| Background and ObjectivesTrichosporon asahii(T.asahii)is an opportunistic pathogenic fungus that often causes invasive fungal infections in people with immune deficiencies such as cancer patients with neutropenia.The patient’s mortality rate is still above 50%,even if they receive antifungal treatment.Unfortunately,people have little knowledge about the related molecular mechanisms of trichosporon asahii at present,such as pathogenic mechanism,drug resistance mechanism,and evolutionary mechanism within the hosts.We found a patient with chronic trichosporon asahii infection for 14 years.We successively isolated the primary strain AS2.2174 and the recurrent strain BM1403 from the patient’s facial skin lesions.This rare patient with chronic persistent fungal infection provides a unique advantage to study the evolutionary mechanisms of trichosporon asahii in the host.Methods1.TMT quantitation and bioinformatic analysis:Liquid chromatography tandem-mass spectrometry(LC-MS/MS)combined with tandem mass tag(TMT)labeling was performed to compare the differential expression of proteins in strain AS2.2174 and strain BM1403.Protein quantification data with a fold change≥1.2or≤0.83 and a p value<0.05 were identified as significantly differentially expressed proteins(DEPs).DEPs were analyzed by bioinformatics through Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes(KEGG)online service tool.2.Multiple Reaction Monitoring(MRM):The multiple reaction monitoring technique was used to verify the quantitative proteome data3.Phenotypic research:Growth assays,Melanization assays,Paromomycin assays and Biofilm production assays were performed to verify the correlation between proteomic changes and phenotypic changes,and to reveal the adaptive changes containing physiological characteristics and virulence factors of trichosporon asahii in the host.Results1.TMT quantitation and bioinformatic analysis:A total of 597 significantly differentially expressed proteins(DEPs)were quantitated,of which 293 were significantly upregulated,and 304 were significantly downregulated.Bioinformatics analysis revealed that these DEPs were involved mainly in energy production,protein metabolism and synthesis,drug metabolism,oxidative stress and other important biological processes and pathways.2.MRM analysis:Based on their annotations and different ratios to select proteins,the differential expression changes of 27 candidate proteins were evaluated by MRM analysis to verify the reliability of TMT-based quantitation results.The results of MRM analysis of 21 proteins were consistent with the results of TMT-based quantitation.3.Phenotypic research:The results of the growth assays showed that the growth of the recurrent strain BM1403 was similar to that of the initial strain AS2.2174 at 30℃ but exceeded that at 37℃ and 39℃.Melanin production of BM1403 in both plate and liquid assays was increased compared with AS2.2174 under the same conditions.The results of paromomycin assays showed that a narrow growth inhibition halo was observed in strain BM1403 compared to strain AS2.2174.CLSM images showed that in the recurrent strain,mature biofilm filamentation and thickness were enhanced compared with that of the initial strain(58.83 ±2.79μm vs 48.67±4.50μm,P<0.05)Conclusions1.After the evolution,the proteomics in T.asahii had significant changes,and DEPs were widely involved in a variety of different biological processes and pathways.2.MRM results showed that the candidate proteins exhibit a similar trend to TMT-based quantitation results,which further confirms the authenticity and reliability of the TMT-based quantitation results.3.The strain BM1403 may improve its high temperature tolerance,melanin production,protein translation efficiency and biofilm formation ability to ensure long-term survival in the host. |
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