Posttranscriptional Modification Of Iron Repressor Hap43 Defines A Novel Detoxification Role In Candida Albicans Gastrointestinal Commensalism

Candida albicans is a major opportunistic fungal pathogen of humans.It is capable of causing mucosal infections and systemic lifethreatening infections in immunocompromised individuals.However,it exists as a commensal in the oral cavity,gastrointestinal(GI)tract,and urogenital tract of most healthy individuals.Data showed that C.albicans infection accounts for 81.95% of all fungal infections,and despite the treatment with antifungal drugs,the mortality rate of candidiasis is still as high as 40%-80%.In the commensalism of C.albicans and host,it is critical to obtain iron and avoid immune oxidative burst.Iron is essential for life as it is required for the functionality of energy generation,oxygen transport DNA synthesis and many cellular processes.Because that iron acts as a cofactor within the active site of key enzymes and the mitochondrial electron transport chain subunits.However,excess iron is toxic because ferrous ions promote the Fenton reaction which produces highly toxic reactive oxygen species(ROS).Under endogenous or exogenous stimuli,a rapid increase in ROS can eliminate pathogens by increasing cellular oxidative stress,leading to damage to DNA,proteins or lipids,leading to apoptosis or necrotic cell death.In this study,we focused on the post-transcriptional modification of the ironregulated transcription factor Hap43 under high iron conditions and revealed its detoxification mechanism for high-ROS environment.The microarray test of Sfu1,Sef1 and Hap43 has showed that the number of genes regulated by Hap43 which located in the downstream of the Sfu1-Sef1-Hap43 regulatory circut was significantly more than Sfu1 that located in the upstream of the circut,suggesting that Hap43 may be regulated by post-transcriptional level.We found that the mobility of the Hap43-Myc protein expressed under high-iron conditions was decreased and the protein migration disappeared after treatment with the lambda phosphotase,indicating that Hap43 was phosphorylated under iron-replete conditions.The migration of Hap43 disappeared in the kinase Ssn3 knockout strain compared to wild-type strain.And then we determined that Ssn3 is the kinase of Hap43 by co-immunoprecipitation(Co-IP).Using immunofluorescence microscopy,we also found that the Hap43 in wild-type strains is mainly localized in the cytoplasm under high-iron conditions and localized in the nucleus inder iron-deplete conditions.However,deletion of SSN3 resulted in partial nuclear localization of Hap43-Myc under iron-replete conditions.Considering the Hap43-Myc in wild-type but not in ssn3Δ/Δ showed decrease protein abundance under iron-replete conditions,degradation process of Hap43 protein level was test by the inducible promoter tet O.The immunoblot indicated that Ssn3 negatively regulated the steady state levels of phosphorylated Hap43.Then it was confirmed that phosphorylation Hap43 was degraded by a protease dependent manner by adding the proteasome inhibitor MG132 and co-immunoprecipitation experiments with ubiquitin.In order to study the biological significance of the post-transcriptional modification of Hap43 under iron-replete conditions,we constructed Hap43 phosphorylation sites mutanted strains and found it had growth defects under H2O2 stress conditions.Further studies revealed that Hap43 phosphorylation sites mutanted strains transferred to the nucleus and binded to the promoter of catalase Cat1 and superoxide dismutase Sod2 to inhibit gene expression and repress the decomposition of H2O2.In order to verify it in vivo,a high ROS gastrointestinal infection mouse model was used.Mice were infected by gavage with 1:1 mixture of the wild-type strain and mice were infected by gavage with 1:1 mixture of the wild-type strain and P-mutant strain and the mutant strains exhibited competitive defects compared to wild-type strains.In summary,we identified that Ssn3 promotes Hap43 phosphorylation and leads to its ubiquitin-dependent degradation in iron-replete conditions.Furthermore,the degradation of Hap43 relieved its repression to the antioxidant enzymes so that increased C.albicans survival under oxidative environment.