Haiyan Gong

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.

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.

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

Effects of dextran sulfates on the acute infection and growth stages of Toxoplasma gondii

Toxoplasma gondii is one of the most prevalent parasites, causing toxoplasmosis in various warm-blooded animals, including humans. Because of the broad range of hosts susceptible to T. gondii, it had been postulated that a universal component of the host cell surface, such as glycosaminoglycans (GAGs), may act as a receptor for T. gondii infection. Carruthers et al. (Infect Immun 68:4005–4011, 2000) showed that soluble GAGs have also been shown to disrupt parasite binding to human fibroblasts. Therefore, we investigated the inhibitory effect of GAGs and their analogue dextran sulfate (DS) on T. gondii infection. For up to 24xa0h of incubation after inoculation of T. gondii, the inhibitory effect of GAGs on T. gondii infection and growth inside the host cell was weak. In contrast, DS markedly inhibited T. gondii infection. Moreover, low molecular weight DS particularly slowed the growth of T. gondii inside host cells. DS10 (dextran sulfate MW 10xa0kDa) was the most effective agent in these in vitro experiments and was therefore tested for its inhibitory effects in animal experiments; infection inhibition by DS10 was confirmed under these in vivo conditions. In this report, we showed that DSs, especially DS10, have the potential of a new type of drug for toxoplasmosis.

Characterization of Plasmodium falciparum cdc2-related kinase and the effects of a CDK inhibitor on the parasites in erythrocytic schizogony.

The cell cycle of Plasmodium is unique among major eukaryotic cell cycle models. Cyclin-dependent kinases (CDKs) are thought to be the key molecular switches that regulate cell cycle progression in the parasite. However, little information is available about Plasmodium CDKs. The present study was performed to investigate the effects of a CDK inhibitor, olomoucine, on the erythrocytic growth of Plasmodium falciparum. This agent inhibited the growth of the parasite at the trophozoite/schizont stage. Furthermore, we characterized the Plasmodium CDK homolog, P. falciparum cdc2-related kinase-1 (Pfcrk-1), which is a potential target of olomoucine. We synthesized a functional kinase domain of Pfcrk-1 as a GST fusion protein using a wheat germ protein expression system, and examined its phosphorylation activity. The activity of this catalytic domain was higher than that of GST-GFP control, but the same as that of a kinase-negative mutant of Pfcrk-1. After the phosphatase treatment, the labeling of [γ-(32)P]ATP was abolished. Recombinant human cyclin proteins were added to these kinase reactions, but there were no differences in activity. This report provides important information for the future investigation of Plasmodium CDKs.

Characterization and binding analysis of a microneme adhesive repeat domain-containing protein from Toxoplasma gondii.

The intracellular parasite Toxoplasma gondii invades almost all nucleated cells, and has infected approximately 34% of the worlds population to date. In order to develop effective vaccines against T. gondii infection, understanding of the role of the molecules that are involved in the invasion process is important. For this purpose, we characterized T. gondii proteins that contain microneme adhesive repeats (MARs), which are common in moving junction proteins. T. gondii MAR domain-containing protein 4a (TgMCP4a), which contains repeats of 17-22 amino acid segments at the N-terminus and three putative MAR domains at the C-terminus, is localized near the rhoptry of extracellular parasites. Following infection, TgMCP4a was detected in the parasitophorous vacuole. The recombinant Fc-TgMCP4a N-terminus protein (rTgMCP4a-1/Fc) showed binding activity to the surface proteins of Vero, 293T, and CHO cells. The recombinant GST-TgMCP4a N-terminus protein (rTgMCP4a-1/GST), which exhibited binding activity, was used to pull down the interacting factors from 293T cell lysate, and subsequent mass spectrometry analysis revealed that three types of heat shock proteins (HSPs) interacted with TgMCP4a. Transfection of a FLAG fusion protein of TgMCP4a-1 (rTgMCP4a-1/FLAG) into 293T cell and the following immunoprecipitation with anti-FLAG antibody confirmed the interactions of HSC70 with TgMCP4a. The addition of rTgMCP4a-1/GST into the culture medium significantly affected the growth of the parasite. This study hints that T. gondii may employ HSP proteins of host cell to facilitate their growth.