title: Mutation of G234 amino acid residue in candida albicans drug-resistance-related protein Rta2p is associated with fluconazole resistance and dihydrosphingosine transport.
abstract: Widespread and repeated use of azoles has led to the rapid development of drug resistance in Candida albicans. Our previous study found Rta2p, a membrane protein with 7 transmembrane domains, was involved in calcineurin-mediated azole resistance and sphingoid long-chain base release in C. albicans. Conserved amino acids in the transmembrane domain of Rta2p were subjected to site-directed mutagenesis. The sensitivity of C. albicans to fluconazole in vitro was examined by minimum inhibitory concentration and killing assay, and the therapeutic efficacy of fluconazole in vivo was performed by systemic mice candidiasis model. Furthermore, dihydrosphingosine transport activity was detected by NBD labeled D-erythro-dihydrosphingosine uptake and release assay, and the sensitivity to sphingolipid biosynthesis inhibitors. We successfully constructed 14 mutant strains of Rta2p, screened them by minimum inhibitory concentration and found Ca(2+) did not completely induce fluconazole resistance with G158E and G234S mutations. Furthermore, we confirmed that G234S mutant enhanced the therapeutic efficacy of fluconazole against systemic candidiasis and significantly increased the accumulation of dihydrosphingosine by decreasing its release. However, G158E mutant didn't affect drug therapeutic efficacy in vivo and dihydrosphingosine transport in C. albicans. G234 of Rta2p in C. albicans is crucial in calcineurin-mediated fluconazole resistance and dihydrosphingosine transport.
authors.affiliation: a Research and Develop Center of New Drug ; School of Pharmacyl Second Military Medical University ; Shanghai , China.|b Department of Pharmacology ; School of Medcine ; Tongji University ; Shanghai , China.
authors.forename: Shi-Qun
authors.initials: SQ
authors.lastname: Zhang
authors.affiliation: c Department of Laboratory Diagnosis ; Changhai Hospital ; Second Military Medical University ; Shanghai , China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Miao
authors.affiliation: b Department of Pharmacology ; School of Medcine ; Tongji University ; Shanghai , China.
authors.forename: Li-Ping
authors.initials: LP
authors.lastname: Li
authors.affiliation: a Research and Develop Center of New Drug ; School of Pharmacyl Second Military Medical University ; Shanghai , China.
authors.forename: Lu-Lu
authors.initials: LL
authors.lastname: Zhang
authors.affiliation: a Research and Develop Center of New Drug ; School of Pharmacyl Second Military Medical University ; Shanghai , China.
authors.forename: Lan
authors.initials: L
authors.lastname: Yan
authors.affiliation: a Research and Develop Center of New Drug ; School of Pharmacyl Second Military Medical University ; Shanghai , China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Jia
authors.affiliation: a Research and Develop Center of New Drug ; School of Pharmacyl Second Military Medical University ; Shanghai , China.
authors.forename: Yong-Bing
authors.initials: YB
authors.lastname: Cao
authors.affiliation: a Research and Develop Center of New Drug ; School of Pharmacyl Second Military Medical University ; Shanghai , China.|b Department of Pharmacology ; School of Medcine ; Tongji University ; Shanghai , China.
authors.forename: Yuan-Ying
authors.initials: YY
authors.lastname: Jiang
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology*; D001692:Biological Transport; D019703:Calcineurin / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002176:Candida albicans / Q000187:drug effects* / Q000235:genetics; D025141:Drug Resistance, Fungal; D005260:Female; D015725:Fluconazole / Q000494:pharmacology*; D005656:Fungal Proteins / Q000235:genetics*; D026901:Membrane Transport Proteins / Q000235:genetics; D008813:Mice, Inbred ICR; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000187:drug effects; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D000935:Antifungal Agents; D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D050505:Mutant Proteins; D015725:Fluconazole; D019703:Calcineurin; D013110:Sphingosine; C005682:safingol; D002118:Calcium
keywords: Candida albicans; Rta2p; calcineurin pathway; dihydrosphingosine; drug resistance; functional sites