Aspergillus Fumigatus Drug Stress Response Proteins Dap, McuA And AgcA Participate In The Study Of Azole Drug Tolerance Mechanism

Aspergillus fumigatus is able to cause a wide range of diseases,including allergic syndromes,aspergilloma,and invasive aspergillosis.The frequency of A.fumigatus infections worldwide has increased in recent decades.Recognition of the importance of A.fumigatus infections has led to a dramatic rise in the application of antifungal agents for the treatment and prevention of infection.Unfortunately,A.fumigatus share a close evolutionary relationship with their human hosts,minimizing the number of antifungal drugs with distinct targets for the treatment of fungal infections.Current treatment for invasive aspergillosis(IA)typically involves utilization of the triazole class of antifungal drugs.In addition,resistance to the triazoles has recently been documented and appears to be becoming more prevalent in some areas of the world.The mechanisms contributing to azole resistance are now well characterized and include reducing drug-target interactions either by modifying the target(drug affinity changes or target abundance)or by reducing the effective cellular content of drug(the upregulation of drug transporters).In addition to these mechanisms,A.fumigatus has evolved stress response pathways that enable the cell to cope with the stress induced by antifungal drugs.The immediate response of A.fumigatus to antifungals may be drug tolerance,which can lead to drug resistance.Thus,a prospect for resistance prevention is to abrogate the initial stress response that permits tolerance and the development of resistance.However,little is known about azole stress responses pathway in A.fumigatus.So,this paper will focus specifically on how Dap protein,McuA and AgcA responds to antifungal stress in A.fumigatus,providing a foundation for preventing the development of drug resistance.1 Stress response protein Dap coordinately regulates ergosterol biosynthesis and azole susceptibilityErgosterol 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.In Saccharomyces cerevisiae,Dapl(damage resistance protein 1)positively regulates some reactions catalyzed by cytochrome P450 proteins.Based on a genome-scale homologue search,we find that A.fumigatus genomes contain three Dap proteins.Moreover,expressions of all three genes are induced in an azole concentration-dependent way,which indicate that Dap proteins in A.fumigatus respond to azole stress.We confirm that deletion of dapA or dapC but not dapB causes differing azole susceptibilities by homologous recombination.Next,overexpression and double deletion experiments demonstrate that DapA dominantly contributes to azole resistance,while DapB and DapC negatively mediate this process,suggesting that Dap proteins coordinately affected azole susceptibility in A.fumigatus.Dap family coordinately affected azole susceptibility,which most likely results from abnormal membrane lipid compositions of fungal cells,because ergosterol biosynthesis is a drug target of ITZ and VOR.So,we confirm that DapA is required for the normal function of Erg5 and Erg 11 by LC-MS/MS and genetic rescue.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 △dapA to azoles,which suggest that DapA,B and C coordinately regulates functionality of cytochrome P450 enzymes Erg5 and Erg11 and oppositely affects susceptibility to azoles.In order to further explore how DapA regulate Erg5 and Erg 11 function,we demonstrate that the requirement of DapA for the normal protein expression of Erg5 and Erg11A/B is most likely due to an effect on protein stabilization by Western blots.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 the end,in vivo data demonstrate that inactivated DapA combined with activated DapB yields an A.fumigatus mutant easily treatable with azoles in an immunocompromised mice model of invasive pulmonary aspergillosis.Compared to only one Dap protein included in Saccaromyces cerevisiae and in Schizosaccharomyces pombe,we suggest that complex Dap family regulatory system emerged during evolution of fungi as an adapted means to regulate ergosterol synthesis in response to environmental stimuli.2 Stress response protein McuA and AgcA contributes the mitochondrial Ca2+homeostasis and the azole stress responsesCalcium signaling is an important regulator for responding to diverse environmental stresses.Cells have evolved mechanisms to change the free Ca2+concentration in the cytoplasm([Ca2+]c)to eventually convert original environmental stimuli into cellular responses.In eukaryotic system,the cytosolic free Ca2+homeostasis is strictly and precisely regulated by various calcium-channels,calcium-pumps and calcium-antiporters dwelling in intracellular organelles in response to environmental stimuli.In mammalian cells,mitochondria are essential intracellular organelles for energy production,and play essential roles in buffering cytosolic Ca2+ transiently increases.Mitochondria store large amounts of calcium taken up through a channel called the calcium uniporter(MCU)located at the mitochondria.To date,the mitochondrial Ca2+ uptake system and its physiological implications has not been reported yet in fungi.In this paper,based on the genome-scale homolog search,we found that A.fumigatus genome encodes a homolog of a putative MCU and a mitochondrial carrier protein AGC1/MICU1 homolog.Through GFP-tagged,gene deletion and overexpression experiments showed that both putative homologs are mitochondrial-localized and required for response to azole and oxidative stress such that loss of either McuA or AgcA results in reduced azole and oxidative stress susceptibilities,suggesting the functions involved in the environmental stress adaptation.In order to further explore the genetic interaction between McuA and AgcA in A.fumigatus,we first transformed the full-length open reading frame(ORF)of McuA under the control of the gpdA promoter into the agcA deletion strain,and we found that overexpressed mcuA restores the azole resistance phenotype of the △agcA strain to that of wild-type strain,but not vice versa,indicating McuA plays a dominant role during these stress responses.Through a calcium-sensitive photoprotein aequorin probe guided with a specific mitochondrial targeted sequence,we identified that only deletion of mcuA causes the mitochondrial and cytoplasm Ca2+ homeostasis dysfunction,suggesting that McuA,but not AgcA,contributes to mitochondrial calcium(Ca2+)uptake.Further site-mutated experiments combined with effects of extracellular calcium chelator verified that two predicted Ca2+-binding sites in McuA are required for the mitochondrial calcium uptake and against the stress responses through regulating cytoplasmic Ca2+ homeostasis.