Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fulfills various physiological roles that are unrelated to its glycolytic function. However, to date, the nonglycolytic function of GAPDH in trypanosomal parasites is absent from the literature. Exosomes secreted from Leishmania, like entire parasites, were found to have a significant impact on macrophage cell signaling and function, indicating cross talk with the host immune system. In this study, we demonstrate that the Leishmania GAPDH (LmGAPDH) protein is highly enriched within the extracellular vesicles (EVs) secreted during infection. To understand the function of LmGAPDH in EVs, we generated control, overexpressed, half-knockout (HKO), and complement cell lines. HKO cells displayed lower virulence compared with control cells when macrophages and BALB/c mice were infected with them, implying a crucial role for LmGAPDH in Leishmania infection and disease progression. Furthermore, upon infection of macrophages with HKO mutant Leishmania and its EVs, despite no differences in TNFA mRNA expression, there was a considerable increase in TNF-α protein expression compared with control, overexpressed, and complement parasites as determined by ELISA, RT-PCR, and immunoblot data. In vitro protein translation studies suggest that LmGAPDH-mediated TNF-α suppression occurs in a concentration-dependent manner. Moreover, mRNA binding assays also verified that LmGAPDH binds to the AU-rich 3'-UTR region of TNFA mRNA, limiting its production. Together, these findings confirmed that the LmGAPDH contained in EVs inhibits TNF-α expression in macrophages during infection via posttranscriptional repression.

中文翻译：

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存在于主要利什曼原虫细胞外囊泡中的 3-磷酸甘油醛脱氢酶可抑制宿主 TNF-α 的表达。

3-磷酸​​甘油醛脱氢酶 (GAPDH) 履行与其糖酵解功能无关的各种生理作用。然而，迄今为止，文献中还没有 GAPDH 在锥虫寄生虫中的非糖酵解功能。发现从利什曼原虫分泌的外泌体与整个寄生虫一样，对巨噬细胞信号传导和功能有显着影响，表明与宿主免疫系统存在串扰。在这项研究中，我们证明了利什曼原虫 GAPDH (LmGAPDH) 蛋白在感染过程中分泌的细胞外囊泡 (EV) 中高度富集。为了了解 LmGAPDH 在 EV 中的功能，我们生成了对照、过表达、半敲除 (HKO) 和补体细胞系。当巨噬细胞和 BALB/c 小鼠感染 HKO 细胞时，与对照细胞相比，它们显示出较低的毒力，暗示 LmGAPDH 在利什曼原虫感染和疾病进展中的关键作用。此外，在用 HKO 突变体利什曼原虫及其 EV 感染巨噬细胞后，尽管 TNFA mRNA 表达没有差异，但与对照、过表达和补体寄生虫相比，TNF-α 蛋白表达显着增加，如通过 ELISA、RT-PCR 确定的和免疫印迹数据。体外蛋白质翻译研究表明 LmGAPDH 介导的 TNF-α 抑制以浓度依赖性方式发生。此外，mRNA 结合测定还证实 LmGAPDH 与 TNFA mRNA 的富含 AU 的 3'-UTR 区域结合，从而限制了其产生。总之，这些发现证实了 EV 中包含的 LmGAPDH 通过转录后抑制抑制感染期间巨噬细胞中 TNF-α 的表达。