ABSTRACT
East Coast fever (ECF) is a tick-borne disease caused by the parasite Theileria parva which infects cattle. In Sub-Saharan Africa it leads to enormous economic costs. After a bite of a tick, sporozoites invade the host lymphocytes and develop into schizonts. At this stage the parasite transforms host lymphocytes resulting in the clonal expansion of infected lymphocytes. Animals develop a lymphoma like disorder after infection which is rapidly fatal. Hitherto, a few drugs of the quinone type can cure the disease. However, therapy can only be successful after early diagnosis. The genera Theileria and Plasmodium, which includes the causative agent of human malaria, are closely related apicomplexan parasites. Enzymes of the hypusine pathway, a posttranslational modification in eukaryotic initiation factor EIF-5A, have shown to be druggable targets in Plasmodium. We identified the first enzyme of the hypusine pathway from T. parva, the deoxyhypusine synthase (DHS), which is located on chromosome 2 of the Muguga strain. Transcription is significantly increased in schizonts. The expressed T. parva DHS reveals an open reading frame (ORF) of 370 amino acids after expression in Escherichia coli Rosetta cells with a molecular size of 41.26 kDa and a theoretical pI of 5.26. Screening of the Malaria Box which consists of 400 active compounds resulted in a novel heterocyclic compound with a guanyl spacer which reduced the activity of T. parva DHS to 45%. In sum, the guanyl residue seems to be an important lead structure for inhibition of Theileria DHS. Currently, more different guanyl analogues from the Malaria Box are tested in inhibitor experiments to determine their efficacy.
Subject(s)
Oxidoreductases Acting on CH-NH Group Donors/antagonists & inhibitors , Plasmodium/enzymology , Theileria parva/enzymology , Amino Acid Sequence , Animals , Cattle , Cloning, Molecular , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Escherichia coli/metabolism , Guanine/chemistry , Heterocyclic Compounds/chemistry , Heterocyclic Compounds/metabolism , Humans , Kinetics , Lymphocytes/parasitology , Molecular Sequence Data , Oxidoreductases Acting on CH-NH Group Donors/genetics , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Plasmodium/genetics , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Sequence Alignment , Theileria parva/geneticsABSTRACT
A study on different Theileria annulata and Theileria parva infected cell lines was performed in order to evaluate the general relevance of the MAP-kinase activation status for Theileria- mediated transformation.
Subject(s)
Mitogen-Activated Protein Kinases/metabolism , Theileria annulata/enzymology , Theileria parva/enzymology , Animals , Blotting, Western , Cell Line , Enzyme Activation , Extracellular Signal-Regulated MAP Kinases/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
The apicomplexan parasites Theileria annulata and Theileria parva cause severe lymphoproliferative disorders in cattle. Disease pathogenesis is linked to the ability of the parasite to transform the infected host cell (leukocyte) and induce uncontrolled proliferation. It is known that transformation involves parasite dependent perturbation of leukocyte signal transduction pathways that regulate apoptosis, division and gene expression, and there is evidence for the translocation of Theileria DNA binding proteins to the host cell nucleus. However, the parasite factors responsible for the inhibition of host cell apoptosis, or induction of host cell proliferation are unknown. The recent derivation of the complete genome sequence for both T. annulata and T. parva has provided a wealth of information that can be searched to identify molecules with the potential to subvert host cell regulatory pathways. This review summarizes current knowledge of the mechanisms used by Theileria parasites to transform the host cell, and highlights recent work that has mined the Theileria genomes to identify candidate manipulators of host cell phenotype.
Subject(s)
Cattle Diseases/genetics , Theileria/genetics , Theileriasis/genetics , Amino Acid Sequence , Animals , Cattle/parasitology , Cattle Diseases/enzymology , Cattle Diseases/parasitology , Genes, Protozoan/genetics , Genome, Protozoan/genetics , Host-Parasite Interactions/genetics , Leukocytes/parasitology , Models, Biological , Phenotype , Protozoan Proteins/genetics , Signal Transduction/genetics , Theileria/enzymology , Theileria annulata/enzymology , Theileria annulata/genetics , Theileria parva/enzymology , Theileria parva/genetics , Theileriasis/enzymology , Theileriasis/parasitologyABSTRACT
Recent studies suggest that peptidyl-prolyl isomerases of the cyclophilin family, that access the secretory pathway, can be involved in the interaction of parasitic protozoa with mammalian host cells. The amino acid sequence of a cDNA encoding a cyclophilin family member of the intracellular protozoan parasite of cattle Theileria parva contains a conserved C-terminal domain that exhibits 70% amino acid identity to cyclophilin proteins from other organisms, and a unique 60 amino acid novel N-terminal extension. Cell-free expression of the cDNA revealed a 26kDa amino translation product, indicating expression of the N-terminal domain. The protein-coding region contains three short introns, less than 100 base pairs in length and Northern blot analysis demonstrates expression of a single 0.9 kb transcript in the piroplasm and schizont stages. The transcript is present in high abundance in the intra-lymphocytic schizont stage. The recombinant protein binds to immobilized cyclosporin A, a finding consistent with peptidyl-prolyl cis-trans isomerase function in vivo. A predicted N-terminal signal peptide was functional for entry into the eukaryotic secretory transport pathway in a cell-free in vitro transcription/translation system. The C-terminal cyclophilin domain was translocated across the membrane of the endoplasmic reticulum and the uncleaved signal peptide functioned as a membrane anchor.
