The Leishmania donovani Lipophosphoglycan Excludes the Vesicular Proton-ATPase from Phagosomes by Impairing the Recruitment of Synaptotagmin V

Abstract

We recently showed that the exocytosis regulator Synaptotagmin (Syt) V is recruited to the nascent phagosome and remains associated throughout the maturation process. In this study, we investigated the possibility that Syt V plays a role in regulating interactions between the phagosome and the endocytic organelles. Silencing of Syt V by RNA interference revealed that Syt V contributes to phagolysosome biogenesis by regulating the acquisition of cathepsin D and the vesicular proton-ATPase. In contrast, recruitment of cathepsin B, the early endosomal marker EEA1 and the lysosomal marker LAMP1 to phagosomes was normal in the absence of Syt V. As Leishmania donovani promastigotes inhibit phagosome maturation, we investigated their potential impact on the phagosomal association of Syt V. This inhibition of phagolysosome biogenesis is mediated by the virulence glycolipid lipophosphoglycan, a polymer of the repeating Galβ1,4Manα1-PO ₄ units attached to the promastigote surface via an unusual glycosylphosphatidylinositol anchor. Our results showed that insertion of lipophosphoglycan into ganglioside GM1-containing microdomains excluded or caused dissociation of Syt V from phagosome membranes. As a consequence, L. donovani promatigotes established infection in a phagosome from which the vesicular proton-ATPase was excluded and which failed to acidify. Collectively, these results reveal a novel function for Syt V in phagolysosome biogenesis and provide novel insight into the mechanism of vesicular proton-ATPase recruitment to maturing phagosomes. We also provide novel findings into the mechanism of Leishmania pathogenesis, whereby targeting of Syt V is part of the strategy used by L. donovani promastigotes to prevent phagosome acidification.

Author Summary

Upon their internalization by macrophages, Leishmania donovani promastigotes inhibit phagolysosome biogenesis. This inhibition is mediated by the virulence glycolipid lipophosphoglycan (LPG), attached to the promastigote surface. We recently showed that the exocytosis regulator Synaptotagmin (Syt) V controls early steps of phagocytosis, and remains associated to the phagosome during the maturation process. Here, we show that Syt V contributes to phagolysosome biogenesis by regulating the acquisition of the hydrolase cathepsin D and the vesicular proton-ATPase. Insertion of LPG into lipid microdomains of the phagosome membrane excluded Syt V from phagosomes, enabling L. donovani promatigotes to inhibit the recruitment of the vesicular proton-ATPase to phagosomes, preventing their acidification. Collectively, our results provide novel insight into the mechanism of vesicular proton-ATPase recruitment to maturing phagosomes and reveal how the virulence glycolipid LPG contributes to the mechanism of L. donovani pathogenesis by preventing phagosome acidification.