Biological Functions Of C₂H₂ Transcription Factor SltA And Molecular Chaperone CcsA In Aspergillus Fumigatus

Aspergillus fumigatus is a widespread opportunistic pathogen that causes invasive aspergillosis（IA）in immunocompromised patients.Azole drugs are the primary therapeutic agents for the first-line treatment of IA.Notably,with the continuous application of azole antifungal drugs in medicine and agriculture,azole resistance of clinical A.fumigatus isolates has rapidly emerged over the past two decades,which poses a great challenge to the treatment of IA.Therefore,it is important to study the molecular regulatory mechanisms of azole resistance.The mechanisms of drug resistance in A.fumigatus can be classified into three categories:1)drug target Erg11 mutation reducing its affinity with the drug and overexpression of Erg11;2)overexpression of drug pumps decreasing intracellular drug retention;3)drug resistance caused by activation of stress response pathways.Thus,it is very important to understand the drug resistance mechanism of A.fumigatus by studying the expression regulation of drug targets and drug pumps.In addition,the ability to resist oxidative stress is also crucial for the growth and development of A.fumigatus and its adaptation to the host environment.We carried out our research from drug and oxidative stress response pathways.Transcriptome analysis and bioinformatics comparison were used to screen the transcription factor SltA（salt tolerance）involved in azole resistance,and the SOD chaperone protein CcsA（copper chaperone for Sod1）involved in oxidative stress response.Their biological functions were analyzed:Transcriptome analysis were used to identify 16 transcription factors that could respond to the stimulation of azole drugs at the m RNA level.Among them,only deletion of AFUB＿041100 showed hypersensitivity to azole drugs,indicating that this gene is necessary for A.fumigatus to respond to azole drugs.Bioinformatics analysis showed that AFUB＿041100 encodes an SltA protein,which is a C₂H₂transcription factor regulating cytoplasmic calcium balance.In A.fumigatus,we also demonstrated that SltA maintains cytoplasmic calcium balance by negatively regulating vesicular calcium pumps.In addition,lack of SltA is hypersensitive to azole drugs while overexpression of sltA is resistant to azole drugs,suggesting that SltA is a positive regulator involved in azole resistance.Point mutation and truncation experiments showed that the DNA-binding domain and C-terminus of SltA are necessary for SltA-mediated azole resistance.Transcriptional profiling combined with Lac Z reporter analysis and electrophoretic mobility shift assays（EMSA）demonstrated that SltA could directly bind to the conserved AGGCA sequence in the promoter region and regulate the expression of the target enzyme Cyp51A（Erg11A）and the drug efflux pump protein Mdr1.Overexpression of erg11A or mdr1 could rescue the sltA deletion defects under itraconazole conditions,suggesting that SltA contributes to azole resistance by coregulating the expression of the drug target Erg11A（Cyp51A）and the drug efflux pump Mdr1.Importantly,deleting sltA in drug resistant mutants significantly attenuates the degree of azole resistance by downregulating the expression levels of erg11A and mdr1.In addition,SltA is also necessary for A.fumigatus virulence in Galleria mellonella model.On the other hand,superoxide dismutases（SODs）,including Cu/Zn-SOD:Sod1and Mn-SOD:Sod2 and Sod3,play an important role in the resistance to oxidative stress.Although SOD’s functions are well understood,their molecular chaperones are poorly characterized in A.fumigatus.Through bioinformatics analysis,we found that CcsA is a Sod1 molecular chaperone protein.Δccs A mutant exhibited the reduced conidiation and hyphal growth compared to the parental wild-type strain on minimal medium,suggesting that CcsA is required for the vegetative growth and conidiation of A.fumigatus under limited nutritional conditions.Moreover,ccs A null mutant showed increased ROS accumulation and enhanced sensitivity to oxidative stress due to loss of Sod1 activity,indicating that CcsA is involved in resistance to oxidative stress possibly by maintaining Sod1 activity.Point mutation and coimmunoprecipitation assay（Co-IP）indicated that the CXC motif at the C-terminal of CcsA is necessary for the interaction between CcsA and Sod1 and Sod1-mediated oxidative stress response.Collectively,our findings demonstrate that CcsA functions as a Sod1 chaperone that participates in the adaption to oxidative stress in A.fumigatus.Notably,overexpression of sod3 could rescue the defects of ccs A or sod1 deletion mutant,implying that Mn-SOD（Sod3）could replace the enzyme activity of Cu/Zn-SOD（Sod1）and reduce the reactive oxygen species（ROS）accumulation in A.fumigatus.In conclusion,we demonstrated that the C₂H₂transcription factor SltA in A.fumigatus is involved in maintaining the cytoplasmic calcium balance,which is consistent with the results in the model fungi A.nidulans.It is more important that SltA is also involved in azole resistance by coregulating the expression of the drug target Erg11A and the drug pump Mdr1,suggesting that the resistance of drug-resistant strains can be reduced by targeting of SltA.In addition,SltA can serve as a potential drug target for pathogenic fungi in that SltA is a fungal protein which is absent in mammals and necessary for the virulence of A.fumigatus.On the other hand,the SOD molecular chaperone CcsA is involved in oxidative stress response via interactions with Sod1 to maintain the activity of Sod1.This study further broadened the understanding of drug stress response proteins and oxidative stress response proteins in A.fumigatus,and provided a theoretical basis for the treatment of IA caused by azole drug-resistant strains and how A.fumigatus responds to oxidative stress in the hosts.