Department of Dermatology and Venereology, First Affiliated Hospital,Guangxi Medical University, Nanning, Guangxi, 530021, China.
Department of Dermatology and Venereology, First Affiliated Hospital,Guangxi Medical University, Nanning, Guangxi, 530021, China; Guangxi Key Laboratory of AIDS Prevention and Treatment,Nanning, Guangxi, 530021, China.
Microb Pathog. 2021 Jan;150:104701. doi: 10.1016/j.micpath.2020.104701. Epub 2020 Dec 17.
Yeast form of T.marneffei can produce DOPA-melanin which perform an important role in the pathogen surviving in macrophage. So far, the proteomic associated with melanin synthesis remain unclearly in T.marneffei.
The whole yeast cell proteins were extracted from T.marneffei cultured with or without l-DOPA. Using two-dimensional gel electrophoresis combined with MALDI-TOF mass spectrometry, distinguished proteins were identified between T.marneffei cultured with or without l-DOPA. Furthermore, geldanamycin were used to assess the inhibition effect on T.marneffei melanin production in vitro.
16 distinguished proteins were identified in DOPA-melanized yeast cells, as well as 15 triple-up-expressed proteins and 7 triple-down-expressed proteins in comparison with non DOPA-melanized yeast cells. Of note, proteins differentially expressed proteins were predominantly heat shock proteins. HSP90/60/70 genes expressions increased significantly demonstrated by q-RT-PCR, which was consistent with the proteomics changes. GO analysis showed that the majority of differentially expressed proteins including HSPs(especially HSP90) were found enriched in stress response, cellular process, protein folding, stimuli response and biological process. KEGG pathway analysis showed that proteins were enriched predominantly in phagosome. HSP90 inhibitor(Geldanamycin) inhibited the brown-black pigment production of T.marneffei yeast grown on brain heart infusion agar, as well as the inhibition effect was observed by transmission electron microscope.
The results demonstrates that HSP90 palys an essential role in T.marneffei DOPA-melanin synthesis pathway.
新生隐球菌的酵母形式可以产生 DOPA-黑色素，这在病原体在巨噬细胞中存活中起着重要作用。迄今为止，与黑色素合成相关的蛋白质组学在新生隐球菌中仍不清楚。
从用或不用 l-DOPA 培养的新生隐球菌中提取整个酵母细胞蛋白。使用二维凝胶电泳结合 MALDI-TOF 质谱，鉴定出在有或没有 l-DOPA 培养的新生隐球菌之间存在差异的蛋白质。此外，还使用 geldanamycin 评估其对新生隐球菌黑色素生成的体外抑制作用。
在 DOPA-黑色素化的酵母细胞中鉴定出 16 种有差异表达的蛋白质，与非 DOPA-黑色素化的酵母细胞相比，有 15 种三倍表达上调的蛋白质和 7 种三倍表达下调的蛋白质。值得注意的是，差异表达的蛋白质主要是热休克蛋白。q-RT-PCR 显示 HSP90/60/70 基因表达显著增加，与蛋白质组学变化一致。GO 分析表明，大多数差异表达的蛋白质包括 HSPs（特别是 HSP90）在应激反应、细胞过程、蛋白质折叠、刺激反应和生物过程中富集。KEGG 途径分析表明，蛋白质主要富集在吞噬体中。HSP90 抑制剂（geldanamycin）抑制了在脑心浸液琼脂上生长的新生隐球菌酵母的棕黑色色素产生，并且在透射电子显微镜下观察到了抑制作用。
结果表明 HSP90 在新生隐球菌 DOPA-黑色素合成途径中发挥着重要作用。
