Proteomic Analysis On The Regulation Of DOPA-melanin Synthesis In Talaromyces Marneffei

Objective(1)The study was aimed to investigate the proteomics of Talaromyces marneffei(TM)cultured with or without L-DOPA.(2)In order to understand the basic biological process in intracellular,bioinformatics exploration of identified proteins were applied.(3)So as to find a potential therapeutic target for TM infections,we did further study on an important identified protein.Methods(1)The strains of FRR2161 were cultured(without light)on ANM(A.nidulans media solution)medium with or without L-DOPA at 37? for 7 days.(2)Proteins were extracted by Yeast Buster proteins extraction reagent(Novagen),then proteins were quantificated by 2-D Quant-kit.(3)Protein profiles were generated by two-dimensional gel electrophoresis(2DGE)from whole cell proteins extracted from TM with or without melanin generated,protein profiles were analysed by ImageMaster 2D platinum 5.0.(4)Selected proteins were recovered,subjected to sequencing by matrix assisted iaserdesorption/ioninzation time of time of flight mass speetrometry mass spectrometry(MALDI-TOF/TOF MS)and putative identifications derived by searching available databases for homologous sequences in other fungi from Uniprot database.(5)Gene ontology(GO)annotation and metabolic pathways in the Kyoto Encyclopedia of Genes and Genomes(KEGG)were applied to study on identified proteins.(6)Real–Time quantitative PCR(RT-qPCR)was used to verify the selected proteins.(7)The strains of FRR2161 were cultured on BHI(Brain heart infusion agar)with or without GDA(Geldanamycin)at 37? for 7 days,to observe the melanin production in TM and TM growth situation,through naked eye,fluorescence microscope and TEM(transmission electron microscope).Result(1)There were 38 protein spots were discovered,15 protein spots in TM with melanin generated were higher expressed than TM without melanin generated,16 protein spots expressed only in TM with melanin generated,and 7 protein spots in TM with melanin generated were lower expressed than TM without melanin generated.(2)PMF(Peptide Mass Fingerprinting)showed matched proteins were mostly HSPs(heat shock proteins),especially HSP90.(3)RT-qPCR results showed that HSP90,HSP60 and HSP70 mRNA level were up-regulated at TM with melanin generated,and the results were consistent with proteomics.(4)GO analysis indicated that HSPs were enriched in most of the GO functional annotations,and the majority of differentially expressed proteins include HSPs were found enriched in response to stress,cellular process,protein folding,biological process and response to stimulus,KEGG pathway analysis showed that proteins were enriched in phagosome at most.(5)Visual inspection showed that the growth of TM were inhibited,the produce of melanin were reduced at BHI medium with GDA in it,fluorescent microscope showed that the form of TM was kinder changed to hypha at BHI medium with GDA in it.TEM showed that the cell wall of TM was damaged,vesicles were not expanded and less electron dense granules were observed at BHI medium with GDA in it,compared to the control group.Conclusion(1)HSPs paly an important role in TM DOPA-melanin generation,especially HSP90.(2)HSP90 inhibitor has inhibitory effects on TM growth and melanin production.(3)Metabolic pathways of melanin in TM are closely related to phagosome pathway.