Frances C. Recuenco

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.

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

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.

Heparin interacts with elongation factor 1α of Cryptosporidium parvum and inhibits invasion

Cryptosporidium parvum is an apicomplexan parasite that can cause serious watery diarrhea, cryptosporidiosis, in human and other mammals. C. parvum invades gastrointestinal epithelial cells, which have abundant glycosaminoglycans on their cell surface. However, little is known about the interaction between C. parvum and glycosaminoglycans. In this study, we assessed the inhibitory effect of sulfated polysaccharides on C. parvum invasion of host cells and identified the parasite ligands that interact with sulfated polysaccharides. Among five sulfated polysaccharides tested, heparin had the highest, dose-dependent inhibitory effect on parasite invasion. Heparan sulfate-deficient cells were less susceptible to C. parvum infection. We further identified 31 parasite proteins that potentially interact with heparin. Of these, we confirmed that C. parvum elongation factor 1α (CpEF1α), which plays a role in C. parvum invasion, binds to heparin and to the surface of HCT-8 cells. Our results further our understanding of the molecular basis of C. parvum infection and will facilitate the development of anti-cryptosporidial agents.

Lambda-carrageenan treatment exacerbates the severity of cerebral malaria caused by Plasmodium berghei ANKA in BALB/c mice

BackgroundThere is an urgent need to develop and test novel compounds against malaria infection. Carrageenans, sulphated polysaccharides derived from seaweeds, have been previously shown to inhibit Plasmodium falciparum in vitro. However, they are inflammatory and alter the permeability of the blood–brain barrier, raising concerns that their use as a treatment for malaria could lead to cerebral malaria (CM), a severe complication of the disease. In this work, the authors look into the effects of the administration of λ-carrageenan to the development and severity of CM in BALB/c mice, a relatively non-susceptible model, during infection with the ANKA strain of Plasmodium berghei.MethodsFive-week-old female BALB/c mice were infected with P. berghei intraperitoneally. One group was treated with λ-carrageenan (PbCGN) following the 4-day suppressive test protocol, whereas the other group was not treated (PbN). Another group of healthy BALB/c mice was similarly given λ-carrageenan (CGN) for comparison. The following parameters were assessed: parasitaemia, clinical signs of CM, and mortality. Brain and other vital organs were collected and examined for gross and histopathological lesions. Evans blue dye assays were employed to assess blood–brain barrier integrity.ResultsPlasmodium berghei ANKA-infected BALB/c mice treated with λ-carrageenan died earlier than those that received no treatment. Histopathological examination revealed that intracerebral haemorrhages related to CM were present in both groups of infected BALB/c mice, but were more numerous in those treated with λ-carrageenan than in mock-treated animals. Inflammatory lesions were also observed only in the λ-carrageenan-treated mice. These observations are consistent with the clinical signs associated with CM, such as head tilt, convulsions, and coma, which were observed only in this group, and may account for the earlier death of the mice.ConclusionThe results of this study indicate that the administration of λ-carrageenan exacerbates the severe brain lesions and clinical signs associated with CM in BALB/c mice infected with P. berghei ANKA.

Detection and molecular characterization of Cryptosporidium and Eimeria species in Philippine bats.

The genus Cryptosporidium, which is an obligate intracellular parasite, infects various vertebrates and causes a diarrheal disease known as cryptosporidiosis. Bats are naturally infected with zoonotic pathogens; thus, they are potential reservoirs of parasites. We investigated the species and genotype distribution as well as prevalence of Cryptosporidium and Eimeria in Philippine bats. We captured and examined 45 bats; four were positive for Cryptosporidium spp. and seven were positive for Eimeria spp. We detected Cryptosporidium bat genotype II from Ptenochirus jagori. Three other Cryptosporidium sequences, detected from Rhinolophus inops, Cynopterus brachyotis, and Eonycteris spelaea, could not be classified as any known species or genotype; we therefore propose the novel genotype Cryptosporidium bat genotypes V, VI, and VII. Bat genotype V is associated with human cryptosporidiosis clade, and therefore, this genotype may be transmissible to humans. Among the Eimeria sequences, BE3 detected from Scotophilus kuhlii was classified with known bat and rodent clades; however, other sequences detected from C. brachyotis, E. spelaea, Rousettus amplexicaudatus, and R. inops could not be classified with known Eimeria species. These isolates might represent a new genotype. Our findings demonstrate that the bats of the Philippines represent a reservoir of multiple Cryptosporidium and Eimeria spp.

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.