A novel dense granule protein, GRA22, is involved in regulating parasite egress in Toxoplasma gondii

Okada, Tadashi; Marmansari, Dini; Li, Zengmei; Adilbish, Аltanchimeg; Canko, Shishenkov; Ueno, Akira; Shono, Haruhi; Furuoka, Hidefumi; Igarashi, Makoto

doi:10.1016/j.molbiopara.2013.04.005

Cited by 40 publications

(30 citation statements)

References 24 publications

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“…These results indicate that TgCDPK3 likely regulates biological processes during the normal function of intracellular parasites, independent of egress and ionophore treatment. Among the phosphorylation sites that are already different in the absence of ionophore are some on proteins important for ion-homeostasis (P-type ATPase, putative, TGGT1_103910) and a dense granule protein GRA22 (TGGT1_125960) that has recently been shown to play a role in egress [21]. Importantly, many of the differences were observed in the two independently derived TgCDPK3 mutant lines (MBE1.1 and RH Δcdpk3 ) indicating that these changes are a consequence of TgCDPK3 inactivation and not a consequence of the genetic modification of the parasites independent of TgCDPK3 function (Figure 2D).…”

Section: Resultsmentioning

confidence: 99%

The Calcium-Dependent Protein Kinase 3 of Toxoplasma Influences Basal Calcium Levels and Functions beyond Egress as Revealed by Quantitative Phosphoproteome Analysis

et al. 2014

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Calcium-dependent protein kinases (CDPKs) are conserved in plants and apicomplexan parasites. In Toxoplasma gondii, TgCDPK3 regulates parasite egress from the host cell in the presence of a calcium-ionophore. The targets and the pathways that the kinase controls, however, are not known. To identify pathways regulated by TgCDPK3, we measured relative phosphorylation site usage in wild type and TgCDPK3 mutant and knock-out parasites by quantitative mass-spectrometry using stable isotope-labeling with amino acids in cell culture (SILAC). This revealed known and novel phosphorylation events on proteins predicted to play a role in host-cell egress, but also a novel function of TgCDPK3 as an upstream regulator of other calcium-dependent signaling pathways, as we also identified proteins that are differentially phosphorylated prior to egress, including proteins important for ion-homeostasis and metabolism. This observation is supported by the observation that basal calcium levels are increased in parasites where TgCDPK3 has been inactivated. Most of the differential phosphorylation observed in CDPK3 mutants is rescued by complementation of the mutants with a wild type copy of TgCDPK3. Lastly, the TgCDPK3 mutants showed hyperphosphorylation of two targets of a related calcium-dependent kinase (TgCDPK1), as well as TgCDPK1 itself, indicating that this latter kinase appears to play a role downstream of TgCDPK3 function. Overexpression of TgCDPK1 partially rescues the egress phenotype of the TgCDPK3 mutants, reinforcing this conclusion. These results show that TgCDPK3 plays a pivotal role in regulating tachyzoite functions including, but not limited to, egress.

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Section: Resultsmentioning

confidence: 99%

The Calcium-Dependent Protein Kinase 3 of Toxoplasma Influences Basal Calcium Levels and Functions beyond Egress as Revealed by Quantitative Phosphoproteome Analysis

et al. 2014

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“…and targeted to the host nucleus to reprogram host cell activities (68). GRA22 expression peaks at the sporozoite stage, and this protein also functions as a regulator of T. gondii egress (69), which together with TgLCAT establishes an unanticipated role for dense granules in the control of Toxoplasma exit from host cells. As with other unconventional GRA proteins, TgLCAT diverges from conventional GRA proteins based on its homology with LCAT enzymes, higher molecular mass, localization to the parasite plasma membrane, involvement in egress, and secretion into the PV only from 7 h p.i.…”

Section: Discussionmentioning

confidence: 99%

A Lipolytic Lecithin:Cholesterol Acyltransferase Secreted by Toxoplasma Facilitates Parasite Replication and Egress

