Flucytosine Drug-resistant Genetic Screening And Mechanism Studies In Saccharomyces Cerevisiae

Antifungal drug resistance has become an urgent worldwide problem,leading to high mortality and difficult to treat.It was also the main reason for the failure of clinical antifungal drug treatment.Therefore,it is very important for the healthy and harmonious development of the society to control and resolve the occurrence of drug resistance by studying the mechanism of fungal resistance.The clinical fungi are easy to exhibit drug resistance to 5-fluorocytosine(5-FC),however the drug resistance mechanism of 5-FC are very complicated.In this study,we attempted to investigate the new drug resistance mechanism of 5-FC.Firstly,the highly resistant strains were screened and their total protein expression were analyzed by SDS-PAGE.The results showed that the protein contents of these resistant strains were obviously changed compared to the wild type strain.However,these strains were not specific to the resistance to 5-FC.In order to find the specific target genes resistance to 5-FC,an essential gene overexpression pool of S.cerevisiae was used.Next,we found that overexpression of ScGFA1 significantly increased the resistance to 5-FC via spoting test.The gene GFA1 has been known to be involved in the synthesis of chitin in the cell wall.So we measured the contents of chitin after the treatment of 5-FC using the calcofluor white fluorescence(CFW)dye staining and spectrofluorimeter.The results showed that the chitin levels of the yeast overexpressed ScGFA1 was significantly increased,but the chitin content of the yeast treated with 5-FC was significantly reduced.We speculated that the overexpression of Sc GFA1 resulted in resistance to 5-FC may be related to cell chitin synthesis.Then,the chitin synthesis related genes CRH1,GNA1,CHS2,PCM1,QRI1 and CDA1 were cloned and overexpressed in the wild type strain.We found that these strains overexpressed related genes exhibited resistance to 5-FC.It is noted that the chitin synthesis related deletion mutants chs3?,crh1? and chs1? showed more sensitive to 5-FC than the wild type cells.It suggested that the resistance to 5-FC was associated with the chitin synthesis.Additionally,in the salvage pathway of chitin synthesis,glucosamine(Glc-N)exposure induced the resistance of wild type strain of BY4741 to 5-FC,while it inhibited the resistance of the strain overexpressed ScGFA1 to5-FC.In addition,we found that mannose synthetic metabolism also affects the synthesis of chitin.We observed that the deletions of mannoprotein synthesis related genes CWP2,VRG1 and MNT2 increased the content of chitin.The mutants cwp2?,vrg4? and mnt2? overexpressed Sc GFA1 confer to 5-FC resistance.Then,the mannoprotein synthesis related genes CWP2,VRG4 and MNT2 were cloned and overexpressed in the wild type strain.We found that these strains overexpressed related genes exhibited resistance to 5-FC.Furthermore,the contents of sugar nucleotide UDP-GLC was measured via using the high performance liquid chromatography(HPLC),and found that the content of UDP-GLC in the yeast was obviously increased after the treatment of 5-FC.Therefore,it suggested that 5-FC may lead to the reduction of chitin in the cell wall via inhibiting the sugar nucleotides decomposition,and that the overexpression of ScGFA1 can rescue the decrease of chitin caused by 5-FC.Finally,Candida albicans GFA1-CaGFA1 was heterologous expressed in S.Cerevisiae.We found that the strain is also resistant to 5-FC.The chitin content in Candida albicans was obviously decreased after the treatment of 5-FC.In summary,CaGFA1 exhibited similar resistance to 5-FC as GFA1,suggesting that there is a conserved mechanism of 5-FC resistance across species.It is expected that the results lay the foundation for further research on chitin synthetic metabolism mediating resistance mechanism of 5-FC.