A plasma membrane localized protein phosphatase in Toxoplasma gondii, PPM5C, regulates attachment to host cells.
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ABSTRACT:
SUBMITTER: Yang C
PROVIDER: S-EPMC6459975 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Intracellular pathogens have evolved a wide array of mechanisms to invade and co-opt their host cells for intracellular survival. Apicomplexan parasites such as Toxoplasma gondii employ the action of unique secretory organelles named rhoptries for internalization of the parasite and formation of a specialized niche within the host cell. We demonstrate that Toxoplasma gondii also uses secretion from the rhoptries during invasion to deliver a parasite-derived protein phosphatase 2C (PP2C-hn) into the host cell and direct it to the host nucleus. Delivery to the host nucleus does not require completion of invasion, as evidenced by the fact that parasites blocked in the initial stages of invasion with cytochalasin D are able to target PP2C-hn to the host nucleus. We have disrupted the gene encoding PP2C-hn and shown that PP2C-hn-knockout parasites exhibit a mild growth defect that can be rescued by complementation with the wild-type gene. The delivery of parasite effector proteins via the rhoptries provides a novel mechanism for Toxoplasma to directly access the command center of its host cell during infection by the parasite.
| S-EPMC1800361 | biostudies-literature
Project description:The apicomplexan moving junction (MJ) is a highly conserved structure formed during host cell entry that anchors the invading parasite to the host cell and serves as a molecular sieve of host membrane proteins that protects the parasitophorous vacuole from host lysosomal destruction. While recent work in Toxoplasma and Plasmodium has reinforced the composition of the MJ as an important association of rhoptry neck proteins (RONs) with micronemal AMA1, little is known of the precise role of RONs in the junction or how they are targeted to the neck subcompartment. We report the first functional analysis of a MJ/RON protein by disrupting RON8 in T. gondii. Parasites lacking RON8 are severely impaired in both attachment and invasion, indicating that RON8 enables the parasite to establish a firm clasp on the host cell and commit to invasion. The remaining junction components frequently drag in trails behind invading knockout parasites and illustrate a malformed complex without RON8. Complementation of Δron8 parasites restores invasion and reveals a processing event at the RON8 C-terminus. Replacement of an N-terminal region of RON8 with a mCherry reporter separates regions within RON8 that are necessary for rhoptry targeting and complex formation from those required for function during invasion. Finally, the invasion defects in Δron8 parasites seen in vitro translate to radically impaired virulence in infected mice, promoting a model in which RON8 has a crucial and unprecedented task in committing Toxoplasma to host cell entry.
| S-EPMC3053350 | biostudies-literature
Project description:Many obligate intracellular apicomplexan parasites have adapted a distinct invasion mechanism involving a close interaction between the parasite ligands and the sialic acid (SA) receptor. We found that sialic acid binding protein-1 (SABP1), localized on the outer membrane of the zoonotic parasite Toxoplasma gondii, readily binds to sialic acid on the host cell surface. The binding was sensitive to neuraminidase treatment. Cells preincubated with recombinant SABP1 protein resisted parasite invasion in vitro. The parasite lost its invasion capacity and animal infectivity after the SABP1 gene was deleted, whereas complementation of the SABP1 gene restored the virulence of the knockout strain. These data establish the critical role of SABP1 in the invasion process of T. gondii. The previously uncharacterized protein, SABP1, facilitated T. gondii attachment and invasion via sialic acid receptors.
| S-EPMC7296849 | biostudies-literature
Project description:BackgroundThe protozoan parasite Toxoplasma gondii encodes dozens of phosphatases, among which a plant-like phosphatase absent from mammalian genomes named PPKL, which is involved in regulating brassinosteroid signaling in Arabidopsis, was identified in the genome. Among the Apicomplexa parasites, T. gondii is an important and representative pathogen in humans and animals. PPKL was previously identified to modulate the apical integrity and morphology of the ookinetes and parasite motility and transmission in another important parasite, Plasmodium falciparum. However, the exact function of PPKL in the asexual stages of T. gondii remains unknown.MethodsThe plant auxin-inducible degron (AID) system was applied to dissect the phenotypes of PPKL in T. gondii. We first analyzed the phenotypes of the AID parasites at an induction time of 24 h, by staining of different organelles using their corresponding markers. These analyses were further conducted for the parasites grown in auxin for 6 and 12 h using a quantitative approach and for the type II strain ME49 of AID parasites. To further understand the phenotypes, the potential protein interactions were analyzed using a proximity biotin labeling approach. The essential role of PPKL in parasite replication was revealed.ResultsPPKL is localized in the apical region and nucleus and partially distributed in the cytoplasm of the parasite. The phenotyping of PPKL showed its essentiality for parasite replication and morphology. Further dissections demonstrate that PPKL is required for the maturation of daughter parasites in the mother cells, resulting in multiple nuclei in a single parasite. The phenotype of the daughter parasites and parasite morphology were observed in another type of T. gondii strain ME49. The substantial defect in parasite replication and morphology could be rescued by genetic complementation, thus supporting its essential function for PPKL in the formation of parasites. The protein interaction analysis showed the potential interaction of PPKL with diverse proteins, thus explaining the importance of PPKL in the parasite.ConclusionsPPKL plays an important role in the formation of daughter parasites, revealing its subtle involvement in the proper maturation of the daughter parasites during division. Our detailed analysis also demonstrated that depletion of PPKL resulted in elongated tubulin fibers in the parasites. The important roles in the parasites are potentially attributed to the protein interaction mediated by kelch domains on the protein. Taken together, these findings contribute to our understanding of a key phosphatase involved in parasite replication, suggesting the potential of this phosphatase as a pharmaceutic target.
