Research On Mechanism Of Mitochondrial Inner Membrane Genes Phb2 And Mdm28 Involved In Regulation Of Antifungal Drug Resistance In Fission Yeast

Objective:In recent years,the incidence of deep fungal infections has been on the rise,and the increase in drug-resistant strains has brought new severe challenges to clinical treatment,the effective treatment of drug-resistant fungi infection has become a clinical problem to be solved urgently.Currently,the types of antifungal drugs which have been commonly used in the clinic are limited,and the problem of fungal resistance has become increasingly serious.Therefore,it is of great significance to understand the mechanisms that lead to resistance to antifungal drugs.And it is also of great significance for improving the efficacy of antifungal drugs for the treatment of fungal infections,and for providing ideas for the development of new antifungal drugs.This study have used the fission yeast as a model organism to screen and identify two mitochondrial inner membrane genes,phb2⁺and mdm28⁺,which overexpression are resistance to clotrimazole and terbinafine,and we preliminary explored the possible mechanism.The reason that phb2⁺gene was resistant to antifungal drugs when it overexpressed may be mediated by pap1,but the mechanism was still unclear.mdm28⁺may be works together with mba1⁺gene,but the further mechanism of action remains unclear.Mitochondria,as the main organelle of cellular aerobic respiration,are involved in many important life processes,such as cell differentiation,apoptosis,and cell information transmission,in addition to providing energy to cells.They also participate in human and fungal pathogen drug tolerance.Studies have shown that mutations in specific sites in mitochondria DNA can cause changes in mitochondrial function,leading to changes in the sensitivity of fungal pathogens to antifungal agents.In addition to the absence of mitochondrial DNA can lead to drug resistance,the dysfunction of mitochondrial respiratory chain is also related to the drug-resistance.However,we have little known about the mechanism of mitochondrial inner membrane genes involved in the development of antifungal drug resistance,and further exploration is needed.Fission yeast is an ideal model organism for studying cell cycle,apoptosis and cell signal transduction.Its mitochondrial genome is also highly homologous to humans,and its size and structure are also similar to the human mitochondrial genome.Furthermore,fission yeast is an important tool for studying drug targets and mechanisms.Therefore,we used fission yeast to study the reisitance mechanism caused by the overexpression of phb2 and mdm28 genes.The phb2⁺gene of fission yeast encodes Prohibitin 2 and is a member of the prohibitin family.Prohibitin is a multi-functional and conserved protein that can participate in transcriptional regulation,signal transduction,apoptosis,and other physiological processes.It is also important for the maintenance of mitochondrial cristae morphology.However,it has not been reported how to participate in antifungal resistance through affecting mitochondrial function.The mdm28⁺gene in fission yeast encodes a mitochondrial inner membrane protein and its homologue in budding yeast Mdm38 is a protein that interacts with mitochondrial ribosomes and participates in respiratory chain biogenesis.Moreover,Mdm38 also mediates the K⁺/H⁺exchange in the inner mitochondrial membrane and is also a multifunctional protein.Its homolog gene Letm1 deletion in human is associated with Wolf-Hirschhorn syndrome,a congenital multiple malformation.However,it has little report about the mdm28⁺genes participate in drug resistance in fission yeast.This study aims to reveal the mechanism of phb2⁺and mdm28⁺genes involved in the regulation of antifungal drug resistance and their effects on mitochondrial function through the research of the resistance mechanisms of these two genes.It aims to provide new clues and theoretical basis for clinically solving the problem of antifungal drug resistance.Methods:1.In this study,we constructed the phb2⁺-RFP and RFP-mdm28⁺recombinant plasmids with red fluorescent marker,and then we observed the location of the proteins and verified their drug resistance phenotypes.2.We