Study On The Expression Regulation Of Ergosterol Demethylase Erg11A And The Susceptibility To Azole Drugs

The filamentous fungi Aspergillus fumigatus(A.fumigatus)is a kind of opportunistic pathogen,which has strong sporulation ability,is transmitted through the air,and is ubiquitous in the environment.It can cause invasive aspergillosis in immunocompromised patients,once infected,the mortality rate is more than 90%.Anti-aspergillosis drugs include azoles(such as itraconazole),echinocandins(such as caspofungin),and polyenes(such as amphoterin B),among which azoles have become the most common drugs for the treatment of aspergillosis due to their wide antifungal spectrum and small side effects.The widespread use of azole drugs leads to an increase in the occurrence frequency of drug-resistant strains.For such drug-resistant strains,how to choose effective treatment drugs has become a difficult problem.Therefore,it is of great significance to study the mechanism of drug resistance in A.fumigatus and develop new antifungal drugs that can improve the efficacy and reduce drug resistance.Azole drugs mainly acts on the lanosterol 14-?-demethylase Erg11A in A.fumigatus,studies have shown that the overexpression of erg11A is directly related to azole resistance,and that changes in erg11A at the transcription level may be related to the regulation of transcription factors in addition to gene mutations.A.fumigatus erg11A is coordinately regulated by the sterol regulatory element binding protein SrbA and CCAAT binding complex(CBC),in which CBC is composed of three subunits Hap B,Hap C,and Hap E.These three subunits are necessary for the formation of CBC in vitro.Knocking out any of them will cause the same phenotypes,such as slow growth,and reduction of conidia.CBC and SrbA competitively bind to the 34-mer region in the erg11A promoter.In the azole-resistant mutant strain,a tandem repeat mutation(TR34)occurred in the 34-mer,effectively replicating the two sterol regulatory elements SRE1 and SRE2 bound by the SrbA homologous,while the CBC binding site CGAAT was partially duplicated with SRE,greatly reducing the affinity with CBC complex,and finally leading to the increased expression of erg11A.In addition to its important role in regulating the biosynthesis of ergosterol,SrbA is also associated with the hypoxic adaptation of aspergillus fumigatus,and we pay particular attention to its mode of action under hypoxic conditions.Normally,SrbA binds to the membrane in an inactive form.Under hypoxic stimulation,SrbA is subjected to Dsc complex-dependent protein hydrolysis and Spp A protein sequentially cleaved,and is activated to enter the nucleus to bind to the sterol regulatory element(SRE)in the promoter of the target gene,and then activate the expression of the target gene.Since the regulation of erg11A expression by SrbA and CBC is so important,are they only regulated by simple competitive binding to the erg11A promoter region?Are there other more complicated ways of regulation?This paper finds that under the conditions of azole drugs treatment,the SrbA protein is similar to that under hypoxia-induced conditions.The full-length protein localized in the endoplasmic reticulum is cleaved into a mature nuclear protein localized in the nucleus to play the role of transcription factor.The direct expression of SrbA mature nucleoprotein confirmed that SrbA acted in the form of mature nucleoprotein,causing high expression of Erg11A,resulting in azole resistance in srb A~T.In addition,this study was the first to find that the deletion of the Hap B subunit of CBC increased the content of the mature nucleoprotein SrbA and prolonged the retention time of GFP-SrbA in the nucleus.It was also the first to find that the deletion of Hap B increased the expression of Spp A and Dsc B,Dsc C,Dsc D,Dsc E in the Dsc protein complex,which may also be the reason why its deletion affected the expression of SrbA.In summary,in previous studies,it was generally believed that CBC and SrbA competitively combined with the erg11A promoter to coordinately regulate erg11A,but our research found that in addition to the principle of placeholders,Hap B deletion also has an important impact on the cleavage and positioning of SrbA protein.The findings of this study provide a basis for further understanding the mechanism of azole resistance in Aspergillus fumigatus.