The Toxoplasma Adhesive Protein MIC2 Is Proteolytically Processed at Multiple Sites by Two Parasite-derived Proteases

Carruthers, Vern B.; Sherman, Gale D.; Sibley, L. David

doi:10.1074/jbc.275.19.14346

Cited by 176 publications

(199 citation statements)

References 27 publications

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“…This suggests that the PV is largely devoid of proteolytic activity. However, tachyzoites express at least two surface proteases (23) and one ROP-derived protease (TgSUB2) 2 that presumably occupy the PV. Thus, TgPI-1 may serve to inactivate parasite-derived proteases in the PV, thereby preventing them from inappropriately processing vacuolar protein substrates.…”

Section: Discussionmentioning

confidence: 99%

“…Secretion Assays-Secretion analyses were performed essentially as described previously (23). Briefly, 3 ϫ 10 9 tachyzoites were resuspended in invasion media (DMEM, 1.5 g/liter sodium bicarbonate, 20 mM HEPES, pH 7.4, and 3% fetal bovine serum) and incubated for 2 min at 37°C with 100 M BAPTA-AM or invasion media alone before being transferred to an ice water bath.…”

Section: Methodsmentioning

confidence: 99%

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Functional Analysis of Toxoplasma gondii Protease Inhibitor 1

Morris¹,

Coppin²,

Tomavo³

et al. 2002

Journal of Biological Chemistry

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We have characterized a Kazal family serine protease inhibitor, Toxoplasma gondii protease inhibitor 1 (TgPI-1), in the obligate intracellular parasite Toxoplasma gondii. TgPI-1 contains four inhibitor domains predicted to inhibit trypsin, chymotrypsin, and elastase. Antibodies against recombinant TgPI-1 detect two polypeptides, of 43 and 41 kDa, designated TgPI-1 43 and TgPI-1 41 , in tachyzoites, bradyzoites, and sporozoites. TgPI-1 43 and TgPI-1 41 are secreted constitutively from dense granules into the excreted/secreted antigen fraction as well as the parasitophorous vacuole that T. gondii occupies during intracellular replication. Recombinant TgPI-1 inhibits trypsin, chymotrypsin, pancreatic elastase, and neutrophil elastase. Immunoprecipitation studies with anti-rTgPI-1 antibodies reveal that recombinant TgPI-1 forms a complex with trypsin that is dependent on interactions with the active site of the protease. TgPI-1 is the first anti-trypsin/chymotrypsin inhibitor to be identified in bradyzoites and sporozoites, stages of the parasite that would be exposed to proteolytic enzymes in the digestive tract of the host.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Functional Analysis of Toxoplasma gondii Protease Inhibitor 1

Morris¹,

Coppin²,

Tomavo³

et al. 2002

Journal of Biological Chemistry

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“…These organelles play a crucial role in invasion and modulation of the host cell (Dubremetz et al 1993;Carruthers and Sibley, 1997;Ngo et al 2000;Joiner and Roos, 2002). The contents of these 3 secretory organelles are sequentially released in order to establish invasion and to ensure the maintenance of the parasite-host cell interaction (Fourmaux et al 1996;Carruthers et al 1999;Huynh et al 2003;Carruthers and Tomley, 2008). Initially, MIC proteins are secreted either prior to, or immediately upon, contact with the host cell membrane and they mediate adhesion and attachment to the host cell surface.…”

Section: Introductionmentioning

confidence: 99%

Molecular cloning and characterization ofNcROP2Fam-1, a member of the ROP2 family of rhoptry proteins inNeospora caninumthat is targeted by antibodies neutralizing host cell invasionin vitro

et al. 2013

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S U M M A R YRecent publications demonstrated that a fragment of a Neospora caninum ROP2 family member antigen represents a promising vaccine candidate. We here report on the cloning of the cDNA encoding this protein, N. caninum ROP2 family member 1 (NcROP2Fam-1), its molecular characterization and localization. The protein possesses the hallmarks of ROP2 family members and is apparently devoid of catalytic activity. NcROP2Fam-1 is synthesized as a pre-pro-protein that is matured to 2 proteins of 49 and 55 kDa that localize to rhoptry bulbs. Upon invasion the protein is associated with the nascent parasitophorous vacuole membrane (PVM), evacuoles surrounding the host cell nucleus and, in some instances, the surface of intracellular parasites. Staining was also observed within the cyst wall of 'cysts' produced in vitro. Interestingly, NcROP2Fam-1 was also detected on the surface of extracellular parasites entering the host cells and antibodies directed against NcROP2Fam-1-specific peptides partially neutralized invasion in vitro. We conclude that, in spite of the general belief that ROP2 family proteins are intracellular antigens, NcROP2Fam-1 can also be considered as an extracellular antigen, a property that should be taken into account in further experiments employing ROP2 family proteins as vaccines.

