Molecular cloning, characterization and localization of PfPK4, an eIF-2alpha kinase-related enzyme from the malarial parasite Plasmodium falciparum.

PfPK4, a protein kinase gene from the human malarial parasite Plasmodium falciparum, has been cloned utilizing oligonucleotide probing. The gene encodes a protein of a predicted length of 1123 amino acids, and within this amino acid sequence all the conserved regions characteristic of protein kinases can be identified. The catalytic kinase domain possesses highest identities (34-37%) with eukaryotic initiation factor-2alpha (eIF-2alpha) kinases, especially haem-regulated inhibitory (HRI) protein kinases. There are two kinase inserts in PfPK4, located at positions common to eIF-2alpha kinases. The first insert separates kinase subdomains IV and VI by 559 amino acids, and the second subdomains VII and VIII by 41 amino acids. Both inserts are larger than their homologues in eIF-2alpha kinases. The sequence of PfPK4 has one putative haemin-binding site. The recombinant protein, expressed in Escherichia coli, phosphorylates a synthetic peptide representing a substrate of eIF-2alpha kinases. Autophosphorylation and substrate phosphorylation are inhibited by haemin. Thus PfPK4 appears to be the first protozoan protein kinase related to eIF-2alpha kinases and might be the first non-mammalian HRI kinase. Western blots indicated that the protein is expressed as major forms of 80 and 90 kDa. Whereas the 80 kDa form is present throughout the intraerythrocytic development and in merozoites, the two 90 kDa forms are only found in mature parasites. One of the latter is also present in the membrane fraction of erythrocytes harbouring segmenters. Confocal microscopy detected the protein distributed throughout the trophozoite, whereas it was found in discrete foci (punctate distribution) in segmenters. PfPK4 co-localizes with P. falciparum 83 kDa antigen/apical membrane antigen-1 at the apical complex in segmenters and merozoites, but does not co-localize with rhoptry-associated protein-1.

Plasmodium falciparum protein kinase 5 and the malarial nuclear division cycles.

In the course of our studies on cell cycle regulation mechanisms of Plasmodium falciparum, we investigated expression pattern, kinase activity, and localization of PfPK5, a putative malarial member of the family of cyclin-dependent protein kinase (cdks). The kinase was immunoprecipitated from parasites of selected stages and from parasites blocked with the cell-cycle inhibitor aphidicolin. An elevated kinase activity of PfPK5 from aphidicolin-blocked cells suggested that the enzyme might be implicated in the regulation of the parasite's S-phase. To further investigate this hypothetical function, parasite cultures were treated with the specific cdk inhibitors flavopiridol and olomoucine, which act on PfPK5 in vitro at similar concentrations as on other cdks. When applied during the nuclear division cycles of the parasite, both drugs markedly inhibited the DNA synthesis, as predicted from our proposition that PfPK5 is necessary to activate or maintain the parasite S-phase. Immunolocalization studies provide further evidence for this potential role of PfPK5.

Expression and secretion of malarial parasite beta-tubulin in Bacillus brevis.

Microtubule inhibitors are active against the human malarial parasite Plasmodium falciparum, but whether these drugs actually interact with parasite tubulins is not known. It has not previously been possible to produce mg quantities of isolated, soluble tubulin subunits for drug-binding experiments. A cDNA encoding P falciparum beta-tubulin was expressed and the protein secreted in Bacillus brevis. With the addition of EGTA to the culture medium, which increases shedding of proteins from the cell surface, up to 2 mg/l recombinant beta-tubulin was obtained in supernatants. It is not clear why B brevis is able to secrete this normally cytoplasmic protein, but the secretion levels of recombinant proteins may be related to the net charge of the first few residues of the mature polypeptide.

Roles of peptidyl-prolyl cis-trans isomerase and calcineurin in the mechanisms of antimalarial action of cyclosporin A, FK506, and rapamycin.

