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Systematic characterization of all Toxoplasma gondii TBC domain-containing proteins identifies an essential regulator of Rab2 in the secretory pathway [1]

['Justin J. Quan', 'Department Of Microbiology', 'Immunology', 'Molecular Genetics', 'University Of California', 'Los Angeles', 'California', 'United States Of America', 'Lachezar A. Nikolov', 'Department Of Biology']

Date: 2024-05

Toxoplasma gondii resides in its intracellular niche by employing a series of specialized secretory organelles that play roles in invasion, host cell manipulation, and parasite replication. Rab GTPases are major regulators of the parasite’s secretory traffic that function as nucleotide-dependent molecular switches to control vesicle trafficking. While many of the Rab proteins have been characterized in T. gondii, precisely how these Rabs are regulated remains poorly understood. To better understand the parasite’s secretory traffic, we investigated the entire family of Tre2-Bub2-Cdc16 (TBC) domain-containing proteins, which are known to be involved in vesicle fusion and secretory protein trafficking. We first determined the localization of all 18 TBC domain-containing proteins to discrete regions of the secretory pathway or other vesicles in the parasite. Second, we use an auxin-inducible degron approach to demonstrate that the protozoan-specific TgTBC9 protein, which localizes to the endoplasmic reticulum (ER), is essential for parasite survival. Knockdown of TgTBC9 results in parasite growth arrest and affects the organization of the ER and mitochondrial morphology. TgTBC9 knockdown also results in the formation of large lipid droplets (LDs) and multi-membranous structures surrounded by ER membranes, further indicating a disruption of ER functions. We show that the conserved dual-finger active site in the TBC domain of the protein is critical for its GTPase-activating protein (GAP) function and that the Plasmodium falciparum orthologue of TgTBC9 can rescue the lethal knockdown. We additionally show by immunoprecipitation and yeast 2 hybrid analyses that TgTBC9 preferentially binds Rab2, indicating that the TBC9-Rab2 pair controls ER morphology and vesicular trafficking in the parasite. Together, these studies identify the first essential TBC protein described in any protozoan and provide new insight into intracellular vesicle trafficking in T. gondii.

Funding: This work was supported by National Institute of Allergy and Infectious Diseases R01 AI123360 (to P.J.B.) and AI060767 to (I.C.). This work was also supported by National Institute of General Medical Sciences R01 GM089778 (to J.A.W), as well as startup funds from the University of California, Los Angles and Indiana University (to L.A.N.). In addition, J.J.Q was supported by a Milton Gottlieb endowment award and by a Beckman Scholars Award through the Arnold and Mabel Beckman Foundation. The funders had no role in the study design, data collection, interpretation, or decision to submit the work for publication.

Given the gap in our understanding of vesicular transport regulators and GAPs, we performed a systematic analysis of TBC proteins in T. gondii. In this study, we identify 18 TBC proteins and localize them to discrete compartments of the secretory pathway or other vesicles in the parasite. We then focus on the ER-localized TgTBC9 and demonstrate that conditional knockdown of this family member results in a lethal growth arrest, disruption of the ER, aberrant mitochondrial morphology, and the formation of large lipid droplets (LDs) and ER encapsulated structures. We show by mutagenesis that the GAP activity of TgTBC9 is critical for function and that the P. falciparum orthologue of TgTBC9 can rescue the Toxoplasma knockdown. We also show that TgTBC9 directly binds to the ER-Golgi localized Rab2 protein, indicating this is the major target of TgTBC9 [ 17 ]. This work substantially builds on our understanding of vesicular protein targeting and identifies the first essential TBC protein identified in any protozoan, thus revealing a critical player in secretory traffic in T. gondii and related apicomplexan parasites.