| S-EPMC10949797 | biostudies-literature
Project description:We have characterized the Toxoplasma gondii protein phosphatase type 1 (TgPP1) and a potential regulatory binding protein belonging to the leucine-rich repeat protein family, designated TgLRR1. TgLRR1 is capable of binding to TgPP1 to inhibit its activity and to override a G(2)/M cell cycle checkpoint in Xenopus oocytes. In the parasite, TgLRR1 mRNA and protein are both highly expressed in the rapidly replicating and virulent tachyzoites, while only low levels are detected in the slowly dividing and quiescent bradyzoites. TgPP1 mRNA and protein levels are equally abundant in tachyzoites and bradyzoites. Affinity pull down and immunoprecipitation experiments reveal that the TgLRR1-TgPP1 interaction takes place in the nuclear subcompartment of tachyzoites. These results are consistent with those of localization studies using both indirect immunofluorescence with specific polyclonal antibody and transient transfection of T. gondii vector expressing TgLRR1 and TgPP1. The inability to obtain stable transgenic tachyzoites suggested that overexpression of TgLRR1 and TgPP1 may impair the parasite's growth. Together with the activation of Xenopus oocyte meiosis reinitiation, these data indicate that TgLRR1 protein could play a role in the regulation of the T. gondii cell cycle through the modulation of phosphatase activity.
| S-EPMC2043371 | biostudies-literature
Project description:A critical step in the pathogenesis of Toxoplasma gondii is conversion from the fast-replicating tachyzoite form experienced during acute infection to the slow-replicating bradyzoite form that establishes long-lived tissue cysts during chronic infection. Bradyzoite cyst development exhibits a clear tissue tropism in vivo, yet conditions of the host cell environment that influence this tropism remain unclear. Using an in vitro assay of bradyzoite conversion, we have found that cell types differ dramatically in the ability to facilitate differentiation of tachyzoites into bradyzoites. Characterization of cell types that were either resistant or permissive for conversion revealed that resistant cell lines release low molecular weight metabolites that could support tachyzoite growth under metabolic stress conditions and thereby inhibit bradyzoite formation in permissive cells. Biochemical analysis revealed that the glycolytic metabolite lactate is an inhibitory component of supernatants from resistant cells. Furthermore, upregulation of glycolysis in permissive cells through the addition of glucose or by overexpression of the host kinase, Akt, was sufficient to convert cells from a permissive to a resistant phenotype. These results suggest that the metabolic state of the host cell may play a role in determining the predilection of the parasite to switch from the tachyzoite to bradyzoite form.
| S-EPMC3458309 | biostudies-literature
Project description:The propagation of the obligate intracellular parasite Toxoplasma gondii is tightly regulated by calcium signaling. However, the mechanisms by which calcium homeostasis and fluxes are regulated in this human pathogen are not fully understood. To identify Toxoplasma's calcium homeostasis network, we have characterized a novel EF-hand domain-containing protein, which we have named TgEFP1. We have determined that TgEFP1 localizes to a previously described compartment known as the plant-like vacuole or the endosomal-like compartment (PLV/ELC), which harbors several proteins related to ionic regulation. Interestingly, partial permeabilization techniques showed that TgEFP1 is also secreted into the parasitophorous vacuole (PV), within which the parasite divides. Ultrastructure expansion microscopy confirmed the unusual dual localization of TgEFP1 at the PLV/ELC and the PV. Furthermore, we determined that the localization of TgEFP1 to the PV, but not to the PLV/ELC, is affected by disruption of Golgi-dependent transport with Brefeldin A. Knockout of TgEFP1 results in faster propagation in tissue culture, hypersensitivity to calcium ionophore-induced egress, and premature natural egress. Thus, our work has revealed an interplay between the PV and the PLV/ELC and a role for TgEFP1 in the regulation of calcium-dependent events.
