Toxoplasma Mechanisms for Delivery of Proteins and Uptake of Nutrients Across the Host-Pathogen Interface

Abstract

Many intracellular pathogens, including the protozoan parasite Toxoplasma gondii, live inside a vacuole that resides in the host cytosol. Vacuolar residence provides these pathogens with a defined niche for replication and protection from detection by host cytosolic pattern recognition receptors. However, the limiting membrane of the vacuole, which constitutes the host-pathogen interface, is also a barrier for pathogen effectors to reach the host cytosol and for the acquisition of host-derived nutrients. This review provides an update on the specialized secretion and trafficking systems used by Toxoplasma to overcome the barrier of the parasitophorous vacuole membrane and thereby allow the delivery of proteins into the host cell and the acquisition of host-derived nutrients.

Keywords: Toxoplasma gondii; nutrient acquisition; parasitophorous vacuole; secreted effectors; trafficking; translocon.

Figure 1.. Trafficking of proteins to Toxoplasma’s specialized secretory organelles.

Vesicles containing GRAs, MICs and RONs/ROPs are trafficked from the ER to the Golgi via the COPII complex (2) while the COPI complex assures retrograde transport (69). At the Golgi, each type of secretory protein localizes to a distinct Golgi region where MICs and RONs/ROPs could bind to the N-terminal domain of the transmembrane Sortilin-Like Receptor (TgSORTLR), which binds to the Adaptor Protein (AP)-1 complex with its C-terminal di-leucine motif (LL) (123). Some MICs (e.g., MIC2, AMA1) possess a transmembrane domain and a tyrosine motif (YxxΦ) (127) that could directly interact with AP1. The hydrophobic domains present in many GRAs are likely kept soluble by a chaperone-like protein, which could interact with an unknown GRA transporter protein. Exported GRAs are cleaved at the TEXEL motif (RRL) by the Golgi-resident ASP5 protease and the resultant N-terminal domain could be acetylated and associate with a chaperone-like protein. The scission of vesicles at the Golgi depends on the dynamin-related protein DrpB (14). MIC- and RON/ROP-containing vesicles are sorted from the Golgi (red and green arrows, respectively) and delivered to Rab5-/Vacuolar Protein Sorting (Vps)9-positive endosomal compartments (114) in a Clathrin/AP1 complex dependent manner (103). It is still unclear how MICs and RONs/ROPs dissociate from TgSORTLR. Subsequently, TgSORTLR could be transported from a Rab5- to a Rab7/VAC-positive endosomal compartment using the Vps11/HOPs complex (87), and finally recycled back to the Golgi by the retromer complex (115). Apical MICs (e.g., MIC3/8) and RONs/ROPs are delivered to their respective pre-organelles via Rab5A/C-positive vesicles (71) while delivery of lateral MICs (e.g., AMA1, MIC2/M2AP) is dependent on the Vps11/HOPs complex (87). In the pre-micronemes, the pro-domain of MICs could be cleaved by the protease TgCPL (37) while in the pro-rhoptries the SUB2 protease could cleave RON/ROP pro-domains (84). It is still unclear how pre-micronemes and pre-rhoptries mature into micronemes and rhoptries. After maturation, lateral and apical micronemes are attached to cortical microtubules using TgCEN2 and TgDLC8a, respectively. The transfer of lateral micronemes to the conoid to get secreted seems to be also dependent on TgDLC8a (74). An unknown protein could traffic RON proteins together from the Golgi, thereby promoting the localization of these proteins at the neck of the rhoptry. Subsequently, TgCA_RP could mediate the fusion of pre-rhoptries containing ROPs with the neck of the rhoptry (22). The mature rhoptry is then transported to the conoid using actin/myosin motors and TgARO. The final attachment of rhoptries to the conoid microtubule depends on TgCSCHAP (86) and TgDLC8a. The sorting of GRAs from the Golgi compartment (purple arrows) is dependent on Vps9 (114) but independent of Rab5-positive vesicles. Rab6 between the Golgi and endosome could be necessary for the formation of a putative pre-dense granule compartment (124). The retromer complex could also recycle the putative GRA transporter from the pre-dense granules to the Golgi, where syntaxin TgSTX6 together with t/v-SNARE assures vesicle fusion to the Golgi (63). It is unclear how GRAs are packed into mature dense granules.

Figure 2.. (A) Model for rhoptry secretion.