Subject(s)
Cyclophilins/genetics , Endoplasmic Reticulum/physiology , Protein Sorting Signals/genetics , Theileria parva/physiology , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Blotting, Southern , Cyclophilins/metabolism , Cyclophilins/physiology , Cyclosporine/metabolism , DNA/chemistry , DNA/genetics , Endoplasmic Reticulum/metabolism , Molecular Sequence Data , Protein Sorting Signals/physiology , RNA/chemistry , RNA/genetics , Sequence Alignment , Theileria parva/enzymology , Theileria parva/geneticsABSTRACT
The protozoan parasite Theileria (spp. parva and annulata) infects bovine leukocytes and provokes a leukaemia-like disease in vivo. In this study, we have detected a type 1 serine/threonine phosphatase activity with phosphorylase a as a substrate, in protein extracts of parasites purified from infected B lymphocytes. In contrast to this type 1 activity, dose response experiments with okadaic acid (OA), a well characterised inhibitor of type 1 and 2A protein phosphatases, indicated that type 2A is the predominant activity detected in host B cells. Furthermore, consistent with polycation-specific activation of the type 2A phosphatase, protamine failed to activate the parasite-associated phosphorylase a phosphatase activity. Moreover, inhibition of phosphorylase a dephosphorylation by phospho-DARPP-32, a specific type 1 inhibitor, clearly demonstrated that a type 1 phosphatase is specifically associated with the parasite, while the type 2A is predominantly expressed in the host lymphocyte. Since an antibody against bovine catalytic protein phosphatase 1 (PP1) subunit only recognised the PP1 in B cells, but not in parasite extracts, we conclude that in parasites the PP1 activity is of parasitic origin. Intriguingly, since type 1 OA-sensitive phosphatase activity has been recently described in Plasmodium falciparum, we can conclude that these medically important parasites produce their one PP1.
Subject(s)
B-Lymphocytes/parasitology , Phosphoprotein Phosphatases/metabolism , Theileria parva/enzymology , Animals , Cattle , Cells, Cultured , Phosphorylase a/metabolism , Protein Phosphatase 1ABSTRACT
Theileria parva schizonts propagated in vitro in peripheral blood lymphocytes were purified and assayed for key enzymes of glucose and glycerol catabolism and the citric acid cycle. The activities of glycolytic enzymes were in the range of 21-100 nmol/min/mg protein. Glycerol kinase and alpha -glycerophosphate dehydrogenase activities were more than 16 times lower than the activities of other enzymes catalysing the oxidation of the triose phosphates to lactate. It was suggested that the catabolism of glycerol is negligible and that glucose is catabolized to lactate via the Embden-Meyerhof pathway. The activities of the enzymes catalysing the section of the citric acid cycle that involves the formation of citrate to succinyl-CoA were consistently very low (less than 2.0 nmol/min/mg protein), indicating that this part of the cycle plays a minor role in this parasite. Enzyme activities of the cycle catalysing the formation of succinate from oxaloacetate were relatively higher than those catalysing other sections of the citric acid cycle, suggesting that this section of the cycle could be important to the parasite. Pyruvate carboxylase activity was more than 10 times that of phosphoenolpyruvate carboxykinase. It was suggested that pyruvate could be carboxylated to oxaloacetate. Taken together, these results suggest that the catabolism of glucose in Theileria parva schizonts is mainly via the Embden-Meyerhof pathway and that the citric acid cycle plays a minor role in energy production.
Subject(s)
Citric Acid Cycle/physiology , Glucose/metabolism , Glycerol/metabolism , Theileria parva/enzymology , Animals , Cell Culture Techniques , Pyruvate Carboxylase/metabolismABSTRACT
Theileria-infected cells are induced to undergo a transformation that is reversible, since their proliferation is inhibited after elimination of the schizonts by the theilericidal drug buparvaquone. The molecular mechanisms of the transformation remain unknown. The experiments described in the present report deal with the role of casein kinase (CK) II, a serine/threonine protein kinase, in the permanent proliferation of the parasitized cells and show that the CK II-alpha subunit is expressed in both T. annulata- and T. parva-infected cells and that its expression is closely related to the presence of the parasites in the host-cell cytoplasm. Thus, elimination of the schizonts by buparvaquone leads to the inhibition of CK II-alpha subunit mRNA expression without affecting the expression of actin. Cells treated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) are inhibited in a dose-dependent manner from under-going DNA synthesis as measured by [3H]-thymidine incorporation and from expressing CK II. Furthermore, a host-cell-specific CK II-alpha antisense inhibits DNA synthesis in a dose-dependent manner. In the present study, 6 microM antisense reduced [3H]-thymidine incorporation by Theileria-infected bovine cells to about 50%. Using a primer derived from T. parva CK II, we detected a parasite-specific CK II mRNA in T. parva-infected cell lines. Interestingly. DRB also inhibited the expression of the parasite-specific CK II. However, to date we have not detected a target sequence for this primer in T. annulata schizonts.