While several Rab GEFs have been studied in T. gondii, the GAP proteins responsible for inactivating Rabs have not been explored. Rab GAPs are typically characterized by the presence of a TBC (Tre-2/Bub2/Cdc16) domain and act by increasing the intrinsic GTPase activity of the Rab protein, resulting in a conversion from the GTP to GDP bound form [ 25 ]. TBC domain-containing proteins (hereafter termed TBC proteins) often contain other functional domains that presumably contribute to their function in various locations within the cell. The TBC family is poorly studied in most systems as there are typically many family members that often have similar localizations, suggesting functional redundancy (e.g., there are 41 TBC proteins in humans) [ 25 , 26 ]. In addition, there are a small number of other proteins that lack a TBC domain but can still function as Rab GAPs [ 16 , 27 ]. The TBC proteins in most parasites remain largely unstudied, and none have been localized or functionally assessed in T. gondii. In addition, phylogenetic analyses suggest that the most recent common ancestor of the eukaryotes contained several TBC proteins, which gave rise to a number of TBC clades in different eukaryotic lineages [ 25 ]. The relationships among clades are poorly resolved likely due to their ancient divergences, which complicates studies of TBC family proteins as well-defined orthologues are frequently difficult to determine.

Similar to other eukaryotic cells, one of the major regulators of Toxoplasma secretory traffic is the Rab family of small GTPases [ 14 ]. Rab proteins are tethered to membranes by C-terminal prenylation and function as molecular switches that cycle between an inactive GDP-bound state and an active GTP-bound state to regulate secretory and vesicular traffic [ 15 ]. Rabs are regulated by guanine nucleotide exchange factors (GEFs) that promote the exchange of GDP for GTP as well as GTPase activating proteins (GAPs), which hydrolyze GTP to GDP [ 16 ]. Twelve Rab proteins have been identified in T. gondii, which appear to be conserved across most of the eukarya [ 17 , 18 ]. The proteins localize to various secretory or vesicular compartments such as the ER/Golgi, Golgi, post Golgi compartments, or cytoplasmic vesicles [ 17 ]. Functional assessment of the Rabs, by dominant negative or overexpression screening, has confirmed their importance in protein trafficking for parasite fitness. For example, overexpression of Rab2, Rab4, Rab5A, Rab5B, and Rab5C dramatically affect parasite growth and overexpression of Rab5A and Rab5C results in mistargeting of rhoptry and microneme cargo into the PV [ 17 , 19 , 20 ]. Rab11A has been shown to regulate dense granule secretion and Rab11B functions in IMC biogenesis [ 21 , 22 ]. In addition, some of the GEFs that activate Rabs have been described including a Vps9 and a Vps11 domain-containing protein, although their target Rabs have not been identified [ 23 , 24 ].

One interesting aspect of T. gondii is its highly polarized secretory pathway, which delivers secretory proteins to organelles at the apical end of the parasite [ 8 , 9 ]. As in other eukaryotic cells, secretory traffic is initiated by co-translational translocation of protein cargo into the endoplasmic reticulum (ER). In T. gondii, this traffic then passes through a single Golgi stack which is positioned just anterior to the parasite’s nucleus [ 7 ]. After exiting the Golgi, vesicle-based trafficking allows the protein cargo to be directed through post-Golgi compartments to the secretory micronemes, rhoptries, and dense granules, which discharge their contents sequentially for motility, host cell invasion, parasitophorous vacuole (PV) formation, and modification of the PV post-invasion [ 7 , 8 ]. In addition to the secretory organelles, protein cargo from the secretory pathway is also targeted to the IMC, apicoplast, plant-like vacuolar compartment (PLVAC), endosome-like compartment (ELC), or parasite’s surface [ 7 , 10 – 12 ]. Specific signals have been identified for targeting to most of these compartments, as has some of the vesicle trafficking machinery [ 10 , 13 ]. However, a detailed mechanistic understanding of vesicle sorting as well as the identification of many components of the trafficking machinery has yet to be completed.

