The Regulating Mechanism Of PHO4 And HAP2 Involved In The Azole Resistance Of Candida Albicans

Research objectives: To study the influence of Candida albicans transcription factors(TF)on drug susceptibility and the relevant regulating mechanisms,a screening of a TF deletion mutant library has been conducted.Methods: The broth microdilution method and spot assay were used to determine drug susceptibility of TF deletion strains.The expression of multidrug transporter genes were examined by real-time PCR,and the analysis of rhodamine 6G efflux was performed to evaluate the efflux pump activity.The intracellular ATP levels and mitochondrial membrane potential measuring were two important ways to measure if the change of efflux pump activity was related to disfunction of mitochondrial respiratory and metabolism.Growth curve analysis was used to observe the strain sensitivity to H2O2 and the contents of the intracellular reactive oxygen species were measured.RT-PCR technology was used to assess the transcription level of antioxidant-related genes and the assess of glutathione reductase activity was dependent on the detection of intracellular GSH level.Results: The measurement of drug minimal inhibitory concentration and spot assay indicated an increase of susceptibility to fluconazole and miconazole in PHO4 and HAP2 deletion mutants compared to the parental strain.Deletion of PHO4 led to increase of sensitivity to azole antifungal drugs and decrease of rhodamine 6G effux.However,the transcription level of CDR1,MDR1 and ERG11 in pho4? /? mutant decreased.Also,pho4? /? mutant had increased sensitivity to H2O2 and decreased resistance of oxidative stress compared to the parental strain.After the stimulation of miconazole,the intracellular level of reactive oxygen species(ROS)had a slower growth compared to the parental strain.A modest decrease of intracellular ATP level and increase of mitochondrial membrane potential were detected in pho4? /? mutant.HAP2 deletion mutant had significantly increased sensitivity to antifungal drugs,decreased transcription level of drug efflux pump genes(CDR1,MDR1)and drug resistance gene ERG11 as well as obviously decreased effux activity of rhodamine 6G.Succinate dehydrogenase gene(SDH2)had decreased transcription level in HAP2 deletion mutant and the ATP content and intracellular mitochondrial membrane potential both appeared to decrease.Also,the HAP2 mutant showed an increased susceptibility to iron ion and decreased content of iron ion.The intracellular reactive oxygen species(ROS)production decreased in HAP2 deletion mutant and the susceptibility to H2O2 increased in the mutant strain.The expression of antioxidant genes SOD4,GLR1,TRR1 decreased when HAP2 gene was deleted and an increase in intracellular GSH content was also detected in the mutant.Conclusion: Deletion of PHO4 in C.albicans may cause a decrease of oxidative stress resistance and drug efflux ability,finally result in hyper susceptibility to antifungal drugs.More detailed regulation mechanism still needs to be explored.The deletion of HAP2 led to reduction of iron uptake in Candida albicans.The disfuntion of iron metabolism resulted a decrease of intracellular levels of reactive oxygen species(ROS)and an increase of antioxidant gene transcription level.Also,high activity of reductase was detected in HAP2 deletion mutant.Deletion of HAP2 resulted in abnormal function of energy metabolism,decreased ATP production and low transportation capacity.Thus,the activity of ABC transporter protein and facilitated diffusion super family protein decreased in HAP2 deletion mutant,finally resulting in increased sensitivity to azoles.