Subject(s)
Lymphocytes/enzymology , Lymphocytes/parasitology , Protein Serine-Threonine Kinases/genetics , Theileria annulata/physiology , Theileria parva/physiology , Animals , Antiprotozoal Agents/pharmacology , Casein Kinase II , Cattle , Cell Division , Cell Line , Concanavalin A/pharmacology , DNA/biosynthesis , Dichlororibofuranosylbenzimidazole/pharmacology , Gene Expression Regulation , Lymphocytes/cytology , Mitogens/pharmacology , Naphthoquinones/pharmacology , Nucleic Acid Synthesis Inhibitors/pharmacology , Protein Serine-Threonine Kinases/biosynthesis , RNA, Messenger , Theileria annulata/drug effects , Theileria annulata/enzymology , Theileria parva/drug effects , Theileria parva/enzymology , Ticks/parasitologyABSTRACT
Theileria parva sporozoites rapidly enter bovine lymphocytes. Since lymphocytes are normally nonphagocytes sporozoite binding to the host cell surface must initiate events in the host cell, leading to the internalization of the parasite. In the present study inhibitors of various key molecules in cell signal transduction and activation pathways, in combination with a method of quantitation, have been used to examine the possible role(s) of these systems in sporozoite entry. A variety of protein kinase inhibitors caused significant inhibition of sporozoite entry. Moreover, protein kinase activities in both the sporozoite and the host cell were essential to sporozoite invasion. Down-regulation of lymphocyte protein kinase C and inhibitors of phospholipase C but not phospholipase A2 activity also blocked sporozoite entry. Parasite entry could also be blocked by inhibitors of G protein activity. Treatment of sporozoites with AIF(3-5) blocked parasite binding while treatment of host cells inhibited sporozoite internalization. Furthermore, sporozoite entry was dependent on a cholera toxin-inhibitable process(es), whereas mastroparan and pertussis toxin had no significant inhibitory effects. Collectively these results provide initial evidence for both parasite protein kinase- and G protein-dependent processes as well as the participation of a variety of host cell signal transduction pathways in the sporozoite entry process.
Subject(s)
Lymphocytes/parasitology , Signal Transduction , Theileria parva/physiology , Adenosine/analogs & derivatives , Adenosine/pharmacology , Animals , Cattle , Down-Regulation , Enzyme Inhibitors/pharmacology , GTP-Binding Proteins/physiology , Genistein , Isoflavones/pharmacology , Lymphocytes/enzymology , Lymphocytes/physiology , Okadaic Acid/pharmacology , Phospholipases A/antagonists & inhibitors , Phospholipases A/metabolism , Phospholipases A2 , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphoprotein Phosphatases/metabolism , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolism , Staurosporine/pharmacology , Suramin/pharmacology , Tetradecanoylphorbol Acetate/pharmacology , Theileria parva/enzymology , Trypanocidal Agents/pharmacology , Type C Phospholipases/antagonists & inhibitors , Type C Phospholipases/metabolismSubject(s)
Protein Serine-Threonine Kinases/metabolism , T-Lymphocytes/parasitology , Theileria parva/physiology , Theileriasis/enzymology , Animals , Casein Kinase II , Cattle , Cell Division , Cell Transformation, Neoplastic , Lymphocyte Activation , Lymphoma/etiology , Mice , Mice, Transgenic , Signal Transduction , T-Lymphocytes/enzymology , T-Lymphocytes/physiology , Theileria parva/enzymology , Theileriasis/parasitologyABSTRACT
Dinucleotide frequency (DiF) and codon usage (cu) were analysed in gene sequences from four parasitic protozoa, Babesia bovis, Theileria parva, Toxoplasma gondii and Eimeria tenella, of the phylum Apicomplexa. In keeping with the 'genome hypothesis', cu was found to be non-random and species specific in these organisms, although cu among members of the same subclass was found to be very similar. Several low-usage (lu) codons were identified, and the usage of lu codons appears to be related to the taxonomic position of the organisms under study. A comparison of the observed/expected DiF ratios obtained from gene coding regions revealed a low frequency of the TA and CG dinucleotides in all organisms studied. A comparison of these DiF ratios with those found in rRNA-encoding genes and in introns, showed that in the parasites, B. bovis and Th. parva (representing the piroplasms), the low frequency of dinucleotides appeared to be the result of coding pressure alone. In T. gondii and E. tenella (representing the coccidia), however, coding pressure could not completely explain differences in DiF.