Toxoplasma gondii is an obligate intracellular parasite in the phylum Apicomplexa that causes the disease toxoplasmosis and infects all mammals including approximately one-third of the human population [ 1 , 2 ]. Although most human infections remain asymptomatic, immunocompromised individuals or congenitally infected neonates are vulnerable to more severe symptoms such as cardiomyopathy, blurred vision, encephalitis, or fatality if left untreated [ 3 ]. T. gondii is the most widespread apicomplexan and serves as a model system for studying other apicomplexan parasites, such as Cryptosporidium spp., which causes diarrheal disease, and Plasmodium falciparum, which causes malaria [ 4 , 5 ]. In addition to the standard eukaryotic organelles, apicomplexans share a number of unique organelles such as the inner membrane complex (IMC), micronemes, rhoptries, apicoplast, and dense granules that facilitate host cell invasion and intracellular survival [ 6 , 7 ]. As these unique organelles often contain parasite-specific proteins, they are considered excellent targets for therapeutic intervention.

Results

Toxoplasma TBC proteins localize to the secretory pathway and cytoplasmic vesicles To determine the localization of TBC proteins in T. gondii, we endogenously tagged the C-terminus of each TBC protein with an epitope tag in tachyzoites (Fig 2A) [37]. Proteins with higher expression levels were tagged with a 3xHA, 3xTy, or 3xMyc tag, while those that were likely to have low levels of expression were tagged with either a spaghetti monster HA (smHA) or spaghetti monster OLLAS (smOLLAS) epitope tag [37,38]. Using this approach, we found that TgTBC1, 2, 3, and 18 localizes to a bar-like shape that is positioned anterior to the nucleus, which suggests localization to the Golgi or Golgi-adjacent compartments. We co-stained these 4 proteins with the cis-Golgi marker GRASP55 and observed varying degrees of overlap as assessed by Pearson’s correlation coefficients (Fig 2B) [39]. TgTBC1 and 2 colocalize best with GRASP55, while TgTBC3 and 18 appear slightly more posterior to GRASP55, indicating that these proteins localize to trans-Golgi or post-Golgi compartments [39]. Eight of the TBC proteins (TgTBC5, 6, 9, 12, 13, 14, 16, and 17) showed a nuclear-excluded, reticular or spotted cytoplasmic staining indicative of the ER or cytoplasmic vesicles. We co-stained these with the ER marker SERCA which showed that TgTBC6, 9, and 14 colocalized best and were most likely ER-resident proteins (Fig 2C) [40,41]. The remaining 5 were denoted as cytoplasmic vesicles (Fig 2D), some of which likely represent different levels of intersection of the endocytic and exocytic pathways downstream of the Golgi [13], although localization to other types of cytoplasmic vesicles is also possible. TgTBC10 localizes to spots in the cytoplasm, but it is not nuclear-excluded as assessed by SERCA co-staining (Fig 2E). PPT PowerPoint slide

