Molecular Mechanism Of Fluconazole Resistance In Trichosporon Asahii

Backgrounds and Objectives:Trichosporon asahii(T.asahii)is one of the most common(third)pathogenic fungi that cause clinical invasive fungal infections.Due to the increase of cancer,AIDS and other patients,the abuse of antifungal drugs,and the large number of applications of immunosuppressive agents,the incidence rate is increasing year by year.Disseminated infection caused by T.asahii,which can quickly involve multiple organs,causing bacteremia,shock,etc.at its acute phase.Also,the mortality rate is as high as 80%.Considering fluconazole as the representative for the azole antifungal agent,it has become the most commonly used antifungal drug in clinical practice because of its high safety and good curative effect.Since T.asahii is not sensitive to most antifungal drugs other than azoles,fluconazole is also one of the first choice drugs for the treatment of its infection.However,during the long-term treatment and repeated drug administration,the degree of drug resistance has become more and more serious,and related research and reports have been increasing.We have treated the first disseminated trichosporosis patient in China,and isolated the fluconazole-sensitive T.asahii strain from her skin,liver and other diseased tissues.After that,the patient received long-term and repeated antifungal drugs such as fluconazole.treatment.Fifteen years later,we isolated the fluconazole-resistant T.asahii strain in her new facial lesions,which makes our treatment of T.asahii infection more difficult.But the current mechanism of resistance to T.asahii is still unclear.So it is of great significance to accomplish the research on the fluconazole resistance mechanism of the bacteria,to find and develop new antifungal agents,also to reduce the mortality rate of T.asahii infection.Methods:Part I Isolation and Identification of a Drug-resistant Strain of T.asahii1.Isolation and identification of clinically resistant strains in the body: The patient's facial new skin lesions were taken.Then,pathological examination and fungal culture were performed respectively.Microscopic examination and IGS region PCR amplification and sequencing were performed on the obtained strains.The MIC value of fluconazole was determined by reference to the susceptibility M27-A3 protocol.2.Isolation and identification of drug-resistant strains in vitro: The first isolated fluconazole-sensitive strain A01 S was induced in vitro by increasing the concentration of fluconazole.Until after the drug concentration was ?128?g/ml,and the MIC value of the strain was ?64?g,the drug-free culture was carried out for 30 generations.Part II Screening analysis of drug resistance-related genes in T.asahii1.Construction of DNA library and genome sequencing: DNA of fluconazole-sensitive and drug-resistant strains isolated from the same patient was extracted.A paired-end library was constructed by using the KAPA DNA sample preparation kit.Genomic high-throughput sequencing was completed by following standard sample preparation protocol and using Illumina HiSeq 3000.2.Construction of RNA library and transcriptome sequencing: RNA from fluconazole-sensitive and drug-resistant strains isolated from the same patient was extracted.And RNA-Seq library was constructed by using KAPA-strand RNA-Seq kit.Highthroughput sequencing of transcriptomes was performed by following standard sample preparation protocol and using Illumina HiSeq 3000.3.Bioinformatical analysis: through using TopHat,FPKM,R package edgeR,ABySS,KOBAS,MEME,TOMTOM,PyMOL,TMpred,InterProScan,Clustal Omega(v1.1.0)and other software,formula mapping,evaluation,filtration,assembly,we can compare and predict related gene functions and structures.Part III ERG11 gene resistance function verification1.The gene analysis and verification of in vitro induced drug-resistant strains transcriptome ERG11: RNA of fluconazole-resistant strains was extracted in vitro.RNA-Seq library was constructed by using KAPA strand RNA-Seq kit.High-throughput sequencing of transcriptomes was performed by following standard sample preparation protocol and using Illumina HiSeq 3000.The ERG11 gene expression changes were analyzed and compared by using relevant bioinformatics software.2.Test verification of ERG11 gene Saccharomyces cerevisiae transfection drug sensitivity: RNA of sensitive and resistant strains was extracted in vivo.Then,we reversed the above mentioned transcription into cDNA,also expanded the full length of ERG11 gene.The recombinant plasmid of ERG11 gene was constructed by cloning and extraction.And the recombinant plasmid and empty plasmid were transfected into Saccharomyces cerevisiae by using a high-efficiency yeast transformation kit.The monoclonal colonies of the S.cerevisiae genetically engineered bacteria which contained the recombinant plasmid can be screened.Finally,the susceptibility test was carried out according to the M27-A3 protocol.Part IV The effect of a new antibacterial agent-nanosilver on T.asahii1.Detecting the drug sensitivity of nanosilver to T.asahii: The MIC values of 17 clinical and environmental sources of T.asahii for nanosilver and 7 commonly used antifungal drugs(fluconazole,itraconazole,amphotericinB,caspofungin,terbinaphine,5fluorocytosine and voriconazole)were determined by the M27-A3 protocol in the CLSI guidelines.2.To observe the effect of nano-silver on the morphology of T.asahii: the colony size was observed after 7 days of culture on 7 concentrations of PDA medium with a final concentration of 0-32 ?g/ml.Then,the clinical standard strains treated with 4?g/ml nano-silver were selected under SEM and TEM to observe their gross morphology and ultrastructural changes.Results:Part I Isolation and identification of resistant strains of T.asahii1.Isolation and