1) The process of host cell invasion raises the parasite internal [Ca2+] and the upper polar ring and conoid are extended until the base of the conoid protrudes beyond the lower polar ring (62). MICs are secreted, and MIC8/CLAMP could bind to the parasite and host plasma membrane (67, 120). The increase in [Ca2+] or the presence of MICs on the PPM could trigger a signaling cascade that changes the parasite phospholipid composition. The retracted conoid with rhoptry organelle moves through the cortical microtubules towards the apical polar ring. TgRASP and a putative v-SNARE protein are present at the membrane of the apical end of the rhoptry. A putative t-SNARE and TgFER2 at the PPM could bind to the membrane upon sensing a calcium signal. 2) When the conoid and the apical end of the rhoptry (containing RON proteins) are close to the apical polar ring, TgFER2 could mediate the interaction of the t/v-SNARE and bring the rhoptry membrane closer to the PPM, which could allow TgRASP2 to interact with PA/PIP2 (Phosphatidic Acid/Phosphatidylinositol (4,5)-bisphosphate) on the PPM. 3) The conoid ultimately passes through the apical polar ring, and the t/v-SNARE/TgFER2/TgRASP2 complex could mediate the fusion of the rhoptry membrane and the PPM allowing RONs/ROPs to be secreted. Possibly MIC/ROP/RON secretion changes the permeability of the host plasma membrane at the attachment site allowing RONs and ROPs to pass through and reach the cytosol. (B) Model for trafficking of GRAs from dense granules to their destinations. (1) Inside the dense granules, hydrophobic GRAs are packed as soluble oligomers via shielding of their hydrophobic domains by putative chaperone-like GRAs to avoid fusion with the dense granule membrane. Mature dense granules bind to myosin and/or Rab11A-positive vesicles, which mediate the transport of dense granules via actin tracks to the parasite’s periphery where they dock at IMC gaps. (2) Docking of dense granules at an IMC gap allows their fusion with the PPM via the NSF/SNAP/SNARE/Rab machinery resulting in exocytosis of GRAs into the PV lumen. (3) Once secreted into the PV lumen, ASP5-processed TEXEL-containing exported GRAs with potential N-terminal acylation are escorted by unknown chaperone GRAs to the PVM translocon, of which MYR1/2/3/4 and GRA44 may be components. GRA45 likely functions as a chaperone helping the translocon components traffic to and insert into the PVM. GRA24 is a TEXEL-negative exported protein and traffics to the membrane translocon via an unknown mechanism followed by translocation into the host cell. (4) Hydrophobic GRAs (transmembrane, amphipathic helices or α-helices) are escorted by chaperone GRAs to traffic inside the PV lumen and reach their destinations through an unknown mechanism. WNG kinase (WNG1) is involved in the eventual insertion of GRAs into the PVM or IVN via either directly phosphorylating the hydrophobic GRAs or phosphorylating the chaperone GRAs leading to the dissociation of hydrophobic GRA cargo from the chaperone. (C) Nutrient and small molecule import across the PVM. Host organelles, such as endoplasmic reticulum (ER), Golgi apparatus and the microtubule organizing center (MTOC), are recruited to the PVM. The relocation of the MTOC leads to the conversion of the microtubules network around the PVM bringing vesicular organelles, such as endolysosomal vesicles, to its vicinity where they can be used as nutrient resources for Toxoplasma. Host Golgi is retained in PVM invaginations by GRA3 whereas ER and MTOC are recruited via unknown PVM proteins. Host microtubules are recruited to invaginations of the PVM, mediated by GRA7, where they form a structure named H.O.S.T. responsible for cholesterol uptake derived from host lysosomes. Small nutrients are likely acquired through a membrane pore formed by GRA17/23 and/or other unknown PVM transporters. Host lysosomes, lipid droplets, Rab-positive vesicles derived from the endolysosomal system as well as polypeptides are invaginated into the PV and transiently stored at the IVN and intravacuolar vesicles of which the outer membrane can be digested by TgLCAT resulting in the release of nutrients into the PV lumen. GRA2 and GRA6 are present in the IVN and mediate the uptake of host cytosolic proteins. Host mitochondria are anchored to the PVM by MAF1. ABCG₁₀₇ is a potential PVM transporter for scavenging host lipids and maintaining lipid homeostasis inside the PV lumen.