Screening Of Resistance Genes Of Fumonazole And Study Of Resistance Mechanism

The frequently implement of the immunosuppressive therapies results in dramatic increase in the incidence of invasive fungal infections（IFIs）with a high mortality rate caused by one of major pathogens-Aspergillus fumigatus.Currently,according to different drug targets,there are three classes of antifungal agents,including azoles,polyenes and echinocandins that are used in the clinical treatment of IFIs.Azoles belong to major guideline-recommended therapy agents for the first-line treatment and prevention of IFIs.However,more and more commonly usages in medicine and agricultural settings have caused the azole-resistant isolates emerged very often in environment,resulting a great threaten to the human’s health.To date,the general molecular mechanisms leading to azole resistance can be divided into following three categories:（ⅰ）alteration or overexpression of the drug target,（ii）activation of the drug efflux pump,（ⅲ）modification of the cellular stress-response pathway.Whereas,our knowledge of the molecular mechanisms underlying azole resistance in A.fumigatus is relatively limited and superficial.Besides,studies involving only cyp51A-based mutations are inadequate.Thus,we using A.fumigatus and itraconazole（ITZ）pressure to isolate resistant strains.Through whole genome resequencing analysis and reversed genetics approaches,we aimed to identification of genes whose mutations lead to drug resistance and increase understanding on drug resistance mechanism.Previous study has indicated that loss of algA,a new putative calcium signaling pathway component in A.fumigatus,resulted in an increased frequency of azole-resistant isolates.In this study,we used an experimental evolution approach starting with AalgA strain to isolate ITZ resistant strains.Among our 105 stable azole-resistant A.fumigatus strains isolated in background of algA deletion strain,only one isolate emerges mutation in cyp51A（F219L mutation）,which is remarkable difference from previous reported clinical azole resistance isolates related to cyp51A mutation.In addition,by using whole genome resequencing analysis combined with reversed genetics approaches,a new mutation at R243Q in cox 10 gene（AFUB₀65450）and an amino acid substitution F219L in cyp51A were found to contribute to high-level resistance to ITZ respectively.HPLC analysis verified that the decreased absorption of ITZ in related cox10 mutants is a mainly reason for the ITZ resistance.Our finding indicates there are existed many unexplored non-cyp51A mutations conferring azole resistance in A.fumigatus.Furthermore,loss of algA results in increased non-cyp51A mutation emergence frequency of azole-resistance isolates.We demonstrated that an amino acid substitution F219L in Cyp51A and an amino acid substitution G243Q in Cox 10 could cause azole resistance respectively.Taken together,our study suggests that genome-wide sequencing combined with alignment comparison analysis is an efficiency approach to identify the contribution of SNP diversity to the drug resistance.