title: The Aspergillus fumigatus Damage Resistance Protein Family Coordinately Regulates Ergosterol Biosynthesis and Azole Susceptibility.
abstract: Ergosterol is a major and specific component of the fungal plasma membrane, and thus, the cytochrome P450 enzymes (Erg proteins) that catalyze ergosterol synthesis have been selected as valuable targets of azole antifungals. However, the opportunistic pathogen Aspergillus fumigatus has developed worldwide resistance to azoles largely through mutations in the cytochrome P450 enzyme Cyp51 (Erg11). In this study, we demonstrate that a cytochrome b5-like heme-binding damage resistance protein (Dap) family, comprised of DapA, DapB, and DapC, coordinately regulates the functionality of cytochrome P450 enzymes Erg5 and Erg11 and oppositely affects susceptibility to azoles. The expression of all three genes is induced in an azole concentration-dependent way, and the decreased susceptibility to azoles requires DapA stabilization of cytochrome P450 protein activity. In contrast, overexpression of DapB and DapC causes dysfunction of Erg5 and Erg11, resulting in abnormal accumulation of sterol intermediates and further accentuating the sensitivity of ΔdapA strains to azoles. The results of exogenous-hemin rescue and heme-binding-site mutagenesis experiments demonstrate that the heme binding of DapA contributes the decreased azole susceptibility, while DapB and -C are capable of reducing the activities of Erg5 and Erg11 through depletion of heme. In vivo data demonstrate that inactivated DapA combined with activated DapB yields an A. fumigatus mutant that is easily treatable with azoles in an immunocompromised mouse model of invasive pulmonary aspergillosis. Compared to the single Dap proteins found in Saccharomyces cerevisiae and Schizosaccharomyces pombe, we suggest that this complex Dap family regulatory system emerged during the evolution of fungi as an adaptive means to regulate ergosterol synthesis in response to environmental stimuli.
  
  
  OBJECTIVE
  Knowledge of the ergosterol biosynthesis route in fungal pathogens is useful in the design of new antifungal drugs and could aid in the study of antifungal-drug resistance mechanisms. In this study, we demonstrate that three cytochrome b5-like Dap proteins coordinately regulate the azole resistance and ergosterol biosynthesis catalyzed by cytochrome P450 proteins. Our new insights into the Dap regulatory system in fungal pathogens may have broad therapeutic ramifications beyond their usefulness for classic azole antifungals. Moreover, our elucidation of the molecular mechanism of Dap regulation of cytochrome P450 protein functionality through heme-binding activity may extend beyond the Kingdom Fungi with applicability toward Dap protein regulation of mammalian sterol synthesis.
authors.affiliation: Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Microbiology, College of Life Sciences, Nanjing Normal University, Nanjing, China.
authors.forename: Jinxing
authors.initials: J
authors.lastname: Song
authors.affiliation: Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Microbiology, College of Life Sciences, Nanjing Normal University, Nanjing, China.
authors.forename: Pengfei
authors.initials: P
authors.lastname: Zhai
authors.affiliation: Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Microbiology, College of Life Sciences, Nanjing Normal University, Nanjing, China.
authors.forename: Yuanwei
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Dermatology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, China.
authors.forename: Caiyun
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Department of Dermatology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Sang
authors.affiliation: Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Microbiology, College of Life Sciences, Nanjing Normal University, Nanjing, China.
authors.forename: Guanzhu
authors.initials: G
authors.lastname: Han
authors.affiliation: Department of Bacteriology and Department of Medical Microbiology and Immunology, UW-Madison, Madison, Wisconsin, USA.
authors.forename: Nancy P
authors.initials: NP
authors.lastname: Keller
authors.affiliation: Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Microbiology, College of Life Sciences, Nanjing Normal University, Nanjing, China linglu@njnu.edu.cn.
authors.forename: Ling
authors.initials: L
authors.lastname: Lu
mesh_terms: D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology*; D001232:Aspergillus fumigatus / Q000187:drug effects / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D001393:Azoles / Q000494:pharmacology*; D002352:Carrier Proteins / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D004271:DNA, Fungal / Q000378:metabolism; D004195:Disease Models, Animal; D025141:Drug Resistance, Fungal / Q000235:genetics; D004875:Ergosterol / Q000096:biosynthesis*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D006418:Heme / Q000378:metabolism; D000081062:Heme-Binding Proteins; D006420:Hemeproteins / Q000378:metabolism; D016867:Immunocompromised Host; D051379:Mice; D009154:Mutation; D055732:Pulmonary Aspergillosis / Q000382:microbiology
chemical_list: D000935:Antifungal Agents; D001393:Azoles; D002352:Carrier Proteins; C000631028:Cyb5 protein, rat; D004271:DNA, Fungal; D005656:Fungal Proteins; D000081062:Heme-Binding Proteins; D006420:Hemeproteins; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D004875:Ergosterol