TOP2 gene disruption reduces drug susceptibility by increasing intracellular ergosterol biosynthesis in Candida albicans.
In this study the role of the TOP2 gene in fungal drug susceptibility was investigated by disrupting and overexpressing the gene in Candida albicans. MIC determination and a spot assay showed that a top2Delta/Delta null mutant (strain T2bc) was more resistant to the antifungals tested than the wild-type (strain CAI4). Real-time RT-PCR and rhodamine 6G efflux examination showed that TOP2 did not influence the activity of drug efflux pumps. Sterol analysis with GC/high-resolution MS indicated that the intracellular ergosterol composition of the top2Delta/Delta mutant was significantly increased. Subsequently, fluorescence polarization measurements also revealed that Top2-deprived cells displayed a decrease in membrane fluidity, resulting in enhanced passive diffusion of the drugs. Quantitative real-time RT-PCR analysis further confirmed that the ERG11 gene, an essential gene in ergosterol biosynthesis, was upregulated. These results demonstrate a close relationship between the TOP2 gene and drug susceptibility in C. albicans.
School of Pharmacy, Second Military Medical University, Shanghai, PR China.|Department of Pharmacology, China Pharmaceutical University, Nanjing, PR China.
Hao
H
Zheng
School of Pharmacy, Second Military Medical University, Shanghai, PR China.
Yuan-Ying
YY
Jiang
School of Pharmacy, Second Military Medical University, Shanghai, PR China.
Yan
Y
Wang
School of Pharmacy, Second Military Medical University, Shanghai, PR China.
Xin-Ming
XM
Jia
Department of Pharmacology, China Pharmaceutical University, Nanjing, PR China.
Tian-Hua
TH
Yan
School of Pharmacy, Second Military Medical University, Shanghai, PR China.
Ping-Hui
PH
Gao
School of Pharmacy, Second Military Medical University, Shanghai, PR China.
Lan
L
Yan
School of Pharmaceutical Science and Technology, Tianjin University, PR China.
Ling-Huo
LH
Jiang
Department of Pharmacology, China Pharmaceutical University, Nanjing, PR China.
Hui
H
Ji
School of Pharmacy, Second Military Medical University, Shanghai, PR China.
Yong-Bing
YB
Cao
D000935:Antifungal Agents / Q000494:pharmacology*; D002176:Candida albicans / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000235:genetics* / Q000378:metabolism; D025141:Drug Resistance, Fungal / Q000235:genetics; D004875:Ergosterol / Q000096:biosynthesis* / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal / Q000502:physiology*; D008826:Microbial Sensitivity Tests; D009154:Mutation
D000935:Antifungal Agents; D005656:Fungal Proteins; D004250:DNA Topoisomerases, Type II; D004875:Ergosterol