Modulation of TLR4 Sialylation Mediated by a Sialidase Neu1 and Impairment of Its Signaling in Leishmania donovani Infected Macrophages.

Abstract

Altered sialylation is generally maintained by a fine balance between sialidases and sialyltransferases, which plays an essential role during disease pathogenesis. TLR4 is a membrane-bound highly sialylated glycoprotein predominantly having α2,3-linked sialic acids. It is one of the most important client molecules in the anti-leishmanial innate immune arm. Here, we initiated a comprehensive study on the modulation of TLR4 sialylation in Leishmania donovani (L. d)-infected macrophages by a mammalian sialidase/neuraminidase-1 (Neu1) having substrate specificity toward α2,3-linked sialic acids. We observed reduced membrane-associated Neu1 with its decreased enzyme activity in infected macrophages. Moreover, we demonstrated reduced association of Neu1 with TLR4 leading to enhanced sialylation of TLR4 in these infected cells. Conversely, Neu1 over expression exhibited enhanced association of TLR4 with Neu1 leading to reduced sialylation which possibly linked to increased association of TLR4 with its downstream adaptor protein, MyD88. This, in turn, activated downstream MAP kinase signaling pathway, with enhanced nuclear translocation of NFκB that resulted in increased genetic and protein levels expression of Th1 cytokines and effector molecule nitric oxide secretion which ultimately leads to reduced parasite burden in macrophages. This was further validated by Neu1 silencing in infected macrophages which reversed such a situation. Such events strongly confirm the importance of Neu1 in modulation of TLR4 sialylation during parasite infection resulting in impairment of innate immune response. Furthermore, decreased membrane-bound Neu1 in infected macrophages could be attributed to its reduced tyrosine-phosphorylation as well as diminished association with cathepsin A. Both these phenomenon possibly play significant roles in inhibiting translocation of the sialidase from cytosol to membrane. Taken together, our study first time demonstrated impaired translocation of cytosolic Neu1 to the membrane of L. donovani-infected macrophages due to impaired phosphorylation of this enzyme. This novel finding establishes a link between enhanced α2,3-linked sialic acids on TLR4 and reduced membrane-bound Neu1 which plays a significant role for inhibiting downstream signaling to establish successful infection in the host cells.