Exclusion of synaptotagmin V at the phagocytic cup by Leishmania donovani lipophosphoglycan results in decreased promastigote internalization.

Regulators of membrane fusion play an important role in phagocytosis, as they regulate the focal delivery of endomembrane that is required for optimal internalization of large particles. During internalization of Leishmania promastigotes, the surface glycolipid lipophosphoglycan (LPG) is transferred to the macrophage membrane and modifies its fusogenic properties. In this study, we investigated the impact of LPG on the recruitment of the exocytosis regulator synaptotagmin V (Syt V) at the area of internalization and on the early steps of phagocytosis. Using Leishmania donovani LPG-defective mutants and LPG-coated particles, we established that LPG reduces the phagocytic capacity of macrophages and showed that it causes exclusion of Syt V from the nascent phagosome. Silencing of Syt V inhibited phagocytosis to the same extent as LPG, and these effects were not cumulative, consistent with a Syt V-dependent mechanism for the inhibition of phagocytosis by LPG. Previous work has revealed that LPG-mediated exclusion of Syt V from phagosomes prevents the recruitment of the vacuolar ATPase and acidification. Thus, whereas exclusion of Syt V from phagosomes in the process of formation may be beneficial for the creation of a hospitable intracellular niche, it reduces the phagocytic capacity of macrophages. We propose that the cost associated with a reduced internalization rate may be compensated by increased survival, and could lead to a greater overall parasite fitness.

The Leishmania donovani lipophosphoglycan excludes the vesicular proton-ATPase from phagosomes by impairing the recruitment of synaptotagmin V.

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 Galbeta1,4Manalpha1-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.

Malarial hemozoin activates the NLRP3 inflammasome through Lyn and Syk kinases.

The intraerythrocytic parasite Plasmodium -- the causative agent of malaria -- produces an inorganic crystal called hemozoin (Hz) during the heme detoxification process, which is released into the circulation during erythrocyte lysis. Hz is rapidly ingested by phagocytes and induces the production of several pro-inflammatory mediators such as interleukin-1beta (IL-1beta). However, the mechanism regulating Hz recognition and IL-1beta maturation has not been identified. Here, we show that Hz induces IL-1beta production. Using knockout mice, we showed that Hz-induced IL-1beta and inflammation are dependent on NOD-like receptor containing pyrin domain 3 (NLRP3), ASC and caspase-1, but not NLRC4 (NLR containing CARD domain). Furthermore, the absence of NLRP3 or IL-1beta augmented survival to malaria caused by P. chabaudi adami DS. Although much has been discovered regarding the NLRP3 inflammasome induction, the mechanism whereby this intracellular multimolecular complex is activated remains unclear. We further demonstrate, using pharmacological and genetic intervention, that the tyrosine kinases Syk and Lyn play a critical role in activation of this inflammasome. These findings not only identify one way by which the immune system is alerted to malarial infection but also are one of the first to suggest a role for tyrosine kinase signaling pathways in regulation of the NLRP3 inflammasome.

The exocytosis regulator synaptotagmin V controls phagocytosis in macrophages.

Synaptotagmins (Syts) play a key role in the regulation of Ca(2+)-triggered exocytosis and membrane fusion events, two crucial events associated to the phagocytic process. In the present study, we investigated the role of Syt V, a regulator of focal exocytosis, in phagocytosis. In macrophages, Syt V is localized on recycling endosomes and on filopodia-like structures and is recruited to the nascent phagosomes independently of the phagocytic receptor engaged. Silencing of Syt V by RNA interference revealed a role for this protein for phagocytosis, particularly under conditions of high membrane demand. In contrast, silencing of Syt V had no effect on the recruitment of the lysosomal marker LAMP1 to phagosomes, indicating that phagosome maturation is not regulated by Syt V. Collectively, these results illustrate the importance of Syt V in the regulation of an important innate function of macrophages. Furthermore, our results are consistent with the concept that focal exocytosis of endocytic organelles is a key event in phagocytosis and suggest that Syt V regulates this process.

Large scale phagosome preparation.

Phagocytosis is the process by which cells engulf and destroy large particles such as pathogens or apoptotic cells. In this way, macrophages play a pivotal role in the resolution of microbial infections. However, many microorganisms have evolved efficient strategies to preempt the weaponry of macrophages. A better understanding of the components engaged in the phagosome formation and maturation is necessary to devise novel approaches aimed at counteracting these microbial strategies. Recently, large-scale approaches have been used to improve our understanding of phagosome functional properties by the identification of hundreds of proteins and by studying each of them. Presently, purification of pathogen-containing phagosomes presents several technical challenges, whereas the use of latex beads to isolate phagosomes presents many advantages because this system can mimic host-pathogen interactions during phagocytosis. This system thus remains the best approach to advance our knowledge of phagosome biology, notably when used in conjunction with functional approaches. In this chapter, we outline an approach for the isolation of large-scale phagosome preparations with high degrees of purity.

Initiation of multicellular differentiation in Dictyostelium discoideum is regulated by coronin A.

Many biological systems respond to environmental changes by activating intracellular signaling cascades, resulting in an appropriate response. One such system is represented by the social amoeba Dictyostelium discoideum. When food sources become scarce, these unicellular cells can initiate a cAMP-driven multicellular aggregation program to ensure long-term survival. On starvation, the cells secrete conditioned medium factors that initiate cAMP signal transduction by inducing expression of genes such as cAMP receptors and adenylate cyclase. The mechanisms involved in the activation of the first pulses of cAMP release have been unclear. We here show a crucial role for the evolutionarily conserved protein coronin A in the initiation of the cAMP response. On starvation, coronin A-deficient cells failed to up-regulate the expression of cAMP-regulated genes, thereby failing to initiate development, despite a normal prestarvation response. Of importance, external addition of cAMP to coronin A-deficient cells resulted in normal chemotaxis and aggregate formation, thereby restoring the developmental program and suggesting a functional cAMP relay in the absence of coronin A. These results suggest that coronin A is dispensable for cAMP sensing, chemotaxis, and development per se but is part of a signal transduction cascade essential for system initiation leading to multicellular development in Dictyostelium.

Leishmania donovani lipophosphoglycan inhibits phagosomal maturation via action on membrane rafts.

Lipophosphoglycan (LPG), the major surface glycoconjugate on Leishmania donovani promastigotes, is crucial for the establishment of infection inside macrophages. LPG comprises a polymer of repeating Galbeta1,4Manalpha-PO(4) attached to a lysophosphatidylinositol membrane anchor. LPG is transferred from the parasite to the host macrophage membrane during phagocytosis and induces periphagosomal F-actin accumulation correlating with an inhibition of phagosomal maturation. The biophysical properties of LPG suggest that it may be intercalated into membrane rafts of the host-cell membrane. The aim of this study was to investigate if the effects of LPG on phagosomal maturation are mediated via action on membrane rafts. We show that LPG accumulates in rafts during phagocytosis of L. donovani and that disruption of membrane rafts abolished the effects of LPG on periphagosomal F-actin and phagosomal maturation, indicating that LPG requires intact membrane rafts to manipulate host-cell functions. We conclude that LPG associates with membrane rafts in the host cell and exert its actions on host-cell actin and phagosomal maturation through subversion of raft function.