The immunosuppressive peptide cyclosporin A inhibits the growth of malaria parasites in vitro and in vivo, but little is known about its mechanism of antimalarial action. The immunosuppressive action of cyclosporin A is believed to result from binding of the drug to cyclophilins (intracellular peptidyl-prolyl cis-trans isomerases), and inhibition of the protein phosphatase calcineurin by the cyclosporin A-cyclophilin complex. Two immunosuppressive macrolides, FK506 and rapamycin, bind to a distinct isomerase, FKBP12, and the FK506-FKBP complex also inhibits calcineurin. Calcineurin itself is apparently involved in signal transduction between the T-cell membrane and nucleus, and its inhibition blocks T-cell activation. Rapamycin inhibits a later step in T-cell proliferation. Peptidyl-propyl cis-trans isomerase activity was detected in extracts of Plasmodium falciparum. It was completely inhibited by concentrations of cyclosporin A above 0.1 microM, but not by FK506 or rapamycin, and probably represented one or more cyclophilins. Comparison of the antimalarial and anti-isomerase activities of a series of cyclosporin analogues failed to reveal a correlation between the two properties. Cyclosporin A and its more active 8'-oxymethyl-dihydro-derivative, in combination with the cyclophilin-containing P. falciparum extract, inhibited the protein phosphatase activity of bovine calcineurin. Therefore inhibition of a putative P. falciparum calcineurin by a complex of CsA and cyclophilin might be responsible for the antimalarial action of the drug. The most active cyclosporin, however, was a 3'-keto-derivative of cyclosporin D (SDZ PSC-833) which inhibited P. falciparum growth with a 50% inhibitory concentration (IC50) of 0.032 microM (compared with 0.30 microM for cyclosporin A), but was a poor inhibitor of the parasite isomerase. 3'-Keto-cyclosporin D has negligible immunosuppressive activity, but it strongly inhibits the P-glycoprotein of multi-drug resistant mammalian tumour cells. FK506 and rapamycin were also active antimalarials (IC50 of 1.9 and 2.6 microM, respectively) but in the absence of detectable FKBP in P. falciparum extracts, their mechanisms of antimalarial action remain unclear.

Effects of microtubule inhibitors on protein synthesis in Plasmodium falciparum.

At low concentrations, both isomers of tubulozole (C, T) inhibit Plasmodium falciparum but only tubulozole C inhibits mammalian cells. Since tubulozole C prevents polymerization of mammalian tubulin whereas tubulozole T does not, the antimalarial action of tubulozoles may not involve microtubules. The present study concerns the inhibition of parasite protein synthesis by the tubulozoles. While tubulozoles took 3-4 h to kill parasites in erythrocytic culture, they inhibited protein synthesis within 10 min. The concentrations of the drug required were, however, too high for this to account for their antimalarial action. The microtubule inhibitor colcemid inhibited protein synthesis rapidly and at relevant concentrations, but vinblastine did not inhibit protein synthesis. Tubulozole T and colcemid inhibited protein synthesis posttranscriptionally since they had little effect on RNA synthesis. Analysis of labelled parasite proteins by two-dimensional gel electrophoresis showed that while it inhibited synthesis of most proteins to the same degree, tubulozole T super-inhibited the synthesis of certain proteins. This may cause its antimalarial effect at low concentrations.

Characterization of a specific inhibitor of IL2-mediated proliferation from serum of Plasmodium berghei infected mice.

Serum pools from mice undergoing lethal infection with Plasmodium berghei inhibit the growth of an IL2-dependent mouse cytotoxic T cell line (CTLL). A partially purified preparation of the inhibitory factor specifically inhibited IL2-mediated events such as IL2-dependent CTLL growth and the Con A mitogenic response of normal mouse spleen cells. Production of and response to IL1, as well as growth of myeloma lines, was not affected. Administration of the partially purified preparation to normal mice resulted in a significant depression in IL2 production, thereby indicating a role for the inhibitory factor in maintaining immune depression in malaria-infected mice.

Autoantibodies in humans with cystic or alveolar echinococcosis.

Sera from 16 echinococcosis patients were analyzed with respect to polyclonal B cell activation and autoantibody formation. At least 8 of the sera were from patients who were never in tropical countries and therefore their cases were not complicated by other parasitic diseases. In comparison with a group of 52 healthy controls, these patients had significant levels of antibodies to DNP and haemocyanin, indicators of polyclonal B cell activation. There were also significant differences between control and patient groups with respect to antibodies to dsDNA, histones, actin, vimentin, and desmin. This is the first report of autoantibodies in echinococcosis.