| Trichosporon asahii is now emerging as an important life-threatening opportunistic systemic pathogen, beyond these three important pathogenic fungi,including Candida spp., Aspergillus spp. and Cryptococcusspp. Trichosporon asahii can cause invasive trichosporonosis in immuno-compromised individuals and critically ill patients, resulting in high mortality rates (50-80%). Triazoles seem to have better in vitro and in vivo antifungal activities against Trichosporon spp. than the otherantifungal agents. Unfortunately, the over-prescription of these drugsfor prophylaxis or treatment led to an increase in resistance to azole drugs.ObjectiveThe aims were to analyze the overall cellular response to fluconazole, and to find fungal proteins involved in this response that might be useful targets inexploring the possible mechanism.Materials and Methods1 T. asahii drug resistance strains were induced in vitroDrug resistance was induced in vitro by a multi-step method forT. asahii strains CBS 2479 with FLC. We got the stable fluconazole-resistant T. asahii isolates,2479R.2Differential proteins identified by ITRAQWe used high throughput iTRAQ technology combined with mass spectrometry identification method,to determine and screene related proteins of differentiation in T. asahiiclinical isolates and in vitro fluconazole induced resistance and in response to fluconazole.3 Bioinformatics analysis of differential proteinsBioinformation analysis for the differentially expressed proteins including molecular function, cluster of orthologous groups of proteins and cluster of GO, in order to detect the biological function of differential proteins. Protein-protein interaction analysis and enrichment analysis of pathway were conducted to determine the metabolic pathway and the signal transduction pathways which differential proteins involved.4Differential proteins identified involved influconazoleresistance mechanisms of Trichosporon asahii.According to the results of bioinformatics analysis and integrated document analysis, differential proteins identified involved in fluconazole resistance mechanisms of Trichosporon asahii.Result:1 The resistant Trichosporon asahii strain was successfully induced in vitro.2 Differential protein identified by ITRAQFrom quantitative proteomic analysis of Trichosporon asahiiCSB8904, total of 77320 MS/MS spectra acquired from LC MS/MS analysis resulted in the identidication of 30682 peptieds and unique peptieds were 26847 corresponding to 7174 proteins. From quantitative proteomic analysis of Trichosporon asahiiCSB2479, total of 35653MS/MS spectra acquired from LC MS/MS analysis resulted in the identidication of 12746 peptieds and unique peptieds were12022 corresponding to 4585 proteins.3 Result of bioinformatics analysis of differential proteins3.1 Result of bioinformatics analysis of differential proteins for CBS8904 group:68 proteins differentially expressed between CBS8904 vs8904R by≥1.5-fold,34 were up and 34 were down regulated.33 proteins differentially expressed between CBS8904 vs8904F by≥1.5-fold,9 were up and 24 were down regulated; 38 proteins differentially expressed between CBS8904F vs8904RF by≥1.5-fold,31 were up and 7 were down regulated. Proteins differentially expressed in association with azole resistance included E g2、E g4、 E g5、E g7、E g11、E g26 involved in the ergosterol biosynthesis pathway, and Ste12、 TOR1、DDR48、HSP70、Calcineurin related protein involved in cellular Stress Responses.3.2 Result of bioinformatics analysis of differential proteins for CBS2479 group:167 proteins differentially expressed between CBS2479 vs2479R by≥1.5-fold,91 were up and76 were down regulated.55 proteins differentially expressed between CBS2479 vs2479F by≥1.5-fold,32were up and 23 were down regulated; 159 proteins differentially expressed between CBS2479F vs2479RF by≥1.5-fold,78 were up and 81 were down regulated. Proteins differentially expressed in association with azole resistance included E’g4、E’g5、E’g6、E g7、Eg11、E g25、E g26 involved in the ergosterol biosynthesis pathway, and DDR48、HSP70、Calcineurin related protein involved in cellular stress responses.Conclusion1 The resistant Trichosporon asahii strains were successfully induced in vitro and used for future resistance-related research.2 Differential protein identified between Trichosporon asahii and resistant Trichosporon asahii, changes in the proteome of Trichosporon asahii and resistant-Trichosporon asahii in response to fluconazole. Differential proteins identified involved in Trichosporon asahii with azole resistance mechanisms which were in involved in the ergosterol biosynthesis and cellular stress responses... |
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