constructed phb1⁺recombinant plasmid,and verified the drug phenotype of its overexpression or deletion by spot culture method.3.We detected the mitochondrial membrane potential of the dysfunctional Phb2,Phb1, Mdm28 and Mba1 proteins,and detected the phenotypes of these dysfunctional proteins on YPG or H₂O₂ medium by spot culture method.4.We constructed theΔpap1Δphb2 andΔpap1Δphb1 double-knockout strains by random spore analysis.And detected the effect of pap1 gene deletion on drug resistance phenotypes or H₂O₂ resistance caused by phb2 or phb1 gene deletion by spot culture method,and the the effect of pap1 gene deletion on drug resistance phenotypes5.Detected Rst2 transcriptional level of the dysfunctional Phb2,Mdm28 and Mba1 proteins,and the effect of pap1 gene deletion on Rst2 transcriptional level caused by dysfunctional Phb2 protein.6.We used the NO and ROS probes to detect NO or ROS level in dysfunctional Phb2,Phb1,Mdm28 and Mba1 cells,and the effect of pap1 gene deletion on it.7.Real-time PCR was used to detect and Pap1 mRNA levels when phb2 gene was dysfunctional or phb1 gene deleted.And detected the clotrimazole,terbinafine,and phenylglyoxal sensitivity phenotypes of pap1⁺overexpression strains.Results:1.The Phb2-RFP and RFP-mdm28 were localized in the mitochondria of the strain and the mitochondrial morphology was normal.When the two proteins were overexpressed,the mitochondrial morphology changed from a continuous tubular structure to a fragmented fragment-like structure.Both proteins are present in the cytoplasm in an aggregated state.2.We constructed the phb1⁺recombinant plasmid of fission yeast successfully,and its overexpression could not cause antifungal drug resistance,but its deletion strain could.3.The dysfunctional Phb2 and the deletion of mba1 could increase the mitochondrial membrane potential.The phb2⁺gene overexpression could contribute to the use of non-fermented carbon sources for yeast.The dysfuntion of phb2 and deletion of phb1 gene could resist the oxidative stress produced by H₂O₂ on the strains,but mdm28⁺gene overexpression,mba1 gene overexpression and knockout are not related to oxidative stress reaction.4.The drug or H₂O₂ resistance caused by phb2⁺gene overexpressed was disappeared when the pap1 gene was deleted.We constructed theΔpap1Δphb2 andΔpap1Δphb2 double deletion strains successfully,but the drug or H₂O₂ resistance caused by phb2 or phb1 gene deletion was disappeared when the pap1 gene was deleted.5.Dysfunctional Phb2 or mba1 gene deletion could increase Rst2 transcription level, but mdm28⁺or mba1⁺gene overexpression could not increase Rst2 transcription level.Moreover,the pap1 gene deletion had no effect on the dysfunctional Phb2.6.The dysfunctional Phb2 cells or Phb1 deletion cells could stimulate the NO and ROS synthesis.The NO level was not changed when the mdm28⁺or mba1⁺gene overexpressed and mba1 gene deleted,but their ROS level was increased.Moreover, the pap1 gene deletion had no effect on NO and ROS level of the dysfunctional Phb2.7.Dysfunctional Phb2 and Phb1 deletion stains could increase Pap1 mRNA level,and overexpression of Pap1 exhibited drug resistance phenotypes,similar to dysfunction of Phb2.Conclusion:1.Between the prohibitin complex in fission yeast,only phb2⁺gene overexpression could cause antifungal resistance,while phb1⁺gene overexpression could not,but deletion of phb1 or phb2 gene both could cause aintifungal resistance.2.The Phb2 and Mdm28 proteins were both localized in mitochondria,and their overexpression could cause mitochondrial morphological changes,and both of them existed in the cytoplasm in an aggregated form.3.Both overexpression and deletion of phb2 gene and the mba1 deletion strains could increase the mitochondrial membrane potential.The overexpression of phb2⁺gene could contribute the use of non-fermented carbon sources.Dysfunctional Phb2 protein and loss of Phb1 protein could resist the damage of oxidative stress generated by H₂O₂.4.Pap1 did not mediate the drug resistance caused by overexpression of mdm28⁺genes in fission yeast.5.Dysfunction of phb2 results in multidrug resistance via activating the oxidative stress responsive transcription factor Pap1 in fission yeast,and Pap1 mediated the multidrug resistance.