Construction of af-fadO gene knockout strain of Aspergillus fumigatus to investigate the role of the gene in the synergistic effect of pyrvinium pamoate(PP)and azole against A.fumigatus
Pyrvinium pamoate(PP)is an antiparasitic drug,which has been found to exert synergistic effect with azoles against Aspergillus fumigatus.The putative protein encoded by the af-fadO gene in A.fumigatus is a FAD binding oxidoreductase,which might be involved in the respiration chain in the mitochondria.af-fadO knockout strain was constructed to investigate the role of the gene in antifungal susceptibility,osmotic and oxidative stress adaption of A.fumigatus,and the synergistic effect of PP and azoles.af-fadO knockout strain was constructed via homologous gene replacement method.The effect of af-fadO disruption on the in vitro antifungal susceptibility,oxidative and osmotic stress adaption as well as the effect on the combinational interactions between PP and azoles(posaconazole,itraconazole,voriconazole)in the mutant Δ af-fadO and wild-type(WT)(ku80,pyrG+)strains was observed.The effect of the gene on synergism of PP and azoles was investigated via broth microdilution checkerboard technique and disk diffusion method.In vitro antifungal susceptibility assay revealed that fractional inhibitory concentration index(FICI)value of PP combined with posaconazole against Δ af-fadO was 0.563,indicating no interaction,whereas the FICI against WT was 0.5,indicating synergistic effect.The results of disk diffusion method showed that the inhibitory zone diameter of PP combined with posaconazole against Δ af-fadO was significantly smaller as compared with that against WT.In addition,Δ af-fadO was more sensitive to the oxidants H2O2 and menaphthoquinone,osmotic agents NaCl and D-sorbitol as compared with WT.The disruption of af-fadO gene in A.fumigatus results in mutant Δ af-fadO showing more sensitivity to oxidative and osmotic stress,reversing the synergistic effect of PP and posaconazole.The present study enhances the knowledge of PP and azole antifungal mechanism,and is valuable for future clinical application.
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