Hitoshi Takemae

Use of the kinase inhibitor analog 1NM-PP1 reveals a role for Toxoplasma gondii CDPK1 in the invasion step.

ABSTRACT Toxoplasma gondii CDPK1 (TgCDPK1) was found to be the target of the toxoplasmocidal compound 1NM-PP1. When TgCDPK1 was mutated at position 128 from glycine to methionine, resistance was gained. Inhibition of gliding motility without inhibition of micronemal secretion by 1NM-PP1 suggests a function for TgCDPK1 in gliding motility.

Plasmodium falciparum BAEBL Binds to Heparan Sulfate Proteoglycans on the Human Erythrocyte Surface

Erythrocyte invasion is critical to the pathogenesis and survival of the malarial parasite, Plasmodium falciparum. This process is partly mediated by proteins that belong to the Duffy binding-like family, which are expressed on the merozoite surface. One of these proteins, BAEBL (also known as EBA-140), is thought to bind to glycophorin C in a sialic acid-dependent manner. In this report, by the binding assay between recombinant BAEBL protein and enzyme-treated erythrocytes, we show that the binding of BAEBL to erythrocytes is mediated primarily by sialic acid and partially through heparan sulfate (HS). Because BAEBL binds to several kinds of HS proteoglycans or purified HS, the BAEBL-HS binding was found to be independent of the HS proteoglycan peptide backbone and the presence of sialic acid moieties. Furthermore, both the sialic acid- and HS-dependent binding were disrupted by the addition of soluble heparin. This inhibition may be the result of binding between BAEBL and heparin. Invasion assays demonstrated that HS-dependent binding was related to the efficiency of merozoite invasion. These results suggest that HS functions as a factor that promotes the binding of BAEBL and merozoite invasion. Moreover, these findings may explain the invasion inhibition mechanisms observed following the addition of heparin and other sulfated glycoconjugates.

Roles of 1-Cys peroxiredoxin in haem detoxification in the human malaria parasite Plasmodium falciparum

In the present study, we investigated whether Plasmodium falciparum 1‐Cys peroxiredoxin (Prx) (Pf1‐Cys‐Prx), a cytosolic protein expressed at high levels during the haem‐digesting stage, can act as an antioxidant to cope with the oxidative burden of haem (ferriprotoporphyrin IX; FP). Recombinant Pf1‐Cys‐Prx protein (rPf1‐Cys‐Prx) competed with glutathione (GSH) for FP and inhibited FP degradation by GSH. When rPf1‐Cys‐Prx was added to GSH‐mediated FP degradation, the amount of iron released was reduced to 23% of the reaction without the protein (P < 0.01). The rPf1‐Cys‐Prx bound to FP–agarose at pH 7.4, which is the pH of the parasite cytosol. The rPf1‐Cys‐Prx could completely protect glutamine synthetase from inactivation by the dithiothreitol–Fe3+‐dependent mixed‐function oxidation system, and it also protected enolase from inactivation by coincubation with FP/GSH. Incubation of white ghosts of human red blood cells and FP with rPf1‐Cys‐Prx reduced formation of membrane associations with FP to 75% of the incubation without the protein (P < 0.01). The findings of the present study suggest that Pf1‐Cys‐Prx protects the parasite against oxidative stresses by binding to FP, slowing the rate of GSH‐mediated FP degradation and consequent iron generation, protecting proteins from iron‐derived reactive oxygen species, and interfering with formation of membrane‐associated FP.

Identification of Toxoplasma gondii cAMP Dependent Protein Kinase and Its Role in the Tachyzoite Growth

Background cAMP-dependent protein kinase (PKA) has been implicated in the asexual stage of the Toxoplasma gondii life cycle through assaying the effect of a PKA-specific inhibitor on its growth rate. Since inhibition of the host cell PKA cannot be ruled out, a more precise evaluation of the role of PKA, as well as characterization of the kinase itself, is necessary. Methodology/Principal Finding The inhibitory effects of two PKA inhibitors, H89, an ATP-competitive chemical inhibitor, and PKI, a substrate-competitive mammalian natural peptide inhibitor, were estimated. In the in vitro kinase assay, the inhibitory effect of PKI on a recombinant T. gondii PKA catalytic subunit (TgPKA-C) was weaker compared to that on mammalian PKA-C. In a tachyzoite growth assay, PKI had little effect on the growth of tachyzoites, whereas H89 strongly inhibited it. Moreover, T. gondii PKA regulatory subunit (TgPKA-R)-overexpressing tachyzoites showed a significant growth defect. Conclusions/Significance Our data suggest that PKA plays an important role in the growth of tachyzoites, and the inhibitory effect of substrate-competitive inhibitor PKI on T. gondii PKA was low compared to that of the ATP competitive inhibitor H89.

Molecular analyses of Toxoplasma gondii calmodulin-like domain protein kinase isoform 3.

Ca(2+) signaling is thought to play an important role in Toxoplasma gondii motility, including invasion of and egress from host cells. Recently, it has been reported that phosphorylation of the glideosome apparatus components of T. gondii occurs during invasion. To elucidate the role of T. gondii calmodulin-like domain protein kinase in the signaling pathway that bridges Ca(2+) stimulation and motility, we characterized T. gondii calmodulin-like domain protein kinase isoform 3 (TgCDPKif3). TgCDPKif3 is homologous to Plasmodium falciparum calcium-dependent protein kinase 1, which has been reported to phosphorylate P. falciparum glideosome components. TgCDPKif3 was purified as a fusion protein that was labeled with [gamma-(32)P]ATP, and the label was subsequently removed by phosphatase treatment. Phosphorylation was eliminated when the putative catalytic lysine residue of TgCDPKif3 was replaced with alanine. TgCDPKif3 phosphorylated Histone II(AS) as a representative substrate in a Ca(2+)-dependent manner at a high Ca(2+) concentration. TgCDPKif3 was localized to the apical ends of tachyzoites. TgCDPKif3 showed the translocation between intra- and extracellular tachyzoites. TgCDPKif3 could phosphorylate T. gondii aldolase 1 (TgALD1) in vitro. The interaction between TgCDPKif3 and TgALD1 was confirmed by the co-immunoprecipitation assay in mammal cells. We suggested that TgCDPKif3 could participate in the motility of T. gondii through the phosphorylation of glideosome complex member.

