Screening, Identification And Related Mechanism Research Of Non-drug Target Cyp51A Mutant Resistant Strains Of Aspergillus Fumigatus

The opportunistic fungal pathogen Aspergillus fumigatus,which produces a large number of air borne spores and survives under a wide range of environmental conditions,causes aspergillosis in immunocompromised individuals.Millions of people with weakened immune systems are at a high（30%to 95%）mortality rate due to the limited specific diagnoses and treatment options,with an increasing threat of fungal infection arising from the more frequent use of invasive surgeries,chemotherapy for the treatment of cancer,and organ transplantation.Currently,triazole antifungals are recommended as the first line agent for the primary treatment of invasive fungal infections（IFIs）by Infectious Diseases Society of America guideline.However,drug resistance in fungal pathogens has risen steadily over the last decades due to long-term azole therapy or triazole usage in agriculture.Previous studies identified there were three major azole resistance mechanisms in A.fumigatus:changes in the drug target Cyp51,activation of drug efflux pumps and induction of cellular stress responses.Among them,the modification of the drug target protein to prevent drug binding is a major recognized route to induce drug resistance.Moreover,cyp51A-related azole-resistant strains of A.fumigatus have been reported frequently in the last decade.However,there are a growing number of A.fumigatus isolates with non-cyp51A mutations which resistant mechanisms remain only loosely defined.In our previous work,104 non-cyp51A mutation isolates conferring azole resistance are obtained by mimicking a long-term azole exposure environment.Among them,a new mutation conferring azole resistance related to active domain of yeast homolog farnesyltransferase Cox10 is found through screening in azole resistant isolates.In this thesis,the biological characteristics of Cox10 and the relationship between mitochondrial function and drug susceptibility were further explored in A.fumigatus.The relationship between 34 lab-isolated strains with mitochondrial defect or clinical isolates and drug resistance was roundly analyzed.Moreover,this thesis illuminates that the fungal pathogen is adapt to drug environment by regulating the mitochondrial function.Taken together,the conclusions are summarized as follows:1.The enzyme activity of farnesyltransferase Cox10 is required for radial growth and virulence in A.fumigatus,and is also involved in drug susceptibility.The conserved site mutations of Cox10 E230A,D234A,R243Q and loss of Cox10 all results in the decreased growth and virulence,but increased drug resistance.2.The loss of Cox10 causes the defect of mitochondrial Heme A biosynthesis,which influences the usage of nonfermentable carbon source and the decreased mitochondrial membrane potential,suggesting that the defect of Cox10 results in mitochondrial defect.3.The loss function of Cox11,Cox15,Cox17 and Cox19 involved in the cofactor biosynthesis in core subunit Cox1 of cytochrome c oxidase all causes the similar phenotype of drug susceptibility with Cox10 deficiency.4.The enhanced activity of multidrug transports in cox10 mutants due to the overexpression of multidrug transport genes causes the reduced intercellular drug contents.5.The upregulation of chitin synthase encoding genes in cox10 mutants leads to the change of chitin contents and the increased thickness of cell wall,which influences the import of drug.6.The loss of Cox10 results in reduced mitochondrial Ca²⁺transients while enhanced cytosolic Ca²⁺transients which cause persistent nuclear localization of transcription factor CrzA by dephosphorylation possibly.7.CrzA can bind the promoters of multidrug transport gens mdr1,atr B and abc E,and is required for the overexpression of major multidrug transport genes and most chitin synthase genes in cox10 mutants.Moreover,the down-regulated crz A in cox10 mutants results in the increased cytosolic drug contents.8.The multidrug transport gene atrF is up-regulated in cox10 mutants,but the promoter region of atrF can not be binded by CrzA,suggesting that atrF is not directly regulated by CrzA.9.The transcription factor ZfpA is the downstream target of CrzA and nuclear localization,which influences the drug susceptibility and the cell wall construction.Moreover,ZfpA is involved in the drug susceptibility by regulating a series of multidrug transport genes,and is the negative control factor of multidrug transport gene atrF.10.The calcium chelator prompts the cytoplasmic localization of CrzA in cox10mutants and coincidentally reduces the azole resistance of cox10-related mutants.Among the 34 mitochondrial defective isolates in 89 lab-induced isolates,calcium chelator reduces MIC values in 14 independent isolates and significantly increases the azole efficacy in these A.fumigatus clinic isolates.In conclusion,this work illuminates that drug resistant mutations are an efficient adaptation strategy of fungal pathogens under long-term drug treatment conditions.Resistant mutations can normal growth and sporulation under drug environment,but the resistant isolates have a lower fitness than wild-type isolates in the absence of the antifungal drug.The mitochondrial dysfunction as a fitness cost can trigger calcium signalling and therefore globally upregulates a series of embedding calcineurin-dependent-response-element genes,leading to antifungal resistance.These findings illuminate the drug resistant mechanism resulted from the mitochondrial defect in non-cyp51A mutation isolates,suggesting the synergistic combinations of antifungals and calcium chelator might be a compromising strategy.