Phenotypic And Genotypic Studies Of Trichophyton Xurans And Its Genomic And Transcriptomic Studies

Part I A Study on Phenotype and Genotype of Trichophyton schoenleinii Objectives:To investigate the molecular epidemiological characteristics of Trichophyton schoenleinii in Xinjiang and East China,and to analyze the relationship between the phenotypic characteristics,geographical origin and genotypic characteristics.Methods:(1)We collected 53 clinical isolates of T.schoenleinii from patients with tinea capitis in Xinjiang and East China.PCR amplification,sequencing,identification and phylogenetic analysis were performed on the rDNA internal transcribed spacer region(ITS),translation elongation factor 1-α(TEF1-α),calmodulin(CaM)and D1/D2 region of the large subunit(LSU).(2)The morphological characteristics of the colonies were observed,which were cultured on Sabauraud dextrose agar(SDA)at 28℃ for 14 days.The ability of producing urease,protease and lipase was tested respectively by urea medium,bovine serum albumin medium,Tween 80 medium and Tween 20 medium.(3)The genetic diversity of T.schoenleinii clinical strains was evaluated by amplified fragment length polymorphism(AFLP),and possible relationships between AFLP clusters and phenotypic traits and geographical origins were investigated.Results:(1)There was no difference in ITS,TEF1-a,CaM and LSU gene sequences among the T.schoenleinii clinical strains collected from different regions in China.ITS gene had the highest level of interspecific polymorphism among the T.schoenleinii and its related species.(2)T.schoenleinii clinical strains were divided into three groups according to the colony morphology.The results of urease test showed that 16.7%were positive,31.5%were weak positive and 51.8%were negative.The results of Tween 80 and Tween 20 lipase tests showed that 85.2%and 92.6%were positive,respectively.The protease activities were all positive.(3)The T.schoenleinii clinical strains were divided into 6 clusters by UPGMA cluster analysis based on AFLP data.Conclusions:The results of this study showed that ITS gene was more suitable for molecular identification and classification of T.schoenleinii and its related species.T.schoenleinii exhibited phenotypic and genotypic diversity,but there was no significant correlation between the genotypic and phenotypic characteristics or geographical origins.Part II Genome and Transcriptome study of Trichophyton schoenleiniiObjectives:To study the key genes and pathways of Trichophyton schoenleinii degrading keratin(a major constituent of the keratinized tissue)by genome and transcriptome,and to predict potential virulence factors and new therapeutic targets.Methods:(1)The whole genome of T.schoenleinii strain T2s was obtained by combining data from second-generation sequencing and third-generation sequencing.Predict and annotate the coding gene and non-coding RNA,and predict the secretory proteome.(2)RNA-seq technique was used to detect the mRNA expression profile of T.schoenleinii in keratin medium,soybean protein medium and Sabouraud dextrose broth.(3)The differentially expressed genes were analyzed,and the key genes and pathways for hydrolyzing and absorbing keratin were preliminarily inferred to develop new therapeutic targets.Results:(1)The whole genome contains 16 scaffolds with a total length of 23.4mb.7474 protein-coding genes,167 non-coding genes and 424 secretory protein genes were predicted in T2s genome.(2)The Sabouraud dextrose broth as a control group,2394 genes were up-regulated and 614 genes were down-regulated in keratin medium,1943 genes were up-regulated and 356 genes were down-regulated in soybean protein medium.(3)When T.schoenleinii was cultured in keratin medium,the expression of genes encoding protease,amino acid permease and peptide transporter was significantly up-regulated.In addition,the potential virulence factors ABC transporters and MFS transporters were strongly activated.Meanwhile,the genes related to carbon assimilation were inhibited and the genes related to nitrogen assimilation were activated.As the keratin medium became alkaline,the pH response signal transduction pathway was activated.ERG3,ERG4,ERG6,ERG27 and ERG11/CYP51,which are key enzymes in ergosterol biosynthesis pathway,were significantly up-regulated.Conclusions:A high-quality genome of T.schoenleinii was obtained by combining the second and third generation sequencing data.However,the results of comparative genomics study of dermatophytes showed small differences in genome organization and genetic content,which suggested that the difference in gene regulation and transcription mechanism might be the reason for their phenotypic differences.T.schoenleinii hydrolyzes protein into amino acids and oligopeptides by secreting proteases,and then transports them into cells through transporters.Therefore,proteolytic enzymes and membrane transporters may be potential virulence factors of T.schoenleinii.The energy metabolism of T.schoenleinii is beneficial to its successful adaptation and colonization in keratinized tissues.