| S-EPMC9139715 | biostudies-literature
Project description:The phylum Apicomplexa comprises a group of obligate intracellular parasites of broad medical and agricultural significance, including Toxoplasma gondii and the malaria-causing Plasmodium spp. Key to their parasitic lifestyle is the need to egress from an infected cell, actively move through tissue, and reinvade another cell, thus perpetuating infection. Ca(2+)-mediated signaling events modulate key steps required for host cell egress, invasion and motility, including secretion of microneme organelles and activation of the force-generating actomyosin-based motor. Here we show that a plant-like Calcium-Dependent Protein Kinase (CDPK) in T. gondii, TgCDPK3, which localizes to the inner side of the plasma membrane, is not essential to the parasite but is required for optimal in vitro growth. We demonstrate that TgCDPK3, the orthologue of Plasmodium PfCDPK1, regulates Ca(2+) ionophore- and DTT-induced host cell egress, but not motility or invasion. Furthermore, we show that targeting to the inner side of the plasma membrane by dual acylation is required for its activity. Interestingly, TgCDPK3 regulates microneme secretion when parasites are intracellular but not extracellular. Indeed, the requirement for TgCDPK3 is most likely determined by the high K(+) concentration of the host cell. Our results therefore suggest that TgCDPK3's role differs from that previously hypothesized, and rather support a model where this kinase plays a role in rapidly responding to Ca(2+) signaling in specific ionic environments to upregulate multiple processes required for gliding motility.
| S-EPMC3514314 | biostudies-literature
Project description:The intracellular parasite Toxoplasma gondii infects a large proportion of humans worldwide and can cause adverse complications in the settings of immune-compromise and pregnancy. T. gondii thrives within many different cell types due in part to its residence within a specialized and heavily modified compartment in which the parasite divides, termed the parasitophorous vacuole. Within this vacuole, numerous proteins optimize intracellular survival following their secretion by the parasite. We investigated the contribution of one of these proteins, TgPPM3C, predicted to contain a PP2C-class serine/threonine phosphatase domain and previously shown to interact with the protein MYR1, an essential component of a putative vacuolar translocon that mediates effector export into the host cell. Parasites lacking the TgPPM3C gene exhibit a minor growth defect in vitro, are avirulent during acute infection in mice, and form fewer cysts in mouse brain during chronic infection. Phosphoproteomic assessment of TgPPM3C deleted parasite cultures demonstrated alterations in the phosphorylation status of many secreted vacuolar proteins including two exported effector proteins, GRA16 and GRA28, as well as MYR1. Parasites lacking TgPPM3C are defective in GRA16 and GRA28 export, but not in the export of other MYR1-dependant effectors. Phosphomimetic mutation of two GRA16 serine residues results in export defects, suggesting that de-phosphorylation is a critical step in the process of GRA16 export. These findings provide another example of the emerging role of phosphatases in regulating the complex environment of the T. gondii parasitophorous vacuole and influencing the export of specific effector proteins from the vacuolar lumen into the host cell.
| S-EPMC7793252 | biostudies-literature
Project description:The phylum Apicomplexa comprises a group of obligate intracellular parasites that alternate between intracellular replicating stages and actively motile extracellular forms that move through tissue. Parasite cytosolic Ca2+ signalling activates motility, but how this is switched off after invasion is complete to allow for replication to begin is not understood. Here, we show that the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A catalytic subunit 1 (PKAc1) of Toxoplasma is responsible for suppression of Ca2+ signalling upon host cell invasion. We demonstrate that PKAc1 is sequestered to the parasite periphery by dual acylation of PKA regulatory subunit 1 (PKAr1). Upon genetic depletion of PKAc1 we show that newly invaded parasites exit host cells shortly thereafter, in a perforin-like protein 1 (PLP-1)-dependent fashion. Furthermore, we demonstrate that loss of PKAc1 prevents rapid down-regulation of cytosolic [Ca2+] levels shortly after invasion. We also provide evidence that loss of PKAc1 sensitises parasites to cyclic GMP (cGMP)-induced Ca2+ signalling, thus demonstrating a functional link between cAMP and these other signalling modalities. Together, this work provides a new paradigm in understanding how Toxoplasma and related apicomplexan parasites regulate infectivity.
| S-EPMC6152992 | biostudies-literature