ABSTRACT
Objective To compare and analyze the differences in the transcriptomics between mycelium and early yeast phases of Sporothrix schenckii (S.schenckii),and to realize the changes in transcriptome expression profiles during mycelium-to-yeast transformation.Methods A standard strain of S.schenckii (ATCC 10268) was subjected to 96-hour culture with Sabouraud medium at 25 ℃ or 36-hour culture with brain-heart infusion medium at 37 ℃ to obtain the mycelium and yeast form of S.schenckii,and then,their transcriptomes were sequenced.Functional annotation was performed for screened unigenes by comparison using several databases (such as NR,Swiss-Prot,KEGG,COG,KOG,GO and Pfam),coding sequence prediction,and gene expression analysis in each sample.Finally,the differentially expressed genes were subjected to pattern clustering,functional annotation and enrichment analysis.Results A total of 14.76 Gb valid data (clean data) were obtained,and functional annotation results were acquired in 28 094 of 43 863 assembled unigene clusters.Compared with S.schenckii in mycelium phase,there were 10 969 up-regulated genes and 199 down-regulated genes in S.schenckii in yeast phase.These differentially expressed genes were involved in protein phosphorylation,intracellular protein transport,cellular protein modification,small guanosine triphosphate-mediated signal transduction,vesicle-mediated transport,translation,intracellular signal transduction,microtubule formation,adenosine triphosphate synthesis,coupled proton transport and so on.Sixteen genes in the mitogen-activated protein kinase (MAPK) pathway and two-component signaling pathway,which were two important signal transduction pathways involved in fungal morphogenesis,and 16 genes involved in chitin synthesis and metabolism were all confirmed to be up-regulated in S.schenckii in yeast phase.Conclusions Compared with S.schenckii in mycelium phase,great changes in gene expression profiles were observed in S.schenckii in yeast phase.These differentially expressed genes are involved in many functions,suggesting that the dimorphic transition of S.schenckii is regulated by a multi-gene network system.
ABSTRACT
Objective To investigate differences in the expression of Ras 1,Rac1 and Rho1 genes between yeast and hyphal phases of Trichosporon asahii (T.asahii),and to explore their roles in the formation of hyphae.Methods The yeast phase and hyphal phase of T.asahii were cultured and served as yeast phase group and hyphal phase group respectively.Total RNA was extracted from the 2 groups,and real -time fluorescence-based quantitative PCR (RT-PCR) was performed to measure the mRNA expression of Ras1,Rac1 and Rho1.Results The hyphal formation rate was significantly lower in the yeast phase group than in the hyphal phase group (0.40％ ± 0.53％ vs.99.33％ ± 0.57％,t =13.93,P ＜ 0.05).When the mRNA expression of Ras1,Rac1 and Rho1 in the yeast phase group was all set as 1,that in the hyphal phase group was 25.17 ± 10.99,16.81 ± 7.80,42.61 ± 18.50,respectively,with significant differences between the two groups in the three parameters (t =3.81,3.51,3.90,respectively,all P ＜ 0.05).Conclusion Ras1,Rac1 and Rho1 genes may participate in the regulation of hyphal formation in T.asahii.
ABSTRACT
Antigenic preparations from Sporothrix schenckii usually involve materials from mixed cultures of yeast and mycelia presenting cross-reactions with other deep mycoses. We have standardized pure yeast phase with high viability of the cells suitable to obtain specific excretion-secretion products without somatic contaminations. These excretion-secretion products were highly immunogenic and did not produce noticeable cross-reactions in either double immunodiffusion or Western blot. The antigenic preparation consists mainly of proteins with molecular weights between 40 and 70 kDa, some of them with proteolytic activity in mild acidic conditions. We also observed cathepsin-like activity at two days of culture and chymotrypsin-like activity at four days of culture consistent with the change in concentration of different secreted proteins. The proteases were able to cleave different subclasses of human IgG suggesting a sequential production of antigens and molecules that could interact and interfere with the immune response of the host.
As preparações antigênicas de Sporothrix schenckii provêm geralmente de cultivos mistos de leveduras e micélios e apresentam reações cruzadas com outras micoses profundas. Foi padronizada a obtenção da fase leveduriforme pura, com alto índice de células viáveis, o que permite, por sua vez, obter produtos específicos da excreção-secreção sem contaminantes somáticos. Estes produtos da excreção-secreção são altamente imunogênicos, e não apresentam reações cruzadas visíveis em dupla difusão e sem Western blot. O preparado antigênico consiste principalmente em proteínas com peso molecular entre 40 e 70 kDa, sendo que algumas apresentam atividade proteolítica em meios levemente ácidos. Foi observada atividade do tipo catepsina em produtos da excreção-secreção obtidos a partir de leveduras de dois dias de cultivo, e atividade do tipo quimiotripsina aos quatro dias de cultivo, consistente com a mudança de concentração de proteínas secretadas. As proteases puderam clivar diferentes subclasses de IgG humanas, o que sugere uma produção seqüencial de antígenos e moléculas que podem interagir com a resposta imune do hospedeiro.