Apicomplexan parasite growth and replication relies on nutrient acquisition from host cells, in which intracellular multiplication occurs, yet the mechanisms that underlie the nutrient salvage remain elusive. Numerous ultrastructural studies have documented a plasma membrane invagination with a dense neck, termed the micropore, on the surface of intracellular parasites. However, the function of this structure remains unknown. Here we validate the micropore as an essential organelle for endocytosis of nutrients from the host cell cytosol and Golgi in the model apicomplexan Toxoplasma gondii. Detailed analyses demonstrated that Kelch13 is localized at the dense neck of the organelle and functions as a protein hub at the micropore for endocytic uptake. Intriguingly, maximal activity of the micropore requires the ceramide de novo synthesis pathway in the parasite. Thus, this study provides insights into the machinery underlying acquisition of host cell-derived nutrients by apicomplexan parasites that are otherwise sequestered from host cell compartments.

In the apicomplexans, endocytosed cargos (e.g., hemoglobin) are trafficked to a specialized organelle for digestion. This follows a unique endocytotic process at the micropore/cytostome in these parasites. However, the mechanism underlying endocytic trafficking remains elusive, due to the repurposing of classical endocytic proteins for the biogenesis of apical organelles. To resolve this issue, we have exploited the genetic tractability of the model apicomplexan Toxoplasma gondii , which ingests host cytosolic materials (e.g., green fluorescent protein[GFP]). We determined an association between protein prenylation and endocytic trafficking, and using an alkyne-labeled click chemistry approach, the prenylated proteome was characterized. Genome editing, using clustered regularly interspaced short palindromic repaet/CRISPR-associated nuclease 9 (CRISPR/Cas9), was efficiently utilized to generate genetically modified lines for the functional screening of 23 prenylated candidates. This identified four of these proteins that regulate the trafficking of endocytosed GFP vesicles. Among these proteins, Rab1B and YKT6.1 are highly conserved but are non-classical endocytic proteins in eukaryotes. Confocal imaging analysis showed that Rab1B and Ras are substantially localized to both the trans-Golgi network and the endosome-like compartments in the parasite. Conditional knockdown of Rab1B caused a rapid defect in secretory trafficking to the rhoptry bulb, suggesting a trafficking intersection role for the key regulator Rab1B. Further experiments confirmed a critical role for protein prenylation in regulating the stability/activity of these proteins (i.e., Rab1B and YKT6.1) in the parasite. Our findings define the molecular basis of endocytic trafficking and reveal a potential intersection function of Rab1B on membrane trafficking in T. gondii . This might extend to other related protists, including the malarial parasites. IMPORTANCE: The protozoan Toxoplasma gondii establishes a permissive niche, in host cells, that allows parasites to acquire large molecules such as proteins. Numerous studies have demonstrated that the parasite repurposes the classical endocytic components for secretory sorting to the apical organelles, leaving the question of endocytic transport to the lysosome-like compartment unclear. Recent studies indicated that endocytic trafficking is likely to associate with protein prenylation in malarial parasites. This information promoted us to examine this association in the model apicomplexan T. gondii and to identify the key components of the prenylated proteome that are involved. By exploiting the genetic tractability of T. gondii and a host GFP acquisition assay, we reveal four non-classical endocytic proteins that regulate the transport of endocytosed cargos (e.g., GFP) in T. gondii . Thus, we extend the principle that protein prenylation regulates endocytic trafficking and elucidate the process of non-classical endocytosis in T. gondii and potentially in other related protists.

The apicoplast is a four-membrane plastid found in the apicomplexans, which harbors biosynthesis and organelle housekeeping activities in the matrix. However, the mechanism driving the flux of metabolites, in and out, remains unknown. Here, we used TurboID and genome engineering to identify apicoplast transporters in Toxoplasma gondii. Among the many novel transporters, we show that one pair of apicomplexan monocarboxylate transporters (AMTs) appears to have evolved from a putative host cell that engulfed a red alga. Protein depletion showed that AMT1 and AMT2 are critical for parasite growth. Metabolite analyses supported the notion that AMT1 and AMT2 are associated with biosynthesis of isoprenoids and fatty acids. However, stronger phenotypic defects were observed for AMT2, including in the inability to establish T. gondii parasite virulence in mice. This study clarifies, significantly, the mystery of apicoplast transporter composition and reveals the importance of the pair of AMTs in maintaining the apicoplast activity in apicomplexans.

Background: The protozoan parasite Toxoplasma gondii encodes a dozen Rab proteins, which are parts of the small GTPase superfamily and regulate intracellular membrane trafficking. Our previous study showed that depletion of Rab1B caused severe defects regarding parasite growth and morphological structure, yet early defects of endocytic trafficking and vesicle sorting to the rhoptry in T. gondii are not expected to have a strong effect. To understand this discrepancy, we performed an integrated analysis at the level of transcriptomics and metabolomics. Methods: In the study, tetracycline-inducible TATi/Ty-Rab1B parasite line treated with ATc at three different time points (0, 18 and 24 h) was used. We first observed the morphological changes caused by Rab1B depletion via transmission electron technology. Then, high-throughput transcriptome along with non-targeted metabolomics were performed to analyze the RNA expression and metabolite changes in the Rab1B-depleted parasite. The essential nature of Rab1B in the parasite was revealed by the integrated omics approach. Results: Transmission electron micrographs showed a strong disorganization of endo-membranes in the Rab1B-depleted parasites. Our deep analysis of transcriptome and metabolome identified 2181 and 2374 differentially expressed genes (DEGs) and 30 and 83 differentially expressed metabolites (DEMs) at 18 and 24 h of induction in the tetracycline-inducible parasite line, respectively. These DEGs included key genes associated with crucial organelles that contain the rhoptry, microneme, endoplasmic reticulum and Golgi apparatus. The analysis of qRT-PCR verified some of the key DEGs identified by RNA-Seq, supporting that the key vesicular regulator Rab1B was involved in biogenesis of multiple parasite organelles. Functional enrichment analyses revealed pathways related to central carbon metabolisms and lipid metabolisms, such as the TCA cycle, glycerophospholipid metabolism and fatty acid biosynthesis and elongation. Further correlation analysis of the major DEMs and DEGs supported the role of Rab1B in biogenesis of fatty acids (e.g. myrisoleic acid and oleic acid) (R > 0.95 and P < 0.05), which was consistent with the scavenging role in biotin via the endocytic process. Conclusions: Rab1B played an important role in parasite growth and morphology, which was supported by the replication assay and transmission electron microscopy observation. Our multi-omics analyses provided detailed insights into the overall impact on the parasite upon depletion of the protein. These analyses reinforced the role of Rab1B in the endocytic process, which has an impact on fatty acid biogenesis and the TCA cycle. Taken together, these findings contribute to our understanding of a key vesicular regulator, Rab1B, on parasite metabolism and morphological formation in T. gondii. Graphical