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TIFF original image Download: Fig 2. T. gondii TBC proteins localize to discrete regions of the secretory pathway and cytoplasmic vesicles. IFAs of endogenously epitope tagged TgTBC1-18 parasites. (A) Diagram of TgTBC1-18 showing the epitope tag and selectable marker. (B) IFA of endogenously tagged TgTBC1, 2, 3, and 18 stained with antibodies against epitope tags and colocalized with GRASP55. The yellow and white arrowheads in the bottom 2 panels denote subtle differences between the TBC protein and GRASP55. Green = endogenously tagged TBC proteins, Magenta = GRASP55-mCherry. Quantification of all colocalizations were quantified by calculating the Pearson’s correlation coefficient (R). Mean values and respective standard deviation of 10–15 parasites are indicated next to the respective image (see also S1 Data). (C) IFA of TgTBC6, 9, and 14 stained with anti-HA and colocalized with SERCA. Green = rabbit anti-HA, Magenta = mouse anti-SERCA. (D) IFA of TgTBC 5, 12, 13, 16, and 17 shown partially colocalizing with SERCA. Green = rabbit anti-HA, Magenta = mouse anti-SERCA. (E) IFA of TgTBC10 shows cytoplasmic and nuclear staining with partial colocalization with SERCA. The white arrowheads show that TgTBC10 is not nuclear excluded. Green = rabbit anti-HA, Magenta = mouse anti-SERCA. (F) IFA of TgTBC11 and TgTBC15 colocalizing with endogenously tagged IMC293xV5 in daughter buds. Magenta = mouse anti-HA, Green = rabbit anti-V5. (G) IFA of TgTBC8 shows staining central portion of the maternal IMC as accessed by IMC6 staining. The white arrowheads indicate basal portions of the parasite where TgTBC8 is absent. Magenta = mouse anti-HA, Green = rabbit anti-IMC6. (H) IFA of TgTBC4 and TgTBC7 with no detectable smHA staining. Magenta = mouse anti-HA, Green = rabbit anti-IMC6. Scale bars for all images, 2 μm. (I) Diagram and PCR of endogenous TgTBC4 and TgTBC7 tagged parasites. Primers labeled as red arrows were used to test gDNA from parental and tagged strains. IFA, indirect immunofluorescence assay; IMC, inner membrane complex; smHA, spaghetti monster HA; TBC, Tre2-Bub2-Cdc16. https://doi.org/10.1371/journal.pbio.3002634.g002 In addition, three of the TBC proteins were found to localize to the parasite-specific IMC. Two of these, TgTBC11 and TgTBC15, localize to the daughter buds of IMC during endodyogeny, as assessed by colocalization with the early daughter bud marker IMC29 (Fig 2F) [42]. In contrast, TgTBC8 appears to localize to the maternal IMC, as it is peripheral but not present in the apical cap or basal portion of the organelle (Fig 2G) [43]. Finally, in agreement with the low expression levels reported in ToxoDB, TgTBC4 and 7 could not be detected by IFA even though integration of the tag was confirmed by PCR, indicating that these family members are not significantly expressed in the parasite’s tachyzoite stage (Fig 2H and 2I) [44,45].

Ultrastructural analysis of TgTBC9 knockdown parasites To further scrutinize the TgTBC9 depletion phenotype, we examined the knockdown parasites using transmission electron microscopy (TEM, Fig 4). In agreement with our IFA analysis, we found aberrant ER structures, swollen mitochondria with anormal electron-dense cristae, and very large LDs (Fig 4A–4C). Other organelles such as the Golgi, apicoplast, rhoptries, acidocalcisomes, dense granules, and intravacuolar network appear unaffected [5,54,55]. We also observed parasites with varying degrees of morphological defects in the shape of the parasite body (Figs 4B and S3A). In addition, we observed amylopectin granules, which usually typifies the parasite cyst forms, suggesting a state of stress (Fig 4B) [56]. In some instances, we noticed several nuclear profiles, suggestive of asynchronous parasite replication (Fig 4C). Most strikingly, knockdown of TgTBC9 resulted in the accumulation of large osmiophilic structures (up to 1 micron in diameter) surrounded by multiple layers of membranes including ER elements (Fig 4D and 4E). These multi-membranous structures that morphologically differ from LD may also be detected by BODIPY staining (Figs 4D, asterisks and S3B). The TEM analysis suggests a progression of these ER encapsulated structures that increase in size and membrane envelopment (Fig 4E, from panel i to iii), although this cannot be definitively determined. Lastly, we also observed clefts in the cytoplasm that appear to originate near the nuclear envelope, events which likely precede parasite death (Figs 4F and S3C, arrowheads). Together, the ultrastructural analysis shows a disruption of the ER and mitochondria, and suggests a dysregulation of ER-based activities such as lipid droplet formation, lipolysis, and autophagy. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Ultrastructural analysis of TgTBC9 knockdown parasites. (A) TEM of intracellular Toxoplasma in HFF for 24 h in the absence of IAA illustrating 4 control (untreated) parasites in a symmetrical organization inside the PV. (B–F) TEM of intracellular Toxoplasma in the presence of IAA. (B) Image showing an example of a PV containing 2 or 3 IAA-treated parasites with disorganized parasite morphology. The TgTBC9 knockdown parasites contain aberrant ER structures, a rounded mitochondrion (mt), and the presence of amylopectin granules (AG). (C) Image showing that TgTBC9 knockdown parasites also contain multiple nuclear profiles within one parasite (n1-3), very large LDs, and a disrupted ER-Golgi (Go) connection with accumulated vesicles of various size and electron density. (D, E) Images showing membranous structures surrounded by ER elements (asterisks) with E illustrating the likely progressive steps of their formation/compaction. (F) Parasites showing cytoplasmic clefts in the cytoplasm (arrowheads in D, F), likely preceding parasite death. Asterisks highlights an ER enveloped structure. Ac, acidocalcisome; ap, apicoplast; DG, dense granule; hc, host cell; IVN, intravacuolar network; mi, microneme; n, nucleus; rh, rhoptry. Scales bar for all images, 500 nm. ER, endoplasmic reticulum; HFF, human foreskin fibroblast; LD, lipid droplet; PV, parasitophorous vacuole; TEM, transmission electron microscopy. https://doi.org/10.1371/journal.pbio.3002634.g004

