Background

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. Methods: 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. Results: 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. Conclusions: The results demonstrates that HSP90 palys an essential role in T.marneffei DOPA-melanin synthesis pathway.

中文翻译：

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蛋白质组学分析马尔尼飞毛藻DOPA-黑色素合成的调控

背景

T.marneffei的酵母形式可产生DOPA-黑色素，其在巨噬细胞中存活的病原体中起重要作用。到目前为止，与黑色素合成相关的蛋白质组学在T.marneffei中仍不清楚。方法：整个酵母细胞蛋白从提取T.marneffei有或没有培养升-DOPA。使用二维凝胶电泳用MALDI-TOF质谱法结合，鉴定了区分蛋白T.marneffei有或没有培养升-DOPA。此外，使用格尔德霉素评估对马尔尼菲菌的抑制作用体外产生黑色素。结果：与非DOPA抑制的酵母细胞相比，在DOPA抑制的酵母细胞中鉴定出16种不同的蛋白质，以及15种三重表达的蛋白质和7种三重表达的蛋白质。值得注意的是，差异表达的蛋白质主要是热激蛋白质。通过q-RT-PCR证实HSP90 / 60/70基因表达显着增加，这与蛋白质组学的变化是一致的。GO分析表明，包括HSPs（尤其是HSP90）在内的大多数差异表达蛋白在应激反应，细胞过程，蛋白质折叠，刺激反应和生物学过程中均富集。KEGG通路分析表明，蛋白质主要富集在吞噬体中。HSP90抑制剂（格达霉素）抑制棕黑色素的产生透射电镜观察了在脑心浸液琼脂上生长的T.marneffei酵母及其抑制作用。结论：结果表明HSP90在T.marneffei DOPA-黑色素合成途径中发挥重要作用。