identification of clinically resistant strains in vivo: pathological results are consistent with fungal infection,and fungal-like structures can be seen by PAS staining.Tissue can be cultured to gray-white,brain-like fungal colonies,microscopically visible joint spores,hyphae and other T.asahii morphological structure.The IGS region alignment results were consistent with T.asahii.The fluoroconazole MIC value was in accordance with the drug-resistant strain and was numbered as A15 R.2.Isolation and identification of drug-resistant strains in vitro: the concentration of fluconazole induced drug reached 64 ?g/ml,the MIC value of the induced strain reached 64 ?g/ml for the first time.When the drug concentration was ?128 ?g/ml,its MIC value was ?64 ?g/ml.After 30 generations of drug-free subculture,the MIC value was stable at 64 ?g/ml,and it was confirmed that a stable resistant strain was obtained which was numbered as A01 R.Part II Screening analysis of drug resistance-related genes in T.asahii1.Construction of DNA library and genome sequencing: The DNA library was successfully constructed,and the genome sequencing was of high quality(Q30 and above).The total amount of 2G data was measured in sensitive strains.Among them,the high-quality data volume was 1.7G,the total data volume of the resistant strains was 1.5G and the high-quality data volume was 1.3G.The availability rate was 84% and 88% respectively,and the coverage was 70 X and 56 X respectively.The amount of data is sufficient for genome splicing and mutation detection analysis.2.Construction of RNA library and transcriptome sequencing: RNA library was successfully constructed.And three biological replicates were performed by transcriptome sequencing.The data saturation and quality were good.A total of 24.7 Gb of high quality data was found to be sufficient for differential expression gene analysis.3.Bioinformatical analysis: The genome of the drug-resistant strain and the sensitive strain showed a deletion of 34 amino acids in the ERG11 gene.The genome of the drug-resistant strain ergosterol biosynthesis pathway-related gene mutation was found,in which the ERG11 gene intron 4 was lost,the first intron increased by 346 nt,and thirty-six new mutations were found.It has been experimentally verified that these mutations can be predicted to alter the structure of protein-related.Three point mutations(V458L,D457 V and D334S)were found in ERG3.One mutation site(E349D)was found in ERG5.The transcriptome found that the expression of drug transport-related genes in drug-resistant strains was up-regulated,including 6 ABCs,1 MFS and 1 MATE family gene.The expression of sixteen genes related to ERG including ERG11 was up-regulated.The thirteen genes(ERG1-3,5-7,9-11,19,25,26 and HMG)found in the upstream promoter region of the ERG gene contain upc2p-like sequences involved in transcriptional regulation.Several expressions of the antioxidant genes Cu/Zn SOD,GPx,TPx and stress protein DDR48 associated with oxidative stress were found to increase.Part III ERG11 gene resistance function verification1.The gene analysis and verification of In vitro induced drug-resistant strains transcriptome ERG11: transcriptome data of induced drug-resistant strains were successfully obtained.And we found that the expression of ERG11 gene was up-regulated by drug-resistant strains,which was 3.7 times that of sensitive strains.2.The test verification of ERG11 gene Saccharomyces cerevisiae transfection drug sensitivity: the full length of the sensitive and resistant strain ERG11 gene was successfully amplified.Then we constructed it into the ERG11 gene recombinant plasmid.Recombinant Saccharomyces cerevisiae transformants were obtained.The MIC value of the S.cerevisiae transformant containing the sensitive strain ERG11 gene was found to be 8 ?g/ml,while the MIC value of the S.cerevisiae transformant containing the resistant strain ERG11 gene was 64 ?g/ml,which was confirmed as fluconazole-sensitive strain and drug resistant strain.Part IV Effect of a new antibacterial agent-nanosilver on T.asahii1.Results of drug sensitivity test: The MIC values of 17 strains of T.asahii strain nanosilver were 0.5 ?g/ml,which were obviously less than 5 fluorocytosine,fluconazole,itraconazole,amphotericin B,caspofungin and terbinaphine,slightly higher than voriconazole.Meanwhile,it also has a good inhibitory effect on clinical fluconazole resistant strains.2.Morphological observation: Macroscopic observation of nano-silver significantly inhibited the growth of T.asahii colonies.There was no inhibition at ?2?g/ml but a completely inhibition of growth at ?8?g/ml.For environmental strains,clinical strains and fluconazole,there was no difference between clinical resistant strains.Under scanning electron microscopy,the hyphae became sparse,small,shrinkage,and cell membrane damage.Under the transmission electron microscope,the cell wall and cell membrane rupture,loss,content leakage,chromatin condensation,edge collection,ribosome depolymerization,cell matrix lightening,together with ultrastructure of multivesicular and myeloid bodies formed by organelle degradation change.Conclusions:1.Long-term application of fluconazole in vivo can lead to the resistance of T.asahii,and stable drug-resistant strains can be obtained by single drug induction in vitro.2.The molecular mechanism of resistance to T.asahii is complex.The genes in ergosterol synthesis pathway,especially the mutation and expression upregulation of ERG11 gene,are one of the important mechanisms,and also transporter genes and antioxidant stress genes and other genes are involved.3.The new anti-fungal agent nano-silver can effectively inhibit the growth of T.asahii,including fluconazole-resistant strains,and can seriously damage the morphology and ultrastructure of the fungus.