Toward a unified model for T. cruzi invasion

Despite the differences in their surface molecules and signaling pathways they employ for invasion, trypomastigotes, both metacyclic and tissue culture-derived, share the ability to enter a wide variety of mammalian host cells through an actin- independent process which retains in common a similar parasitophorous vacuole formation and maturation, suggesting that shared host cell traits must exist which allow for responsiveness to parasite signals and to support parasite entry.55

Injuries compromising the plasma membrane integrity of mammalian cells are repaired by a mechanism dependent on intracellular free Ca21 signaling, in which membrane wound resealing occurs by targeted lysosome exocytosis.56 Interestingly, the evidence demonstrating that bona fide acidic lysosomes could be mobilized to participate in this wound repair process came from studies of the invasion of nonphagocytic mammalian cells by T. cruzi, and it is believed that the parasite triggers this repair pathway and subsequently subverts it, hijacking the lysosomes to form the vacuole in which it gains access to the host cell.57 More recently, the repair process has been further characterized to demonstrate that pore-forming protein- induced lesions as well as mechanical wounds are removed from the plasma membrane by a coupled endocytosis step58 promoted by lysosomal acid sphingomyelinase, ASM.59 Therefore, Fernandes et al.60 studied whether this enzyme also plays a role during T. cruzi invasion, and showed that blocking ASM activity through inhibitors (desipramine) or RNA interference (RNAi) hampers trypomasti- gote invasion. Additionally, they showed that trypomastigotes cause wounds in the host cells plasma membrane during invasion (presumably through their intense motility and secretion of pore-forming molecules) allowing the flow of extracellular Ca21 into the cell through the lesions. The presence of ceramide in the membranes surrounding >60% of recently invading parasites was shown by immunostaining, and additional lysosomes were shown to progressively fuse with the parasitophor- ous vacuole.60 Therefore, the proposed model for invasion of nonphagocytic cells by T. cruzi has been recently updated as follows. Lysosome exocytosis toward parasite-induced wounds takes place, and fusion of lysosomes with the host cell plasma membrane occurs as part of the repair process. Upon fusion, lysosome content is released toward the cell surface, liberating ASM, which in turn hydrolyzes sphingomyelin on the external layer of the host cell plasma membrane and generating ceramide microdomains, which invaginate and facilitate the formation of the parasitophorous vacuole and the entrance of the parasite into the cell.55,60 This invagination of the plasma membrane, which in the lesion repair removes the membrane lesions by endocytosis is likely to represent the “plasma membrane” invasion route described above.48 Subsequent fusion of lysosomes with the parasitophorous vacuole would ensure parasite retention inside the host cell.55,60 It has been proposed that the frequency with which striated and cardiac muscle cells undergo plasma membrane injury may be related to the parasite tropism toward these cell types.55 This unified model thus reconciles the lysosome-dependent and plasma membrane invasion routes as part of the same invasion process. Additionally, in an attempt to include the knowledge about cruzipain-induced kinin signaling within this unified invasion framework, Scharfstein et al.61 have hypothesized that lipid rafts containing B2R and other G-protein coupled receptors could be internalized during the ASM-mediated invagination of ceramide-rich plasma membrane domains. This hypothesis would predict that during the cell membrane invagination and/or inside the parasitophoruos vacuole the flagellar pocket and the receptors would be in close proximity allowing for the signaling events triggered by the cruzipain-mediated generation of kinins, although experimental support for this is still lacking.

As the unified model of invasion evolves, it is predicted that newer studies will be interpreted within its framework. In some cases, recent findings regarding the T. cruzi invasion process have already been presented by the authors within the framework of this unified invasion model. In one such case, Zhao et al.62 reported that trypomastigotes invade in with parallel orientation to the host cell microtubules. Microtubule cytoplasmic linker associated protein-1 (CLASP1), a plus endtracking protein involved in microtubule stabilization at the cell periphery, was shown to play a key role in trypomastigote internalization, lysosome fusion with the parasitophorous vacuole and postentry parasite localization near the host cell nucleus.62 Furthermore, knocking down CLASP1 impaired all of these events without affecting Ca2+-mediated lysosome exocytosis. Importantly, besides the findings about T. cruzi invasion, this study showed evidence that CLASP1 is involved in the dynamics of intracellular localization of lysosomes in mammalian cells. Fig. 26.2 shows a model for T. cruzi invasion where the different invasion routes are unified. Some of the findings in the study by Zhao et al.62 are also included in Fig. 26.2.

In other cases, even very recent studies are still interpreted within a framework which considers different invasion routes for T. cruzi exist. For example, Cortez et al.63 recently reported that lysosome exocytosis and scattering induced by shortterm nutrient deprivation of the host cells, promotes host cell invasion by metacyclic trypomastigotes while reducing invasion by tissue culture-derived trypomastigotes. Rapamycin treatment of the host cells prior to infection produced the opposite effect, where metacyclics infected less. The interpretation of these findings by the authors was that metacyclic trypomastigotes and tissue culture-derived trypomastigotes invasion occur by different mechanisms, where the former invade mainly through a lysosome-dependent route, while the later invade through a lysosome-independent route.63 These results would argue that even if T. cruzi invasion occurs though a unified mechanism, different parasite life cycle forms could favor the usage of different components of it. If this were true, it is likely that different genetic lineages and isolates of T. cruzi could display such preferences as well.

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