TBC dual-finger active sites are required for TgTBC9 GAP activity TgTBC9 is one of the TBC proteins that contains a conserved dual-finger active site with the IxxDxxR and YxQ motifs (S1 Fig and Table 1). To investigate whether the active site residues are required for TgTBC9’s GAP activity, we generated complementation constructs with the full-length wild-type gene (wt), a mutant of the IxxDxxR motif (R74A), or a mutant of the YxQ motif (Q101A) (Fig 5A), which have previously been shown to disrupt function in other systems [35,57]. Complementation with the wild-type gene (TgTBC9wt) in TgTBC9AID parasites restored the parasite’s ability to form plaques in the presence of IAA (Fig 5B and 5C). In contrast, complementation with either the TgTBC9R74A or TgTBC9Q101A mutant completely failed to rescue the parasite’s ability to form plaques upon knockdown of endogenous TgTBC9 (Fig 5D). Failure of the mutants to complement the knockdown was not due to expression levels, as similar levels of the TgTBC9wt and the TgTBC9R74A and TgTBC9Q101A point mutants were confirmed by western blot analysis (Fig 5E). Quantification of these results confirmed that mutation of the TgTBC9 active sites completely disrupts the protein’s ability to rescue the lethal knockdown (Fig 5F). PPT PowerPoint slide

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TIFF original image Download: Fig 5. TBC dual-finger active sites are required for TgTBC9 GAP activity. (A) Diagram of the dual-finger consensus and highlighting which TgTBC9 residues in the arginine finger and glutamine finger motifs were mutated to alanine. Green boxes depict strictly conserved residues; yellow boxes depict semi-conserved residues; magenta boxes depict residues that were mutated to alanine. (B) Plaque assays showing that complementation with full-length TgTBC9 restores ability to form plaques upon depletion of endogenous TgTBC9. (C) Western blot analysis showing knockdown of endogenous TgTBC9 and complementation with a Ty-tagged TgTBC9wt copy targeted to the UPRT locus. IMC6 is used as a load control. (D) Plaque assays showing complementation with the TgTBC9R74A or TgTBC9Q101A mutants are unable to rescue the TgTBC9AID knockdown. (E) Western blot analysis showing that the TgTBC9wt and TgTBC9R74A and TgTBC9Q101A mutant parasites have similar levels of expression. IMC6 is used as a load control. (F) Quantification of plaque assays at day 7 showing rescue with TgTBC9wt but no plaque formation by TgTBC9AID, TgTBC9R74A, and TgTBC9Q101A mutant parasites +IAA (****, P ≤ 0.0001). All raw data in S1 Data. GAP, GTPase-activating protein; TBC, Tre2-Bub2-Cdc16. https://doi.org/10.1371/journal.pbio.3002634.g005