Functional Analysis of Proteoglycan Galactosyltransferase II RNA Interference Mutant Flies

Heparan sulfate proteoglycan plays an important role in developmental processes by modulating the distribution and stability of the morphogens Wingless, Hedgehog, and Decapentaplegic. Heparan and chondroitin sulfates share a common linkage tetrasaccharide structure, GlcAβ1,3Galβ1,3Galβ1,4Xylβ-O-Ser. In the present study, we identified Drosophila proteoglycan galactosyltransferase II (dβ3GalTII), determined its substrate specificity, and performed its functional analysis by using RNA interference (RNAi) mutant flies. The enzyme transferred a galactose to Galβ1,4Xyl-pMph, confirming that it is the Drosophila ortholog of human proteoglycan galactosyltransferase II. Real-time PCR analyses revealed that dβ3GalTII is expressed in various tissues and throughout development. The dβ3GalTII RNAi mutant flies showed decreased amounts of heparan sulfate proteoglycans. A genetic interaction of dβ3GalTII with Drosophila β1,4-galactoslyltransferase 7 (dβ4GalT7) or with six genes that encode enzymes contributing to the synthesis of glycosaminoglycans indicated that dβ3GalTII is involved in heparan sulfate synthesis for wing and eye development. Moreover, dβ3GalTII knock-down caused a decrease in extracellular Wingless in the wing imaginal disc of the third instar larvae. These results demonstrated that dβ3GalTII contributes to heparan sulfate proteoglycan synthesis in vitro and in vivo and also modulates Wingless distribution.

A Novel PAN/Apple Domain-Containing Protein from Toxoplasma gondii: Characterization and Receptor Identification

Toxoplasma gondii is an intracellular parasite that invades nucleated cells, causing toxoplasmosis in humans and animals worldwide. The extremely wide range of hosts susceptible to T. gondii is thought to be the result of interactions between T. gondii ligands and receptors on its target cells. In this study, a host cell-binding protein from T. gondii was characterized, and one of its receptors was identified. P104 (GenBank Access. No. CAJ20677) is 991 amino acids in length, containing a putative 26 amino acid signal peptide and 10 PAN/apple domains, and shows low homology to other identified PAN/apple domain-containing molecules. A 104-kDa host cell-binding protein was detected in the T. gondii lysate. Immunofluorescence assays detected P104 at the apical end of extracellular T. gondii. An Fc-fusion protein of the P104 N-terminus, which contains two PAN/apple domains, showed strong affinity for the mammalian and insect cells evaluated. This binding was not related to protein-protein or protein-lipid interactions, but to a protein-glycosaminoglycan (GAG) interaction. Chondroitin sulfate (CS), a kind of GAG, was shown to be involved in adhesion of the Fc-P104 N-terminus fusion protein to host cells. These results suggest that P104, expressed at the apical end of the extracellular parasite, may function as a ligand in the attachment of T. gondii to CS or other receptors on the host cell, facilitating invasion by the parasite.

Characterization of the interaction between Toxoplasma gondii rhoptry neck protein 4 and host cellular β-tubulin

Toxoplasma rhoptry neck protein 4 (TgRON4) is a component of the moving junction macromolecular complex that plays a central role during invasion. TgRON4 is exposed on the cytosolic side of the host cell during invasion, but its molecular interactions remain unclear. Here, we identified host cellular β-tubulin as a binding partner of TgRON4, but not Plasmodium RON4. Coimmunoprecipitation studies in mammalian cells demonstrated that the C-terminal 15-kDa region of β-tubulin was sufficient for binding to TgRON4, and that a 17-kDa region in the proximal C-terminus of TgRON4 was required for binding to the C-terminal region of β-tubulin. Analysis of T. gondii-infected lysates from CHO cells expressing the TgRON4-binding region showed that the C-terminal region of β-tubulin interacted with TgRON4 at early invasion step. Our results provide evidence for a parasite-specific interaction between TgRON4 and the host cell cytoskeleton in parasite-infected cells.

Gellan sulfate inhibits Plasmodium falciparum growth and invasion of red blood cells in vitro

Here, we assessed the sulfated derivative of the microbial polysaccharide gellan gum and derivatives of λ and κ-carrageenans for their ability to inhibit Plasmodium falciparum 3D7 and Dd2 growth and invasion of red blood cells in vitro. Growth inhibition was assessed by means of flow cytometry after a 96-h exposure to the inhibitors and invasion inhibition was assessed by counting ring parasites after a 20-h exposure to them. Gellan sulfate strongly inhibited invasion and modestly inhibited growth for both P. falciparum 3D7 and Dd2; both inhibitory effects exceeded those achieved with native gellan gum. The hydrolyzed λ-carrageenan and oversulfated κ-carrageenan were less inhibitory than their native forms. In vitro cytotoxicity and anticoagulation assays performed to determine the suitability of the modified polysaccharides for in vivo studies showed that our synthesized gellan sulfate had low cytotoxicity and anticoagulant activity.

Identification of mutations in TgMAPK1 of Toxoplasma gondii conferring resistance to 1NM-PP1

Graphical abstract