The Correlation Between Multiple Resistance Gene MDR1 And Candida Albicans Virulence And Drugs Combination Therapy Of Disseminated Infected Mice

Objective:Treatment of drug-resistant Candida albicans has been a clinical concern,It’s a controversial subjects that the overexpression of Candida albicans multidrug resistance gene MDR1 cause virulence change. We used drug-resistant Candida albicans Ca R to establish the disseminated resistance Candida albicans infected mice model. On this basis, mice were inected with genetically engineered strain Ca DEL(MDR1 knockout strains), Ca COM-A(MDR1 knockin strain), Ca COM-B(MDR1 knockin strain) and Ca R to investigate the correlation between Candida albicans multidrug resistance gene MDR1 and its virulence and to investigate the effect of overexpression of MDR1 on virulence. Meanwhile, we carried out the screening of resistance antifungal drug and established system of drug screening in vivo.Methods:Drug-resistant Candida albicans Ca R were injected via the lateral tail vein of immunosuppressive ICR mice to establish the disseminated infected mice model. The infected mice’s survival condition and the average survival time were evaluated. Collect kidney, heart, brain, lung, spleen, and liver to test tissue fungal burden and pathologic diagnosis.Test cytokines IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL12p70, IL-15, IL-17, TNF-α, IFN-γ, GM-CSF in blood and kidney tissue by Cytometric bead array(CBA).On this basis, mice were inected with genetically engineered strain Ca DEL, Ca COM-A, Ca COM-B and Ca R to investigate the effect of high expression of MDR1 on virulence. Test the expression of mycelial type genes AFT2, CEK1, CPH1, CPP1, EFG1, FLO8 and adhesion genes ALS1, ALS5, HGC1, HWP1, INT1, MNT1, PMT1, PMT6, TCC1, TEC1, TPK2, TUP1 from Candida albicans infection in kidney tissue. The drug-resistant Candida albicans disseminated infected mice were treated by fluconazole, proflavine and drug combination by oral to evaluate application of the model.Results:1 Establish the drug-resistant Candida albicans disseminated infected mice model.1.0×105CFU Ca R were injected immunosuppressive ICR mice,which injected with CY.Mice mortality rate is about 90% within 14 days. Clinical manifestation: weak, piloerection, eye secretions, spin, partial a head, death. Anatomy manifestation:kidney enlargement, white spots scattered on the surface, the blutene chloaides scattered on the surface of brain. Tissue fungal burden: All tissue were detected of Candida albicans, kidney tissue infection was the highest, followed by heart, brain, lung, spleen, liver; tissue fungal burden decreased gradually as time goes on. Pathologic diagnosis: HE staining shows that inflammatory cell infiltration in tissue, which fungal granuloma pathological change in kidney tissues; PAS staining show Candida albicans scattere in the tissues. Cytokines detection: Infected mice showed acute nonspecific inflammation. Blood cytokines detection showed IL-6, TNF-α,IFN-γ and IL-1β had significantly increased compared with the control group, the others hadn’t marked change; Kidney cytokines detection showed IL-1α, IL-1β, IL-6, IL-15, IL-17, TNF-α, IFN-γ, GM-CSF had highlight rising trend and IL-4, IL-10 had downtrend compared with the control group.2. Evaluate the effects of overexpression of MDR1 on mice virulenceThe immunosuppressive ICR mice showed the typical disseminated Candida infection, which infected with four kinds of Candida albicans. Infected mice mortality rate is about 90% within 14 days. There was no significant difference among four groups infected mice. The detection results of sixth days infected mice showed that no significant difference in kidney, heart, brain, lung, spleen, liver tissue of fungal burden and pathologic diagnosis among four groups infected mice; Cytokines detection showed the Ca DEL group levels of IL-6, IL-10, TNF-α in blood and IL-1α, TNF-α in kidney rise and the levels of IL-4, IL-5, IL-6, IL-15, IL-17 in kidney reduced compared with the others groups, However these differences didn’t affect the virulence; The detection results of the virulence factor showed related virulence genes expression have significant differences between Ca R and Ca DEL, However, there were no statistical significance among Ca DEL, Ca COM-A, Ca COM-B. Although the wild strain Ca R with high expression of MDR1 was tested, which indicate significant difference in 12 virulence genes between Ca R and Ca DEL, there were no statistical significance among Ca DEL, Ca COM-A, Ca COM-B. Accordingly, the overexpression of MDR1 couldn’t cause the changes of virulence.3. Evaluate application of drugs in the drug-resistant Candida albicans disseminated infected mice model.The drug-resistant Candida albicans disseminated infected mice were treated by proflavine,fluconazole and drug combination to evaluate application of the model. The results showed that it was invalid using proflavine to treat mice with drug-resistant Candida albicans disseminated infection, 8mg/kg fluconazole is effective dose for infected mice, neither 100mg/kg proflavine nor 2mg/kg fluconazole and 16mg/kg proflavine combination hadn’t significant effect.Conclusion:1. We successfully established the drug-resistant Candida albicans disseminated infected mice model, and provided the indexs of animal survival, tissue fungal burden, histopathologic and cytokines.2. The indexs of mice infected with Ca R, Ca DEL, Ca COM-A, Ca COM-B showed that the overexpression of MDR1 couldn’t cause the changes of virulence.3. The does of 8mg/kg fluconazole can effectively improve the drug-resistant Candida albicans disseminated infected mice survival, neither 100mg/kg proflavine nor 2mg/kg fluconazole and 16mg/kg proflavine combination can effectively improve infected mice survival.