Relationship Between Drug-resistance-related Protein Rta2p And Lipid Rafts In Candida Aibicans

Candida albicans(C. albicans) is currently one of the most common pathogens ofsystemic fungal infection. Azole drugs are most commonly used in the clinical treatment ofC. albicans infections.With the long term and excessive use of azoles, drug-resistant strainswere constantly produced and led to a growing number of treatment failure.Therefore,looking for new mechanisms of azole resistance in C. albicans to provid thebasis for drug treatment is critical. In C. albicans, lipid rafts (also called detergent-resistantmembranes, DRMs) are involved in many cellular processes and contain many importantproteins. We previously demonstrated that Rta2p was required for calcineurin-mediatedazole resistance and sphingoid long-chain base release in C. albicans.Here, we found that Rta2p was co-localized with raft-constituted ergosterol on theplasma membrane of C. albicans. Furthermore, this membrane expression pattern wastotally disturbed by inhibitors of either ergosterol or sphingolipid synthesis. Biochemicalfractionation of DRMs together with immunoblot uncovered that Rta2p, along withwell-known DRM-associated proteins (Pma1p and Gas1p homologue), was associated withDRMs and their associations were blocked by inhibitors of either ergosterol orsphingolipid synthesis. In order to investigate the effects of Rta2p on lipid raft, we first useGC-MS method to assay the total sterols and ergosterol in lipid rafts areas and non-raftareas. Then we also use Filipin staining to observe the distribution of ergosterol. Resultsshowed that no significant differences between the wild-type and rta2Δ/Δ mutant strain.We found that the disruption of RTA2did not affect the synthesis and distribution ofergosterol in C. albicans.Finally, we used a high-throughput proteomics approach togetherwith immunoblot and identified that Rta2p was required for the association of10proteinswith DRMs. Of these,5proteins (Pma1p, Gas1p homologue, Erg11p, Pmt2p and Ali1p)have been reported to be DRM-associated and Erg11p is a well-known target of azoles in C.albicans. In conclusion, our results showed that Rta2p was predominantly localized in lipidrafts and required for the association of certain membrane proteins with lipid rafts in C.albicans.Apart from inhibition of ergosterol biosynthesis, miconazole induces accumulation ofreactive oxygen species (ROS) in susceptible fungi,leading to fungal cell death.Moreover,we have demonstrated that miconazole induces actin stabilization prior to this ROSaccumulation. In this experiment, we found that the disruption of RTA2did not affect the susceptibility to miconazole in C. albicans.Then We investigated the effect of membraneraft-disturbing compounds on (ⅰ)miconazole antifungal activity,(ⅱ) miconazole-inducedROS accumulation, and (ⅲ) miconazole-induced actin cytoskeleton stabilization inwild-type C. albicans. We have demonstrated that disruption of lipid rafts by treatmentwith edelfosine or MβCD interferes with miconazole antifungal action as well as withmiconazole-induced ROS accumulation and stabilization of the actin cytoskeleton.Toexplore the role of Rta2p in the mode of antifungal action of miconazole,the sameexperiments were performed on rta2null mutant strain(rta2Δ/Δ).We found that there is nosignificant differences between two strains when they treated with miconazole only,butthere is synergic effect between the disruption of RTA2and membrane raft-disturbingcompounds in interference of miconazole antifungal action.