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ABSTRACTAll life cycle stages of the protozoan parasite Trypanosoma cruziare enveloped by mucin-like glycoproteins which, despite major changes in their polypeptide cores, are extensively and similarly O-glycosylated. O-Glycan biosynthesis is initiated by the addition of aGlcNAc to Thr in a reaction catalyzed by Golgi UDP-GlcNAc:polypeptide O-a-N-acetyl-d-glucosaminyltransferases (ppaGlcNAcTs), which are encoded by TcOGNT1and TcOGNT2. We now directly show that TcOGNT2 is associated with the Golgi apparatus of the epimastigote stage and is markedly downregulated in both differentiated metacyclic trypomastigotes (MCTs) and cell culture-derived trypomastigotes (TCTs). The significance of downregulation was examined by forced continued expression of TcOGNT2, which resulted in a substantial increase of TcOGNT2 protein levels but only modestly increased ppaGlcNAcT activity in extracts and altered cell surface glycosylation in TCTs. Constitutive TcOGNT2 overexpression had no discernible effect on proliferating epimastigotes but negatively affected production of both types of trypomastigotes. MCTs differentiated from epimastigotes at a low frequency, though they were apparently normal based on morphological and biochemical criteria. However, these MCTs exhibited an impaired ability to produce amastigotes and TCTs in cell culture monolayers, most likely due to a reduced infection frequency. Remarkably, inhibition of MCT production did not depend on TcOGNT2 catalytic activity, whereas TCT production was inhibited only by active TcOGNT2. These findings indicate that TcOGNT2 downregulation is important for proper differentiation of MCTs and functioning of TCTs and that TcOGNT2 regulates these functions by using both catalytic and noncatalytic mechanisms.

Infection with Leishmania majorelicits myeloid‐derived suppressor cells, which kill Leishmania parasites despite suppressed T‐cell proliferation. Th1/Th2 cytokines play a key role in immune responses to Leishmania majorby controlling macrophage activation for NO production and parasite killing. MDSCs, including myeloid precursors and immature monocytes, produce NO and suppress T cell responses in tumor immunity. We hypothesized that NO‐producing MDSCs could help immunity to L. majorinfection. Gr1hi(Ly6Chi) CD11bhiMDSCs elicited by L. majorinfection suppressed polyclonal and antigen‐specific T cell proliferation. Moreover, L. major‐induced MDSCs killed intracellular parasites in a NO‐dependent manner and reduced parasite burden in vivo. By contrast, treatment with ATRA, which induces MDSCs to differentiate into macrophages, increased development of lesions, parasite load, and T cell proliferation in draining LNs. Altogether, these results indicate that NO‐producing MDSCs help protective immunity to L. majorinfection, despite suppressed T cell proliferation.