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“…Cell pellets were probed with anti-β-galactosidase monoclonal antibody 40a-1 as a control for loading and inadvertent lysis. Secreted MIC2 in the supernatant migrates slightly faster than cellular MIC2 (cMIC2) due to proteolysis during shedding 27 . To test the roles of extracellular and intracellular calcium, respectively, purified parasites were treated with 1 mM of BAPTA or 50 µM of BAPTA-AM for 20 min at 18 °C before treatment with 100 nM of ABA for 5 min, as described previously 7.…”

Section: Protein Secretionmentioning

confidence: 99%

Abscisic acid controls calcium-dependent egress and development in Toxoplasma gondii

Nagamune

Hicks

Fux

et al. 2008

Nature

190

174

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Calcium controls a number of critical events including motility, secretion, cell invasion, and egress by protozoan parasites 1. Compared to animal 2 and plant cells 3 , the molecular mechanisms that govern calcium signaling in parasites are poorly understood. Here we demonstrate that the production of the phytohormone abscisic acid (ABA) controls calcium signaling within the apicomplexan parasite Toxoplasma gondii, an important human pathogen. In plants, ABA controls a number of important events including environmental stress responses, embryo development, and seed dormancy 4 ,5 . ABA induces production of the second-messenger cyclic ADP ribose (cADPR), which controls release of intracellular calcium stores in plants 6 . cADPR also controls intracellular calcium release in the protozoan parasite T. gondii 7,8 ; however, previous studies have not revealed the molecular basis of this pathway 9 . Addition of exogenous ABA induced formation of cADPR in T. gondii, stimulated calcium-dependent protein secretion, and induced parasite egress from the infected host cell in a density-dependent manner. Production of endogenous ABA within the parasite was confirmed by HPLC purification and GC-MS analysis. Selective disruption of ABA synthesis by the inhibitor fluridone (FLU) delayed egress and induced development of the slow-growing, dormant cyst stage of the parasite. Thus, ABA-mediated calcium signaling controls the decision between lytic and chronic stage growth, a developmental switch that is central in pathogenesis and transmission. The pathway for ABA production was likely acquired with an algal endosymbiont that was retained as a non-photosynthetic plastid known as the apicoplast. The plant-like nature of this pathway may be exploited therapeutically as shown by the ability of a specific inhibitor of ABA synthesis to prevent toxoplasmosis in the mouse model.Calcium-mediated secretion in T. gondii controls both motility and cell invasion and previous studies have demonstrated that these processes utilize the second messenger cADPR, yet the signals triggering this pathway remain unresolved 7,8 . In plants 6 , hydra 10 , and sponges 11 , ABA stimulates release of intracellular calcium through elevation of the cyclic nucleotide cADPR. Addition of exogenous ABA proved to be a potent agonist of secretion in T. gondii as shown by the release of the protein MIC2, a parasite adhesin that is discharged into the supernatant in response to increases in intracellular calcium (Fig. 1A). Induction of MIC2 secretion by ABA was highly specific to (±) -ABA and was not induced by (−) -ABA, the precursor β-carotene, or retinoic acid (Fig. 1B). Treatment with ABA lead to a dose-dependent increase in the second messenger cADPR in T. gondii, suggesting ABA may be a natural agonist for calcium signaling in parasites (Fig. 1C). Finally, chelation of intracellular calcium in the parasite blocked secretion induced by ABA, confirming that it acts through release of an intracellular calcium pool (Fig. 1D). Collectively these results ind...

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The Toxoplasma Adhesive Protein MIC2 Is Proteolytically Processed at Multiple Sites by Two Parasite-derived Proteases

Cited by 176 publications

References 27 publications

Functional Analysis of Toxoplasma gondii Protease Inhibitor 1

Functional Analysis of Toxoplasma gondii Protease Inhibitor 1

Molecular cloning and characterization ofNcROP2Fam-1, a member of the ROP2 family of rhoptry proteins inNeospora caninumthat is targeted by antibodies neutralizing host cell invasionin vitro

Abscisic acid controls calcium-dependent egress and development in Toxoplasma gondii

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