Published On: Mon, Mar 18th, 2019

The Toxoplasma gondii dense granule protein TgGRA3 interacts with host Golgi and dysregulates anterograde transport [RESEARCH ARTICLE]


In this study, we revealed one of the molecular mechanisms of host organelle recruitment by the parasite T. gondii. Combining biochemical approaches and mass spectrometry, we showed that TgGRA3 interacts with the host Golgi apparatus. Previously, yeast two-hybrid studies indicated that TgGRA3 binds to the host ER protein calcium-modulating ligand (CAMLG) (Kim et al., 2008). In addition, a retrieval ER motif was found in TgGRA3 C-terminal sequence, suggesting a role of this protein in host organelle hijacking (Henriquez et al., 2005). However, it was reported that TgGRA3-knockout mutants were not impaired in recruitment of host ER and mitochondria (Craver and Knoll, 2007). Instead, we clearly showed that TgGRA3 could bind to host Golgi-enriched fractions. The host Golgi seemed to enter into the PV through TgGRA3 coated invaginations formed at the PVM. We do not know whether TgGRA3 has a direct interaction to host Golgi proteins or lipids or if this binding requires other partners. Nevertheless, we do know that the C-terminal region of TgGRA3 contains a lipid-binding site and a coiled-coil domain that is important for the protein oligomerization. The C-terminal region of TgGRA3 may be required for host Golgi association and binding to another dense granule protein TgGRA23. Based on these findings, we propose that TgGRA3 coats tubules, which are necessary for targeting host organelles to the PV and subsequent membrane scavenging (Fig. 8D).

In addition, we showed that TgGRA3 modulates anterograde trafficking of infected host cells, a function related to the targeting of host Golgi material to the PV. We found that the parasite also secretes a filamentous network containing TgGRA3 into the host cytoplasm. This network connecting the PV is in very close proximity to the host Golgi. TgGRA3-positive filamentous projections were previously observed in T. gondii (Dubremetz et al., 1993) and Plasmodium chabaudi (Lanners et al., 1999), and similar PV projections have been observed in association with TgGRA7 (Dunn et al., 2008; Romano et al., 2013). The composition and role of these projections remain unknown but they could be involved in tethering host organelles including the Golgi apparatus to the PV. Interestingly, filaments originating from vacuoles containing intracellular bacteria such as Salmonella have also been observed and are thought to be important for bacterial replication (Garcia-del Portillo et al., 1993). We hypothesize that, in T. gondii, these filaments are important for delivery of proteins involved in host Golgi fragmentation or targeting of organelles from other host vesicles.

Parasites lacking TgGRA3 also had a defect in host Golgi material entry at the PVM. This result reveals that TgGRA3 may play dual roles: one in host Golgi targeting to the PV and one in Golgi material entry. Indeed, we observed that TgGRA3 coats tubules wrapping host Golgi vesicles in the intravacuolar network. Moreover, TgGRA3 could also influence the shape of these tubules in the intravacuolar network, as evidenced by the abnormal vesicles in TgGRA3-depleted parasites, a phenotype not reported previously (Rommereim et al., 2016). In addition, their disappearance in TgGRA3 knockdown parasites indicated a role of TgGRA3 in their maintenance. Observation of INV tubules fusing with the PVM are not uncommon and were previously visualized with host microtubules (Coppens et al., 2006). Although, we still could not determine if this INV tubules are acting as a common mechanism used by T. gondii to consume host organelles. Purified recombinant TgGRA3 binds lipids and oligomerizes due to its C-terminal region. TgGRA3 binds specifically to PI-(3,4,5) P3, which was found at the host Golgi (Low et al., 2010) but also PI-(3,4) P2 and PI-(4,5) P2 lipids, which are principally found in the host plasma membrane. Given that the PV originates from the host plasma membrane, and considering the proximity of the lipid-binding site with one of TgGRA3 putative transmembrane domain, we suggest that these lipid species may form microdomains at the PVM, which can recruit TgGRA3 to the PVM. Accordingly, we propose a model for TgGRA3 function in host cell Golgi recruitment that requires protein localization to specific lipid domains of the PVM and oligomerization (Fig. 8D). Crystal structure studies on TgGRA3 protein would be necessary to verify TgGRA3 topology and support our finding.

It has been described that intracellular T. gondii sequesters host Golgi Rab vesicles via a phagocytosis-like process and tubules containing two other dense granule proteins, TgGRA2 and TgGRA6 (Romano et al., 2017). This observation confirms the roles played by dense granule proteins in sequestration and engulfment of host organelles. The functional redundancy of these proteins may explain why knockout of only one gene does not have severe deleterious consequences on parasite survival. We conclude that these dense granule proteins, including TgGRA3, act in concert to promote sequestration of host organelles, thereby providing nutrients essential for parasite growth. In addition, we propose that TgGRA3, which binds to specific lipid domains, could cause PVM deformation and modulate the formation of tubules. Host Golgi material interacts with TgGRA3 at these sites and then could induce its own transport inside TgGRA3-coated tubules.

Finally, we observed that depletion of TgGRA3 restored a normal anterograde trafficking in infected host cells, suggesting that the parasite could regulate the host secretory pathway. This observation indicates that, in addition to lipid scavenging, the parasite could recruit host Golgi membranes for other reasons. The ability of T. gondii to divert the host cell’s Rab positive vesicles that are involved in anterograde trafficking (Romano et al., 2013) is in good agreement with our finding. The potential advantages of recruiting host Golgi membranes and modulating anterograde transport could include inhibition of cytokine secretion and the host immune response, as previously proposed by Murray and Stow (2014). As mentioned above, TgGRA3 also plays an important role in parasite virulence (Craver and Knoll, 2007), a phenotype that may be linked to the pre-emption of key host immune molecules involved in controlling the outcome of parasite infection. In line with this point of view, proteomic studies identified TgGRA3 as a protein that was more highly expressed in virulent T. gondii strains than in less virulent strains (Qiu et al., 2016).

In conclusion, this study reveals a new strategy employed by T. gondii to target the host Golgi apparatus and divert its functions. Different parasite mutants, including those lacking multiple dense granule proteins (e.g. simultaneous knockouts of TgGRA2, TgGRA3, and TgGRA6 or others), may completely abrogate host Golgi binding and recruitment. These kind of mutants would provide deeper insight into the role of host organelle hijacking by T. gondii.

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