Effect Of Phospholipase A2(PLA2) Gene On Drug Resistance Of Trichosporon Asahii From Giant Panda

Trichosporon asahii causes severe deep infection and death in humans and animals with immunosuppressed and is resistant to most clinical antifungal agents.The study found that the strain with strong phospholipase activity had more serious drug resistance.In addition,phospholipase belongs to genes involved in lipid metabolism pathway,which is associated with drug resistance.The biological research of fungi has entered the era of functional genome,and the functional analysis of genes can provide theoretical basis for the treatment of T.asahii.According to our previous transcriptomic data analysis,gene phospholipase A2（PLA2）changed significantly in drug-resistant strains,and phospholipase activity of fluconazole induced by T.asahii was also enhanced.In this study,T.asahii PLA2（TaPLA2）is taken as the research object,aiming to explore the effect of its overexpression on the growth of T.asahii and its role in drug resistance.To provide reference for further study of drug resistance mechanism and new drug development of T.asahii.Experiment 1:TaPLA2 cloning and bioinformatics analysis.TaPLA2 was cloned using the genome of wild strain YAN0802 as template to obtain the target fragment.The length of TaPLA2 was 435bp,which was consistent with the gene sequence of T.asahii clinical standard strain CBS2479 published on NCBI.Protein prediction results showed that TaPLA2 is a small molecular weight protein with 144 amino acids.The conserved domain of TaPLA2 is pfam09056.TaPLA2 belongs to the Phospholip＿A2＿3 superfamily.TaPLA2contains a signal peptide at the N-terminal of amino acid sequence and does not exist transmembrane domain.The results showed that the structure of TaPLA2 was consistent with the characteristics of secretory phospholipase A2.The phylogenetic tree showed that it was closely related to BBA＿07804 of Beauveria bassiana and CCM＿07919 of Cordyceps militaris with 98%confidence.Experiment 2:Construction of TaPLA2 overexpression system.In this study,p EGFP-N1 was used as the base vector,the overexpression vector was constructed by homologous recombination,the receptive state of Escherichia coli DH5αwas transformed,and the monoclone was selected for PCR amplification,double digestion and sequencing comparison,to obtain the overexpression vector p EGFP-N1-TaPLA2.The overexpression vector was transferred into wild strain YAN0802 by Agrobacterium tumefaciens transformation method,and the transformers were selected for verification using 200μg/m L G418 as the screening concentration.The overexpressed mutant TaPLA2^OE was successfully constructed by PCR amplification and fluorescence labeling,fluorescence quantitative PCR validation and green fluorescence observation.Experiment 3:Analysis of biological characteristics and drug resistance of TaPLA2^OE.In this experiment,the phospholipase activity of TaPLA2^OE was strongly positive by egg yolk medium.Colony diameter was measured and biological characteristics were observed by copper ring culture method.The results showed that TaPLA2^OE growth slowed down,the colonies were smaller than the wild strains,the middle was raised,the edges were irregular,and the colony surface was not covered by powder.After 3 days of culture,mycelia was observed to reduce spore increase,and a large number of joint spores co-existed with mycelia on the third day.TaPLA2^OE showed resistance to fluconazole,voriconazole,amphotericin B and 5-fluorocytosine by microbroth dilution and agar spot assay.Among them,XTT reduction method showed strong activity of TaPLA2^OE biofilm,and RT-q PCR showed up-regulated expression of TaPLA2^OE drug resistance genes.TaPLA2^OE was not sensitive to Al³⁺/Cu²⁺oxidative stress or temperature stress,but was sensitive to SDS/CR cell wall stress.The expression of chitin synthesis or degradation genes and cell wall integrity related genes ALS and GPI were down-regulated by RT-q PCR.However,HOG-MAPK pathway,one of the signaling pathways of cell wall integrity,was up-regulated to maintain cell survival.Conclusion:TaPLA2 was 435bp long and was predicted to belong to s PLA2 family by bioinformatics analysis,and the overexpressed mutant was constructed by homologous recombination and Agrobacterium tumefaciens transformation.The resistance of TPLA2^OEmutant increased,and the expression of efflux pump gene and biofilm formation increased.Sensitive to cell wall inhibitors,chitin synthesis was slowed down and HOG-MAPK signaling pathway was activated to maintain cell wall integrity,and HOG1 and MAPK were up-regulated to enhance resistance to azole.