The Mechanism Of Synergistic Regulation Of Candida Albicans Transcription Factors Efg1 And Cas5 In Response To Caspofungin

Candida albicans is a major opportunistic human pathogenic fungus which can cause superficial infections and life-threatening systemic infections.It accounted for more than 40%of Candida species infections and caused about 200,000 deaths and serious harm to millions of people per year.Echinocandins have a very favorable safety profile and are recommended as the first-line drugs for the treatment of systemic candidiasis.This drug inhibits the synthesis ofβ-1,3-glucan by non-competitively bindingβ-1,3-glucan synthetase catalytic subunit.In C.albicans,mutations of theβ-1,3-glucan synthase encoding gene FKS1 could lead to the resistance to echinocandins and the most frequent mutation occurred at the S645 amino acid site.Cas5 is an important transcription factor involved in the response to cell wall damage in C.albicans and has a profound impact on global transcriptional responses to cell wall stress caused by caspofungin,but Cas5-independent caspofungin-responsive signaling pathways may also exist.Through screening of C.albicans transcription factor deletion mutants of TRKO library,we found that in addition to the cas5 mutant,deletion of EFG1 which functions as a key central regulator involved in diverse biological processes including hyphal growth,white-opaque phenotypic switch,biofilm formation and so on could also confers hypersensitivity to caspofungin.Efg1 protein level remained largely unchanged regardless of caspofungin or Cas5,although Cas5 protein level increased upon caspofungin induction in an Efg1-dependent manner.However,overexpressing CAS5could not bypass the requirement of Efg1 in caspofungin tolerance indicating that Efg1regulates caspofungin response at least partially independently of Cas5.Based on the role of Efg1 and Cas5 in regulating caspofungin sensitivity,we next created cas5 efg1 double mutant and found that deletion of both EFG1 and CAS5 could exacerbate cell wall stress and lead to increased caspofungin susceptibility.In addition,deleting both EFG1 and CAS5 could also enhance susceptibility to fluconazole.Importantly,single deletion of Efg1 or Cas5 could only partially reduce the resistance of FKS1^S645Fresistant strain to caspofungin,however,only when EFG1 and CAS5 were both deleted could resensitize echinocandin-resistant strains to caspofungin.Thus,these results further confirmed that Efg1 and Cas5 could independently function in regulating caspofungin sensitivity and also provided a novel therapeutic strategy in fungal disease caused by echinocandin-resistant C.albicans.Further transcriptome data showed that the up-regulated genes in wild type cells were mainly associated with cell wall organization or biogenesis,transport and localization,while the down-regulated genes were mainly associated with biosynthesis and metabolic processes of organic acids in response to capofungin.The numbers of caspofungin-responsive genes regulated by Efg1 and Cas5 were 278 and 424respectively.The heatmap and Venn analysis showed that Efg1 and Cas5 could co-regulate the expression of a set of caspofungin-responsive genes but also independently control the induction of some genes.Among those caspofungin-responsive genes that were regulated by Efg1 or Cas5,only 24%with differences in expression were common to both regulators,most of which were involved in cell wall organization or biogenesis.The genes activated only by Cas5 upon caspofungin addition were mainly associated with cell wall organization and organic acid metabolism.Efg1 but not Cas5 regulated members of the SAP gene family and affected chromatin organization,DNA conformation change and iron ion transport processes.Therefore,Efg1 and Cas5 regulate expression of caspofungin-responsive genes in a coordinated and complementary manner.Finally,the accuracy of transcriptome data was verified by q RT-PCR.The yeast two-hybrid and immunoprecipitation assay showed that Efg1 could interact with Cas5 in the presence or absence of caspofungin.Chromatin immunoprecipitation showed Efg1 and Cas5 bind to promoters of caspofungin responsive genes ECM331 and ALS1 to coordinately activate their expression,further supporting the synergistic relationship between Efg1 and Cas5 in regulating the expression of downstream genes.Moreover,Efg1 and Cas5 exhibited different regulatory functions on the expression of different downstream genes.Overall,our study suggests that the C.albicans transcription factor Efg1,together with Cas5synergistically regulates the transcriptional response to cell wall stress induced by caspofungin,deletion of Efg1 and Cas5 could significantly affect the expression of downstream genes,thus leading to increased caspofungin susceptibility and even resensitizing echinocandin-resistant strains to caspofungin.