Date Published: December 8, 2016
Publisher: Public Library of Science
Author(s): Christine Matte, Albert Descoteaux, Laura J Knoll.
Cellular functions such as phagocytosis and cytokine secretion rely heavily on a complex network of vesicle trafficking pathways that interconnect most membrane-bound intracellular compartments . A critical step in the exchange of cargoes between vesicles in this network is the process of membrane fusion, which is mediated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. This superfamily of integral and peripheral membrane proteins displays the distinctive SNARE motif, a stretch of heptad repeats that form a coiled-coil structure with a conserved arginine (R) or glutamine (Q) residue at the central “0” layer. Fusogenic SNARE complex formation requires the parallel association of an R-SNARE domain on the vesicle membrane with three cognate Q-SNARE domains on the target compartment. The subsequent “zippering” of this four-helix bundle from the membrane-distal amino termini towards the membrane-proximal carboxyl termini brings the two apposed membranes into close proximity and provides sufficient mechanical force to overcome the energy barrier for the formation of the fusion pore .
Binding and internalization of infective Leishmania stages to macrophages involves multiple phagocytic receptors . Phagocytosis of those large particles requires the expense of a considerable amount of plasma membrane for pseudopod extension around the prey. Various membrane-bound intracellular compartments lend a hand to this process by fusing with the cell surface and rapidly providing endomembrane required for particle engulfment . Focal exocytosis of recycling endosomes, for instance, contributes not only to phagocytosis but also allows for rapid secretion of preformed inflammatory cytokines including TNF and IL-6 . Fusion of recycling endosomes with the cell surface is mediated by the R-SNARE VAMP3 (vesicle-associated membrane protein 3) [1, 4] and is regulated by Syt V . Interestingly, Syt XI also localizes to recycling endosomes and is recruited to nascent phagosomes, but acts as a negative regulator of phagocytosis and cytokine secretion . Late endosomes and lysosomes assist large particle phagocytosis as well, in a VAMP7- and Syt VII-dependent manner [1, 4, 8]. Contribution of the endoplasmic reticulum (ER) as a source of endomembrane varies according to the nature of the phagocytosed particle and requires the ER Q-SNARE Stx18 [4, 9].
Pathogens use a variety of tactics to manipulate membrane fusion and vesicle trafficking to cause disease . The intracellular bacteria Chlamydia and Legionella, for instance, produce proteins with SNARE-like motifs that interact with host SNAREs and inhibit SNARE-mediated membrane fusion. The best-known example is the specific cleavage of SNAREs by clostridial neurotoxins, which are potent blockers of neurotransmission in peripheral cholinergic nervous system synapses . Leishmania promastigotes use two abundant surface GPI-anchored virulence factors to interfere with vesicle trafficking and fusion: GP63 (glycoprotein 63), a zinc-dependent metalloprotease, and LPG (lipophosphoglycan), a polymer of repeating Galβ1,4Manα1-PO4 units. Upon internalization of the parasites, GP63 and LPG are rapidly redistributed throughout infected cells (Fig 1). Akin to the clostridial neurotoxins, GP63 cleaves components of the host cell membrane fusion machinery, including VAMP3, VAMP8, and Syt XI (Table 1) [10, 15]. The consequences of these cleavage events are diverse. In macrophages and dendritic cells, processing of exogenous antigens for crosspresentation on MHC I molecules is controlled by the NADPH oxidase NOX2: phagosome oxidation prevents excessive acidification and destruction of peptides destined for recognition by T cells . Since VAMP8 is involved in the recruitment of NOX2 to phagosomes, GP63-mediated cleavage of VAMP8 results in increased phagosomal proteolytic activity, ensuing in defective crosspresentation of Leishmania antigens to T cells . In parallel, during a noncanonical autophagic process referred to as LC3-associated phagocytosis (LAP), NOX2-mediated phagosomal oxidation promotes the recruitment of the autophagy-related protein LC3 to a subset of phagosomes. Several roles have been attributed to LAP, including increased phagosomal microbicidal activity and enhanced antigen presentation on MHC II molecules . By cleaving VAMP8 and preventing phagosomal recruitment of NOX2, GP63 allows Leishmania major promastigotes to evade LAP , possibly contributing to the impairment of phagosome maturation and further inhibiting antigen presentation to T cells. Consistent with the role of Syt XI as a negative regulator of cytokine secretion, cleavage of this endosomal protein by GP63 from L. major promastigotes increases the postinfection release of TNF and IL-6 . These proinflammatory cytokines are responsible for the augmentation of neutrophil and inflammatory monocyte influx to the parasite inoculation site, which contributes to the spread and maintenance of infection.
Old World Leishmania species (L. major, L. donovani, and L. tropica) reside in small, tight-fitting PVs that undergo fission shortly after parasite replication, therefore rarely containing more than a single amastigote. On the other hand, the establishment of a successful infection by New World species (L. mexicana, L. amazonensis, and L. pifanoi) requires the formation of spacious, communal vacuoles that can harbour numerous parasites. Our understanding of the mechanisms allowing the development of individual versus communal PVs is very limited. Both types of PVs continuously interact with the host cell reservoir of acidic  and ER-derived vesicles , most likely to accommodate for the high membrane demand. Biogenesis of large communal PVs involves homotypic fusion between smaller PVs (Fig 2) , which may rely on the hijacking of specific components of the membrane fusion machinery. In support of this model, targeting the ERGIC (ER-Golgi intermediate compartment) Q-SNARE Stx5 or the ER R-SNARE Sec22b and its cognate Q-SNARE partners Stx18 and D12 restricts the expansion of L. amazonensis PVs and is detrimental to parasite replication . Whether the molecular basis for this fundamental difference in the lifestyle of these two groups of Leishmania is related to differential expression or activity of virulence factors such as GP63 that directly target specific components of the host cell machinery remains to be investigated.
As our understanding of the function of membrane fusion mediators deepens, we are able to get a better insight into the challenges faced by Leishmania parasites upon entry into host cells and, in parallel, the mechanisms of parasite virulence and pathogenesis. Conversely, Leishmania represents a superb tool for the identification of novel roles for the membrane fusion machinery in macrophages, by investigating the functional consequences of host protein cleavage or intracellular redistribution on cell and immune functions. Components of the membrane fusion machinery might emerge as targets for novel therapeutic interventions in infectious and inflammatory diseases.