The Biological Function Of Aspergillus Fumigatus AfPefA And Its Relationship With Itraconazole Resistance

PEF proteins family is a class of proteins,which have five consecutive EF-hand motifs.PEF protein binds with calcium ions through its Penta EF hand domain and participates in a variety of calcium dependent processes in cell.Human ALG-2 protein is a much conserved PEF protein,its orthologs exist in various of invertebrates,lower plants,animals and fungi.Previous studies verified that ALG-2 is required for the normal cell division and involved in the regulation of endoplasmic reticulum stress-induced apoptosis in mammalian cell.In addition,Peflp,as an ortholog of ALG-2 in yeast,was indicated involving in the controlling for the site selection of bud emergence,and the Peflp deletion strain showed more sensitive to calcium chelating agent EGTA.than that of wild-type strain.Aspergillus fumigatus is widespread existed in the natural environment,can cause immunocompromised patients with invasive aspergillosis.In clinic,A.fumigatus is a second most important pathogenic fungus followed Candida albicans.Based on bioinformatic analysis,we found the ortholog of human ALG-2 in A.fumigatus is PefA,it shows highly conservation with human ALG-2(39%identity),and Saccharomyces cerevisiae Peflp(28%identity).Bioinformatic analysis indicates PefA also includes some of predicted Ca2+-binding motifs,Penta-EF-hand domain.However,whether PefA acts as a Ca2+ sensor that modulate the function of downstream proteins is still unknown yet.In this study,we explored the physiological function of the PEF protein PefA and its relationship with drug resistance to azoles in A.fumigatus.Here,we used gene-targeting techniques to construct the PefA deletion strain Grs01,PefA complemented strain Grs02 and the PefA C-terminal GFP-labeling strain Grs03.We found that loss of PefA lead to A.fumigtus’s producing more resistance to itraconazole(ITZ)than that in wild-type strain though observing the colony phenotype of PefA deletion strain and PefA complemented strain on medium amended with ITZ.In addition,the ergosterol content which is azole drug target were detected by HPLC and the drug efflux pump activity assay was examined using azole mimic reagent-Rh 123 in PefA deletion and wildtype strains.As a result,we didn’t find any difference between them,indicating the azole drugs resistance in PefA deletion strain is not related to the change of ergosterol content or the drug efflux pump activity.Moreover,the PefA-c-terminal GFP labeled strain was observed under fluorescence microscope.We found PefA evenly distributs in the cytoplasm,rather than in nuclei.Interestingly,Colony-forming unit(CFU)assay shows that PefA deletion strain has higher survival rate than that of wild-type under the high concentration of ITZ.The survived PefA deletion strain has cross-resistance to voriconazole(VRC)and miconazole(MCZ),indicating its drug resistance is genetically stable.This is the first report to systematically and comprehensively clarify and reveal the function of PefA in Aspergillus fumigatus,and its relationship with multi-azole resistance.This study can provide some theoretical basis to explain the emergence of azole-resistant strains in nature and in clinic.