Prenylcysteine carboxyl methyltransferase (pcCMT) is the third of three enzymes that posttranslationally modify C-terminal CAAX motifs and thereby target CAAXproteins to the plasma membrane. Here we report the molecular characterization and subcellular localization of the first mammalian (human myeloid) pcCMT. The deduced amino acid sequence of mammalian pcCMT predicts a multiple membrane-spanning protein with homologies to the yeast pcCMT, STE14, and the mammalian band 3 anion transporter. The human gene complemented a ste14 mutant. pcCMT mRNAs were ubiquitously expressed in human tissues. An anti-pcCMT antiserum detected a 33-kDa protein in myeloid cell membranes. Ectopically expressed recombinant pcCMT had enzymatic activity identical to that observed in neutrophil membranes. Mammalian pcCMT was not expressed at the plasma membrane but rather restricted to the endoplasmic reticulum. Thus, the final enzyme in the sequence that modifies CAAX motifs is located in membranes topologically removed from the CAAX protein target membrane.

Toxoplasma gondii possesses sets of dense granule proteins (GRAs) that either assemble at, or cross the parasitophorous vacuole membrane (PVM) and exhibit motifs resembling the HT/PEXEL previously identified in a repertoire of exported Plasmodium proteins. Within Plasmodium spp., cleavage of the HT/PEXEL motif by the endoplasmic reticulum-resident protease Plasmepsin V precedes trafficking to and export across the PVM of proteins involved in pathogenicity and host cell remodelling. Here, we have functionally characterized the T. gondii aspartyl protease 5 (ASP5), a Golgi-resident protease that is phylogenetically related to Plasmepsin V. We show that deletion of ASP5 causes a significant loss in parasite fitness in vitro and an altered virulence in vivo. Furthermore, we reveal that ASP5 is necessary for the cleavage of GRA16, GRA19 and GRA20 at the PEXEL-like motif. In the absence of ASP5, the intravacuolar nanotubular network disappears and several GRAs fail to localize to the PVM, while GRA16 and GRA24, both known to be targeted to the host cell nucleus, are retained within the vacuolar space. Additionally, hypermigration of dendritic cells and bradyzoite cyst wall formation are impaired, critically impacting on parasite dissemination and persistence. Overall, the absence of ASP5 dramatically compromises the parasite’s ability to modulate host signalling pathways and immune responses. The opportunistic pathogen Toxoplasma gondii infects a large range of nucleated cells where it replicates intracellularly within a parasitophorous vacuole (PV) surrounded by a membrane (PVM). Parasites constitutively secrete dense-granule proteins (GRAs) both into and beyond the PV which participate in remodelling of the PVM, recruitment of host organelles, neutralization of the host cellular defences, and subversion of host cell functioning. In addition, the GRAs critically contribute to cyst wall formation, a process that critically ensures parasite persistence and transmission. To act as effector molecules, some of the GRAs must be translocated across the PVM. Within the related apicomplexan parasite P. falciparum, a repertoire of proteins exported beyond the PVM contain a motif cleaved by a specific protease, Plasmepsin V. Examination of the repertoire of GRAs in T. gondii revealed that some proteins exhibit such export-like motifs suggestive of protease involvement. In this study, we have functionally characterized the related aspartyl protease 5 (TgASP5) in both virulent and persistent T. gondii strains, and have investigated the phenotypic consequences of its deletion in the context of overall parasite biology, its intracellular niche, the infected host cells and the murine model. Our findings revealed fundamental roles of TgASP5 at the host-parasite interface.

Toxoplasma is an obligate intracellular parasite that replicates in mammalian cells within a parasitophorous vacuole (PV) that does not fuse with any host organelles. One mechanism developed by the parasite for nutrient acquisition is the attraction of host organelles to the PV. Here, we examined the exploitation of host lipid droplets (LD), ubiquitous fat storage organelles, by Toxoplasma. We show that Toxoplasma replication is reduced in host cells that are depleted of LD, or impaired in TAG lipolysis or fatty acid catabolism. In infected cells, the number of host LD and the expression of host LD-associated genes (ADRP, DGAT2), progressively increase until the onset of parasite replication. Throughout infection, the PV are surrounded by host LD. Toxoplasma is capable of accessing lipids stored in host LD and incorporates these lipids into its own membranes and LD. Exogenous addition of oleic acid stimulates LD biogenesis in the host cell and results in the overaccumulation of neutral lipids in very large LD inside the parasite. To access LD-derived lipids, Toxoplasma intercepts and internalizes within the PV host LD, some of which remaining associated with Rab7, which become wrapped by an intravacuolar network of membranes (IVN). Mutant parasites impaired in IVN formation display diminished capacity of lipid uptake from host LD. Moreover, parasites lacking an IVN-localized phospholipase A2 are less proficient in salvaging lipids from host LD in the PV, suggesting a major contribution of the IVN for host LD processing in the PV and, thus lipid content release. Interestingly, gavage of parasites with lipids unveils, for the first time, the presence in Toxoplasma of endocytic-like structures containing lipidic material originating from the PV lumen. This study highlights the reliance of Toxoplasma on host LD for its intracellular development and the parasite’s capability in scavenging neutral lipids from host LD. Toxoplasma is an obligate intracellular pathogen that multiplies in mammalian cells within a specialized compartment, named the parasitophorous vacuole (PV). While the vacuole does not fuse with host organelles, the parasite scavenges nutrients, including lipids, from these compartments. Present in all mammalian cells, lipid droplets (LD) are dynamic structures that store neutral lipids. Whether Toxoplasma targets host LD for their nutritional content remains to be investigated. We demonstrate that the parasite relies on host LD lipids and their lipolytic enzymatic activities to grow. Toxoplasma salvages lipids from host LD, which surround the PV and, at least partially, accesses these lipids by intercepting and engulfing within the PV host Rab7-associated LD. In the PV lumen, a parasite lipase releases lipids from host LD, thus making them available to the parasite. Exogenous addition of fatty acids stimulates host LD biogenesis and results in the accumulation of enlarged LD containing neutral lipids in Toxoplasma. This study highlights the ability of Toxoplasma to scavenge and store lipids from host LD. Interestingly, exposure of Toxoplasma to excess lipids reveals, for the first time, coated invaginations of the parasite’s plasma membrane and cytoplasmic vesicles containing lipids originating from the PV lumen, potentially involved in endocytosis.

The obligate intracellular protozoan Toxoplasma gondii actively invades mammalian cells and, upon entry, forms its own membrane-bound compartment, named the parasitophorous vacuole (PV). Within the PV, the parasite replicates and scavenges nutrients, including lipids, from host organelles. Although T. gondii can synthesize sphingolipids de novo, it also scavenges these lipids from the host Golgi. How the parasite obtains sphingolipids from the Golgi remains unclear, as the PV avoids fusion with host organelles. In this study, we explore the host Golgi–PV interaction and evaluate the importance of host-derived sphingolipids for parasite growth. We demonstrate that the PV preferentially localizes near the host Golgi early during infection and remains closely associated with this organelle throughout infection. The parasite subverts the structure of the host Golgi, resulting in its fragmentation into numerous ministacks, which surround the PV, and hijacks host Golgi–derived vesicles within the PV. These vesicles, marked with Rab14, Rab30, or Rab43, colocalize with host-derived sphingolipids in the vacuolar space. Scavenged sphingolipids contribute to parasite replication since alterations in host sphingolipid metabolism are detrimental for the parasite's growth. Thus our results reveal that T. gondii relies on host-derived sphingolipids for its development and scavenges these lipids via Golgi-derived vesicles.