Study On Biological Functions And Molecular Mechanisms Of Drug-resistance-related Protein Rta2p In Candida Albicans

Widespread and repeated use of azoles,particularly fluconazole,has led to the rapid development of azole resistance in Candida albicans.Overexpression of CDR1,CDR2, CaMDR1 and FLU1 has been reported contributing to azole resistance in C.albicans.In this study,hyper-resistant C.albicans mutant,with the above 4 genes deleted,was obtained by exposure to fluconazole and fluphenezine for 28 passages.Thirty-five differentially expressed genes were identified in the hyper-resistant mutant by microarray analysis; Among the 35 genes,most were of unknown function(49%),followed by those involved in transcription and RNA processing(14%),calcineurin stress-response pathway(9%), amino acid and carbohydrate metabolism(9%),small molecule transport(9%),energy generation(5%),and those not classified(5%).Moreover,expression pattern of RTA2 was similar to that of other known resistance-related genes(CDR1,CaMDR1,and ERG11).The deletion of RTA2 in C.albicans strains with and without deletions of CDR1,CDR2, CaMDR1 and FLU1,was found to increase their susceptibility to azoles;meanwhile,we found that ectopic overexpression of RTA2 in the rta2 mutants conferred resistance to azoles.RTA2 expression was found elevated in clinical azole-resistant isolates of C. albicans.The calcineurin pathway has also been reported to participate in azole resistance in C.albicans.In the present study,CaCl₂-induced-activation of the calcineurin pathway in wild-type C.albicans promoted resistance to azoles;it was also found that the Ca²⁺ chelator(EGTA),calcineurin inhibitors(FK506 and cyclosporin A) and the deletion of RTA2 blocked the resistance-promoting effects of CaCl₂.Furthermore,we found that the addition of CaCl₂ up-regulated RTA2 in a calcineurin-dependent manner.Ultra-structure analysis by transmission electronic microscopy showed the depletion of RTA2 make the cell membrane of C.albicans liable to be destroyed by fluconazole and Ca²⁺-induced-upregulation of RTA2 attenuated the destroying effects.Bioinformatics assay revealed that Rta2p is a lipid transporter.Our study found that the depletion of RTA2 decreased the translocation of sphingolipid dihydrosphingosine to outside of the cell membrane in C. albicans.In conclusion,our findings suggest that RTA2 is responsible for the development of calcineurin-mediated azole resistance in C.albicans.