Microevolution On Trichosporon Asahii During Chronic Infection

Part I The establishment of a co-culture system for Trichosporon asahiii microevolutionary isolate and murine bone marrow-derived dendritic cellObjective To establish and optimize a co-culture system for Trichosporon asahiii(T.asahii)microevolutionary isolate from an chronic Trichosporonosis patient and bone marrow-derived dendritic cell(BMDC)induced from wild-type mice.Methods In vitro culture of BMDC induced from C57BL/6 mice.Select the most suitable ratio between BMDC and heat-killed T.asahii by co-culturing them in different ratios,select the more suitable assay hour by measuring the concentration of cytokines produced by BMDC in 6h and 24h,and select the more suitable positive control by stimulating BMDC with LPS and?-glucan.Assess the expression of costimulatory molecules and the production of cytokines from BMDC stimulated by T.asahii throuh the optimized co-culture system.Results The most suitable ratio between BMDC and T.asahii should be 1:5,the more suitable assay hour should be in 24h,and ?-glucan should be more suitable as a positive control.The murine BMDC can present the antigens from T.asahii and develop antifungal effects on T.asahii by increasing the production of IL-6 and TNF-a.Conclusion The co-culture system is a reliable way for investigating the immunogenicity of T.asahii to murine BMDC.Part II The effects of microevolution on the immunogenicity and virulence of T.asahii during chronic infectionObjective To investigate the effects of microevolution on the immunogenicity and virulence of T.asahii during chronic infection.Methods Compare the morphological differences between T.asahii oringinal isolate(TO)and microevolutionary isolate(Tevo)from the same chronic Trichosporonosis patient throuh eyesight,optical microscope and scanning electron microscopy.In vitro culture of BMDC induced from C57BL/6 mice and co-culture it with active/heat killed TO and Tevo(1:5)for 24h,then measure the expression of TNF-a and IL-6 by BMDC.Assess the survival rate of mice infected with TO and Tevo.Results We can see obvious hyphal growth in TO and yeast growth in Tevo.Active Tevo induces much lower expression of TNF-? and IL-6 by BMDC than Active TO,but there are no differences when they were heat killed.Mice infected with Tevo show higher survival rate than TO.Conclusion Microevolution during chronic infection may attenuated the immunogenicity and virulence of T.asahii by changing its morphology.Part? Transcriptome analysis of the microevolution of T.asahii during chronic infectionObjective To investigate the molecular basis of the microevolution of T.asahii during chronic infection by comparing the transcripts of TO and Tevo.Methods Extract total RNA from TO and Tevo after they were thawed.Construct cDNA libraries by purified mRNA from total RNA.Sequence the cDNA libraries into RNA-seq reads and then make quality control of them.Compare the transcripts of TO and Tevo by analysing their gene expression levels,differential expressed genes(DEGs),and DEGs enrichment throuh Gene Ontology(GO)database and Kyoto Encyclopedia of Genes and Genomes(KEGG)database.Results All our downstream analyses were based on the clean data with high quality.The gene expression level of Tevo distributes differently compared with T0,totally 2212 DEGs.Among them,the down-regulated genes focus on biological process such as regulation of transcription,RNA biosynthetic or metabolic process and cellular components biosynthetic or metabolic process.While the up-regulated genes scatter on biologicalp rocess,cellular component and molecular function,such as DNA repair,carbonate utilize and host cell.What's more,the metabolic pathways of down-regulated genes most enrich in Valine,leucine and isoleucine degradation,while the up-regulated genes slightly enrich in tryptophan metabolism,nucleotide excision repair,mismatch repair and homologous recombination.Conclusion Microevolution can elicit co-exists with its host of T.asahii during chronic infection in the way of growth or metabolism adaptation directed by gene expression.