Identification of candidate Rabs that are targeted by TgTBC9 Having demonstrated that the catalytic activity of TgTBC9 is required for function, we next sought to determine which of the Rab proteins that are involved in vesicle trafficking are targeted by TgTBC9. To identify candidate Rabs as well as other potential binding partners, we carried out large-scale immunoprecipitations (IP) of TgTBC93xHA via its C-terminal epitope tag. The bound proteins were eluted using high pH and the eluates analyzed by SDS-PAGE, which showed a strong enrichment of the target protein (Fig 7A). The eluted proteins were then identified by mass spectrometry which showed that TgTBC9 was the top hit in the pull down (Fig 7B and S3 Table). We also identified Rab1A, Rab2, Rab5A, Rab6, and Rab11B as candidate small GTPase interactors. Of these candidates, Rab2 stood out as the most likely target of TgTBC9 as it was significantly more enriched in the dataset, localizes to the ER/Golgi, and has been shown to be essential for growth using an overexpression screen [17]. The mass spectrometry dataset also appeared to be enriched in secretory proteins, including a number of rhoptry, dense granule, and IMC proteins, which likely represents cargo trafficking through the secretory pathway that co-immunoprecipitated with TgTBC9 (S3 Table). PPT PowerPoint slide

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TIFF original image Download: Fig 7. IP and pairwise Y2H of TgTBC9 reveals Rab2 an interactor. (A) Western blot analysis of the TgTBC9 IP showing the input (Total) and eluted material (Eluate) probed with mouse anti-HA antibodies. (B) Table showing the rank of TgTBC9 and small GTPase proteins from the IP analysis. The complete dataset is shown in S3 Table. Untagged parasites were used as the control. The ToxoDB-IDs, localization (* determined in [17], ** determined in [21], *** determined in [20]), molecular weight, and GWCS phenotype score are also shown [36]. (C) Spot assays of pairwise Y2H assessing TgTBC9 and Rab2 interaction using either wild-type (wt), GTP-locked mutant (Rab2Q66L), or catalytical inactive mutant (TgTBC9R74A) sequences. Yeast expressing the indicated constructs were grown under permissive (-L/W) or restrictive (-L/W/H) conditions to assess interactions. (D) Y2H assessing the interaction of catalytically inactive mutant TgTBC9 with the indicated mutant Rabs, as described in C. (E) Diagram of Rab2 showing the N-terminal DD and 2xV5 tag (DDV5Rab2). IFA analysis of TgTBC9AID parasites expressing DDV5Rab2 treated for 24 h with 1 μm Shld-1 prior to fixation. Magenta = mouse anti-HA, Green = rabbit anti-V5. Scale bars, 2 μm. Colocalizations were quantified by calculating the Pearson’s correlation coefficient (R). Mean values and standard deviation of 10–15 parasites are indicated next to the image (see also S1 Data). (F) IFA of TgTBC9AID parasites expressing DDV5Rab2 without (-) or with (+) IAA for 24 h (following a 4 h pretreatment ±IAA) and treated for 24 h with 1 μm Shld-1 prior to fixation showing disruption of Rab2 expression and localization. Magenta = mouse anti-V5, Green = rabbit anti-IMC6. Scale bars, 2 μm. (G) Representative IFA showing a field of TgTBC9AID parasites expressing DDV5Rab2 grown in ±IAA as described in F. Magenta = mouse anti-V5, Green = rabbit anti-IMC6. Scale bars, 10 μm. (H) Western blot analysis of TgTBC9AID parasites expressing DDV5Rab2 grown in ±IAA as descried in F showing a 52% decrease of Rab2 in IAA-treated parasites. Quantification was normalized to IMC6, which is used as a load control. GWCS, genome-wide CRIPSR screen; IFA, indirect immunofluorescence assay; IP, immunoprecipitation. https://doi.org/10.1371/journal.pbio.3002634.g007

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002634

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