in Candida albicans.3.Genomic DNA of Candida albicans virulence factor SAP2 and target enzyme gene ERG11 was extracted and sequenced to find mutation sites.The mRNA expression levels of both were detected by RT-PCR to explore the virulence factor SAP2 and azole drug target enzymes.Correlation between mutation / high expression of gene ERG11 and drug resistance of Candida albicans.Results:1.Effect of RIT on the activity of Candida albicans virulence factor SAP2: SAP2 activity of Candida albicans at RIT concentration of 2?g / mL(0.697±0.093),8?g / mL(0.800±0.075),and 32?g / mL(0.856±0.08)were all lower than 0?g / mL(control group:0.631±0.083),the difference was statistically significant,F = 74.06,P <0.05.2.In vitro drug sensitivity test results of RIT combined with FCA: In the free state,the median MIC50 of FCA after combined with RIT decreased from 4 ?g / mL to 2 ?g / mL,the difference was not statistically significant,z = 0.7997,P> 0.05,sensitization The rate was 0;under the condition of biofilm,the median MIC50 of FCA decreased from 32?g /mL to 16?g / mL after combined with RIT,the difference was not statistically significant,z= 0.7342,P> 0.05,and the sensitivity enhancement rate was 0.3.SAP2 gene mutation / expression(1)SAP2 sequencing results and standard sequences were not found;(2)SAP2 mRNA relative expression: 1.715 ± 0.576 in the sensitive strains group,5.380 ± 1.39 in the cross-resistance strains group,The FCA resistant strains group was 3.879±1.125,the ITR resistant strains group was 4.385±1.02,and the VRC resistant strains group was 3.534±1.162.The difference was statistically significant,F = 15.23,P <0.001.4.ERG11 gene mutation / expression:(1)ERG11 sequencing results were compared with the standard sequence and found 23 base mutation sites,including 17 synonymous mutations and 6 missense mutations(D116E,T123 I,K128T,Y132 H,V488I,A516P).The missense mutation D116 E was found in the sensitive group and the drug resistance group,and the remaining missense mutations were found in the drug resistance group,of which A516 P was the newly discovered mutation site in the experiment;(2)Relative expression of ERG11 mRNA: 1.019±0.305 in the sensitive strains group,cross tolerance the drug strains group was 5.117±1.372,the FCA-resistant strains group was 4.712±0.785,the ITR-resistant strains group was 4.498±1.092,and the VRC-resistant strains group was4.102 ± 0.611.The difference was statistically significant,F = 32.58,P <0.001;(3)The relative expression level of ERG11 mRNA(4.06±0.65)in the sense mutation group was lower than that in the non-sense mutation group(5.37 ± 1.06),and Statistically,the difference was significant,t = 4.1842,P <0.05;5.Correlation analysis between SAP2 mRNA expression and ERG11: correlation coefficient r = 0.6655,P <0.001.Conclusion:1.RIT can reduce the virulence of Candida albicans by inhibiting the activity of Candida albicans virulence factor Saps..2.In the liquid medium of RPMI 1640 in vitro,RIT may not increase the sensitivity of FCA to Candida albicans under different conditions.3.In clinical isolates of Candida albicans-resistant azoles drug strains,no mutation in the virulence factor SAP2 gene was found,but SAP2 was overexpressed;the target enzyme gene ERG11 was mutated or overexpressed.High expression of virulence factor SAP2,mutation or high expression of target enzyme gene ERG11 may increase drug resistance of azoles,and there is a positive regulatory effect between SAP2 and ERG11.