New Antifungal Traditional Chinese Medicine Screened By Chemical-Genetics Method And Studies On Its Possible Mechanism Of Action

Clinical infections caused by fungi are increasing.Antifungal resistance in Candida albicans is one of the reasons for treatment failure.Looking for effective antifungal agents is meaningful.The expression peaks of gene ORF19.1985,ORF19.2328,ORF19.3296,ORF19.6219,and ORF19.5322 are all in G1 or G1/S phase of cell cycle;The homologous genes of ORF19.4136,ORF19.4105,ORF19.658,ORF19.2174,ORF19.2440,ORF19.7494 in Saccharomyces cerevisiae are involved in DNA repair.However,the function of these genes in C.albicans is not clear.In this study,homologous recombination knockout cassettes containing the upstream and downstream fragments of target genes were used to transform C.albicans SN152 strain by lithium acetate method,the target genes were replaced by knockout cassettes.All the gene deletion strains were verified by genomic PCR.Following gene deletions in C.albicans SN152 were constructed,including c 19.19S5K2(△orf19.1985/△orfl9.1985),c19.2328K2(△orf19.2328/△orf19.2328),c 19.3296K2(△orfl 9.3296/△orf19.3296),c19.6219K2(△orf19.6219/△orf19.6219),c19.5322K2(△orf19.5322/△orf19.5322),c19.4136K2(△orfl9.4136/△orf19.4136),c 19.4105K2(△orf19.4105/△orf19.4105),c19.658K2(△orf19.658/△orf19.658),c19.2174K2(△orf19.2174/△orf19.2174),c19.2440K2(△orf19.2440/△orf19.2440)and c 19.7494K2/c19.4105K2(△orfl9.19.7494/△orf19.19.7494 △orf19.4105/△orf19.4105),c19.7494K2/c19.658K2(△orf19.19.7494/△orfl9.19.7494 △orfl9.658/△△orf19.658),c 19.7494K2/c19.2174K2(△orf19.19.7494/△orf19.19.7494 △orfl9.2174/△orf19.2174),c 19.7494K2/c19.2440K2(△orfl9.19.7494/△orf19.19.7494 △orf19.2440/△orf19.2440).The biological functions of above gene deletion strains were also studied,which includes the ability of hyphae formation of these strains in different culture conditions;the susceptibility of gene deletion strains to azole drugs,salts,osmotic,hydrogen peroxide and DNA inhibitors by micro-liquid dilution method and point plate method;the growth curve and doubling time of these gene deletion strains;the changes of cell membrane and nuclear morphology which is tested by fluorescence microscopy.The results showed that the deletion strains ofORF19.4136,ORF19.4105,ORF19.658,ORF19.2174,ORF19.2440 and ORF19.7494 were more sensitive to DNA inhibitors than parental strain;the cells became much longer;growth rate slowed down;doubling time were prolonged;the morphology of single colony of ORF19.4105 and ORF19.658 deletion strains had changed.These results suggested that the above genes played important roles in the DNA damage repair process.But the specific mechanism among them is not clear and will be further identified.On the other hand,we did not find any difference between each deletion strain of ORF19.1985,ORF19.2328,ORF19.3296,ORF19.6219,ORF19.5322 and parental strain.This study provides a new manner for looking for new antifungal agents and exploring the resistance mechanism of C.albicans.Then we investigated the susceptibility of eleven Chinese medicine monomer and ten Chinese herb extracts in C.albicans SN152,the above gene deletion strains and several double gene deletion strains by testing the MIC80,in vitro susceptibility testing and checkerboard micro-liquid dilution method.The results showed:all of the target genes do not affect the susceptibility of C.albicans on the above traditional Chinese medicine monomers and Chinese herb extracts,the MIC80 of the same Chinese medicine monomer or Chinese medicine extract in C.albicans SN152(parental strain)and gene deletion strains is consistant;the combined application of Cortex Moutan with fluconazole against clinical isolates of C.albicans had synergy function(FICI<0.1).And we also found that the two clinical isolates of C.albicans 100 and 255 had the same susceptibility on fluconazole,but on Cortex Moutan vary considerably.It suggested that Cortex Moutan might have different target gene C.albicans.