Combination Of Plagiochin E And Azoles Against Resistant Candida Albicans Isolates And Its Mechanisms

In recent two decades,the morbility of deep fungi infections is continuously increasing.Candida albicans remained one of the most common isolates among variety of pathogenic fungi,and led to a high mortality of the systematic infections, especially in cancer and HIV infected patients.Fluconazole was chosed and administrated extensively in clinic,and more and more Candida albicans strains resistant to it by the widespread and long-term use.Moreover,fluconazole resistant strains usually generate cross-resistant to other azole antifungal agents,such as itraconazole,ketoconazole,etc.A great deal of efforts has been undertaken for a practical means to treat fungi infections.There are many advantages of drugs combinations,including wider antimicrobial spectrum,increase of antifungal activity, less incidence of resistance,better safety and tolerance.Drugs combinations generally limited in administration together of antifungal drugs in reports,and not always synergistic effects were produced.Furthermore,rare literatures were reported about treatment of fluoconazole resistant C.albicans by drugs combinations.Plagiochin E(PLE),a bisbibenzyl compound,which was extracted from Marchantia polymorpha.L,showed potential antifungal effects in the primary screen assay.In previous researchs,the synergistic activity of PLE combined with fluconazole against fluconazole resistant strains has been observed,with reduced drug consumption and increased intracellular accumulation of fluconazole.PLE at large dose(32μg/ml)can inhibit the expression of CDR1 gene in resistant Candida albicans isolates.On the basis of these results,antifungal activity of PLE combined with fluoconazole,ketoconazole,itraconazole and voriconazole were studied in this thesis,consequently elucidated the molecular mechanism of reversal resistance of PLE.In vitro susceptibility testing of PLE,fluoconazole,ketoconazole,itraconazole and voriconazole against resistant Candida albicans was carried out by NCCLS M-27A documents,and the results showed that three clinical isolated resistant Candida albicans strains to fluconazole are resistant to other azoles,too.Checkerboard micro-dilution method was applied to determine the combined activity of PLE and ketoconazole,itraconazole,voriconazole,all results suggested that synergistic effects of them with FICI≤0.5.Time-killing assay confirmed the combined synergism of PLE with ketoconazole,itraconazole and voriconazole against CA10,with a decrement of≥2 lgCFU/ml,compared to the related azoles alone;We also found that these synergisms of PLE and other azoles are in a PLE dose-dependent manner. The effect of PLE and fluconazole combination against resistant Candida albicans was significantly higher than fluconazole alone(P＜0.01).The molecular mechanism of PLE on reversal of resistance to azoles is studied. Firstly,the effiux of Rhodamine 6G by ABC transporter in Candida albicans membrane was observed.Rhodamine 6G is a specific substrate of ABC transporter proteins,which can be used for determining the transport ability of protein.Results showed that the effiux of Rhodamine 6G was significantly inhibited by various concentrations of PLE in a dose-dependent manner.At the same time,we found the effiux of Rhodamine 6G was inhibited by azoles,too.This indicated that they shared the same binding sites as R6G to ABC transporter.By detecting the gene expression level of CDR1 and CDR2 of collecting cells by RT-PCR method,we found the mRNA level of CDR1 and CDR2 was not inhibited.These results suggested that the inhibition of PLE on the effiux of Rhodamine 6G was energy related,not dependent on the expression of CDR gene.Secondly,a detection of Pi concentration was carried out to evaluate the effect of PLE on ATPase activity of plasma membranes in resistant Candida albicans isolate CA10.Results showed an obvious inhibition of ATPase activity by 81μg/ml,16μg/ml and 32μg/ml for PLE.HPLC method was established to detect the amount of ATP in culture medium.The ATP efflux was inhibited by PLE at concentration of 16μg/ml. This result suggested that PLE may not be transported by ABC transporters.In R6G effiux experiment,the expression of CDR1 and CDR2 was not inhibited by PLE after incubation with resistant Candida albicans for 100 min.However,we observed that the expression of CDR1 gene was inhibited by PLE at concentration of 128μg/ml over night in previous study.Therefore,we do further study on the mRNA level of CDR1 and CDR2 gene in resistant Candida albicans by incubation with PLE at the concentration of 16μg/ml for 2h,6h and 18h.Results showed that the expression of CDR1 and CDR2 gene in Candida albicans were reduced significantly after incubation with PLE for 6h,18h,while there was no inhibition for 2h.This indicated that PLE could inhibit the mRNA level of CDR1 and CDR2 after incubation for 2 to 6h.In summary,the synergistic activity of PLE combined with fluconazole, ketoconazole,itraconazole and voriconazole against fluconazole resistant strains was confirmed in a dose-dependent manner.The mechanism of this synergism of PLE is related closely to the inhibition of ATPase activity of ABC transporters and the reduction of the mRNA level of CDR1 and CDR2 gene in resistant Candida albicans.