Glyceraldehyde-3-phosphate dehydrogenase present in extracellular vesicles from Leishmania major suppresses host

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ful-fills various physiological roles that are unrelated to its glyco-lytic function. However, to date, the nonglycolytic function of GAPDH in trypanosomal parasites is absent from the litera-ture. 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 im-mune system. In this study, we demonstrate that the Leish-mania 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-alpha protein expression compared with control, overexpressed, and complement para-sites as determined by ELISA, RT-PCR, and immunoblot data. In vitro protein translation studies suggest that LmGAPDH-mediated TNF-alpha 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-alpha expression in macrophages during infection via post-transcriptional repression.