Pszenny

Ehrenman

Romano

et al. 2016

Journal of Biological Chemistry

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The protozoan parasite Toxoplasma gondii develops within a parasitophorous vacuole (PV) in mammalian cells, where it scavenges cholesterol. When cholesterol is present in excess in its environment, the parasite expulses this lipid into the PV or esterifies it for storage in lipid bodies. Here, we characterized a unique T. gondii homologue of mammalian lecithin:cholesterol acyltransferase (LCAT), a key enzyme that produces cholesteryl esters via transfer of acyl groups from phospholipids to the 3-OH of free cholesterol, leading to the removal of excess cholesterol from tissues. TgLCAT contains a motif characteristic of serine lipases "AHSLG" and the catalytic triad consisting of serine, aspartate, and histidine (SDH) from LCAT enzymes. TgL-CAT is secreted by the parasite, but unlike other LCAT enzymes it is cleaved into two proteolytic fragments that share the residues of the catalytic triad and need to be reassembled to reconstitute enzymatic activity. TgLCAT uses phosphatidylcholine as substrate to form lysophosphatidylcholine that has the potential to disrupt membranes. The released fatty acid is transferred to cholesterol, but with a lower transesterification activity than mammalian LCAT. TgLCAT is stored in a subpopulation of dense granule secretory organelles, and following secretion, it localizes to the PV and parasite plasma membrane. LCAT-null parasites have impaired growth in vitro, reduced virulence in animals, and exhibit delays in egress from host cells. Parasites overexpressing LCAT show increased virulence and faster egress. These observations demonstrate that TgLCAT influences the outcome of an infection, presumably by facilitating replication and egress depending on the developmental stage of the parasite.The phospholipase A 2 (PLA 2 ) 2 family of serine lipases comprises more than 30 enzymes in mammals. The PLA 2 members hydrolyze the sn-2 ester of phospholipids, yielding lysophospholipid and releasing a fatty acid (1). Approximately one-third of the PLA 2 family members are secreted from cells and display various localizations post-secretion, reflecting their specialized biological functions (2). Among the secretory PLA 2 , lecithin: cholesterol acyltransferase (LCAT; EC 2.3.1.43) is characterized by dual activity, PLA 2 and acyltransferase. In mammals, this enzyme catalyzes the transacylation of the sn-2 fatty acid liberated from various phospholipids (e.g. phosphatidylcholine or phosphatidylethanolamine) to the 3-␤-hydroxyl group on the A-ring of cholesterol, thereby forming cholesteryl esters (3-5). The primary sequence of LCAT is well conserved between mammalian species (6). A structural model for LCAT predicts the conformation of a catalytic triad formed by SerAsp-His residues involved in the phospholipase reaction (7). Mammalian LCATs are primarily expressed in the liver and secreted to the plasma where they circulate in association with HDL (8). These enzymes are components of the reverse cholesterol transport pathway by which cholesterol from peripheral cells is delivered to the liver f...

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“…The parasites were fixed and permeabilized (4% formaldehyde-0.2% Triton X-100 in PBS, pH 7.0, for 15 min) at indicated time points. Then the parasites were stained using anti-IMC1 polyclonal antisera (1:1000) [28] and the number of parasites per PV was counted. Each time point represents the mean value of about 100 PV of 5 different fields with a standard deviation.…”

Section: Methodsmentioning

confidence: 99%

“…Then the parasites were fixed, permeabilized (4% formaldehyde–0.2% Triton X-100 in PBS, pH 7.0) for 15 min, stained using anti-IMC1 polyclonal antisera (1:1000) [28] and the long axis of plaque was measured. The plaque size represents the mean value of about 30 plaques with a standard deviation.…”

Section: Methodsmentioning

confidence: 99%

Lactate dehydrogenase in Toxoplasma gondii controls virulence, bradyzoite differentiation, and chronic infection

et al. 2017

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In the asexual stages, Toxoplasma gondii stage converts between acute phase rapidly replicating tachyzoites and chronic phase slowly dividing bradyzoites. Correspondingly, T. gondii differentially expresses two distinct genes and isoforms of the lactate dehydrogenase enzyme, expressing LDH1 exclusively in the tachyzoite stage and LDH2 preferentially in the bradyzoite stage. LDH catalyzes the interconversion of pyruvate and lactate in anaerobic growth conditions and is utilized for energy supply, however, the precise role of LDH1 and LDH2 in parasite biology in the asexual stages is still unclear. Here, we investigated the biological role of LDH1 and LDH2 in the asexual stages, and the vaccine strain potential of deletion mutants lacking LDH1, LDH2, or both genes (Δldh1, Δldh2 and Δldh1/2). Deletion of LDH1 reduced acute parasite virulence, impaired bradyzoite differentiation in vitro, and markedly reduced chronic stage cyst burdens in vivo. In contrast, deletion of LDH2 impaired chronic stage cyst burdens without affecting virulence or bradyzoite differentiation. Deletion of both LDH1 and LDH2 induced a more severe defect in chronic stage cyst burdens. These LDH mutant phenotypes were not associated with any growth defect. Vaccination of mice with a low dose of mutants deleted for LDH elicited effective protective immunity to lethal challenge infection, demonstrating the vaccine potential of LDH deletion mutants. These results suggest that lactate dehydrogenase in T. gondii controls virulence, bradyzoite differentiation, and chronic infection and reveals the potential of LDH mutants as vaccine strains.

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A novel dense granule protein, GRA22, is involved in regulating parasite egress in Toxoplasma gondii

Cited by 40 publications

References 24 publications

The Calcium-Dependent Protein Kinase 3 of Toxoplasma Influences Basal Calcium Levels and Functions beyond Egress as Revealed by Quantitative Phosphoproteome Analysis

The Calcium-Dependent Protein Kinase 3 of Toxoplasma Influences Basal Calcium Levels and Functions beyond Egress as Revealed by Quantitative Phosphoproteome Analysis

A Lipolytic Lecithin:Cholesterol Acyltransferase Secreted by Toxoplasma Facilitates Parasite Replication and Egress

Lactate dehydrogenase in Toxoplasma gondii controls virulence, bradyzoite differentiation, and chronic infection

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