PKA and Apicomplexan Parasite Diseases.

The cAMP-dependent protein kinase PKA is a well-characterized member of the serine-threonine protein AGC kinase family and is the effector kinase of cAMP signaling. As such, PKA is involved in the control of a wide variety of cellular processes including metabolism, cell growth, gene expression and apoptosis. cAMP-dependent PKA signaling pathways play important roles during infection and virulence of various pathogens. Since fluxes in cAMP are involved in multiple intracellular functions, a variety of different pathological infectious processes can be affected by PKA signaling pathways. Here, we highlight some features of cAMP-PKA signaling that are relevant to Plasmodium falciparum-infection of erythrocytes and present an update on AKAP targeting of PKA in PGE2 signaling via EP4 in Theileria annulata-infection of leukocytes and discuss cAMP-PKA signling in Toxoplasma.

Factors of occurrence of ocular toxoplasmosis. A review.

Acquired and congenital toxoplasmosis are frequently complicated by ocular toxoplasmosis. The diagnosis relies on clinical aspects, response to specific treatment and results of biological assays. The incidence and the prevalence of this complication are difficult to establish precisely and depend on the prevalence of the parasite infection in the general population, and are affected by factors such as type of exposure to the parasite, genetic backgrounds of the parasite and the host, and type of immune response elicited by the parasite.

Chromosome-sized DNA molecules of Plasmodium falciparum.

At least seven chromosome-sized DNA molecules (750 to 2000 kilobases in length and one fraction of undetermined molecular weight) from cultured clones and isolates of Plasmodium falciparum have been separated by pulsed-field gradient gel electrophoresis. Whereas asexual blood stages and sexual stages of the same line have identical molecular karyotypes, the length of chromosome-sized DNA molecules among different geographical isolates and several clones derived from a single patient is different. These length alterations of chromosomes are the result of DNA rearrangements that must occur unrelated to sexual differentiation.

Signalling in malaria parasites. The MALSIG consortium.

Depending on their developmental stage in the life cycle, malaria parasites develop within or outside host cells, and in extremely diverse contexts such as the vertebrate liver and blood circulation, or the insect midgut and hemocoel. Cellular and molecular mechanisms enabling the parasite to sense and respond to the intra- and the extra-cellular environments are therefore key elements for the proliferation and transmission of Plasmodium, and therefore are, from a public health perspective, strategic targets in the fight against this deadly disease. The MALSIG consortium, which was initiated in February 2009, was designed with the primary objective to integrate research ongoing in Europe and India on i) the properties of Plasmodium signalling molecules, and ii) developmental processes occurring at various points of the parasite life cycle. On one hand, functional studies of individual genes and their products in Plasmodium falciparum (and in the technically more manageable rodent model Plasmodium berghei) are providing information on parasite protein kinases and phosphatases, and of the molecules governing cyclic nucleotide metabolism and calcium signalling. On the other hand, cellular and molecular studies are elucidating key steps of parasite development such as merozoite invasion and egress in blood and liver parasite stages, control of DNA replication in asexual and sexual development, membrane dynamics and trafficking, production of gametocytes in the vertebrate host and further parasite development in the mosquito. This article, which synthetically reviews such signalling molecules and cellular processes, aims to provide a glimpse of the global frame in which the activities of the MALSIG consortium will develop over the next three years.

A primary malarial infection is composed of a very wide range of genetically diverse but related parasites.

To address the question of how many distinct parasites are injected when a mosquito bites, we have characterized isolates resulting most probably from a single sporozoite inoculum. We describe the direct and immediate cloning on hepatocyte feeder layers of a Thai and an African Plasmodium falciparum primary isolate and the characterization of 67 independent clones by four techniques totaling nine different markers. This led to three main conclusions: (a) both the phenotypic and genotypic markers revealed an unexpectedly large degree of diversity within the clones from a single isolate; (b) the clones are nonetheless genetically related; and (c) a single mosquito inoculum would most likely be sufficient to generate considerable isolate complexity in the absence of repeated exposure. This diversity, which has been greatly underestimated in previous studies, does not bode well for the development of successful malaria control means.

Notch is constitutively active in Theileria-transformed B cells and can be further stimulated by the filarial nematode-secreted product, ES-62.

Theileria parva-infected B cells express Jagged-1 and activate Notch signalling in a parasite-dependent manner. ES-62, a filarial nematode-secreted phosphorylcholine-containing glycoprotein, is able to further stimulate Notch-mediated signalling in parasitized cells. Notch is also activated to a similar extent by addition of exogenous IL-10, and this occurs prior to any increase in proliferation in T. parva-infected B cells.

TaCRK3 encodes a novel Theileria annulata protein kinase with motifs characteristic of the family of eukaryotic cyclin dependent kinases: a comparative analysis of its expression with TaCRK2 during the parasite life cycle.

The TaCRK3 gene from the bovine apicomplexan parasite Theileria annulata, encodes a 46 kDa polypeptide with strong homology to the eukaryotic family of cyclin-dependent kinases. TaCRK3 does not show significant alignment with any particular CDK group, other than the Pfmrk kinases from the related apicomplexans Plasmodium falciparum and Plasmodium yoelii. It has a putative bipartite nuclear localization signal and is located to parasite nuclei by IFAT. Protein levels are constitutive throughout differentiation of the intra-lymphocytic macroschizont. This contrasts with the expression pattern of TaCRK2 (Kinnaird et al., 1996, Mol. Microbiol., 22, 293-302) which is closely related to the eukaryotic CDK1 /2 families involved in regulation of cell cycle progression. TaCRK2 is also located to the parasite nuclei but has no nuclear localization signal and exhibits transient up-regulation in protein levels during mid-merogony. However compared to TaCRK3, it shows down-regulation near the end of merogony. We predict that TaCRK3 may have a role in regulation of gene transcription while TaCRK2 is more likely to be involved in control of parasite nuclear division.

A MAP kinase homologue from the human malaria parasite, Plasmodium falciparum.

Pfmap-1, a gene encoding a novel protein kinase, has been identified in the human malaria parasite Plasmodium falciparum, using the polymerase chain reaction with degenerate oligodeoxyribonucleotides designed to hybridise to conserved regions of cdc2-related kinases. Computer comparison with other protein kinases strongly suggests that the protein encoded by this gene is closely related to mitogen-activated protein (MAP) kinases, which play important roles in eukaryotic adaptative response and signal transduction. In addition to the conserved MAP kinase catalytic domain, Pfmap-1 contains a highly charged C-terminal extension that includes two sets of repeated amino acid motifs. Pfmap-1 is located on chromosome 14 of P.falciparum, and its mRNA has a size of 3.7 kb.

A Plasmodium falciparum novel gene encoding a coronin-like protein which associates with actin filaments.

Plasmodium falciparum, the major causative agent of human malaria, is an Apicomplexa protozoan parasite which invades in its intermediate host hepatocytes and erythrocytes. The driving force underlying internalization into the host cell is thought to involve both polymerization of parasite actin, as entry is inhibited by the cytochalasins, and an actin motor-associated protein. In the related Apicomplexa parasite, Toxoplasma gondii, the involvement of parasite actin during both processes of motility and host cell entry has been genetically established. In a search for molecules that can regulate actin dynamics within Apicomplexa parasites, we have identified a P. falciparum homologue of the actin associated protein called coronin originally described in the amoeba Dictyostelium discoideum. The single copy gene displays a strong homology with the amoeba sequence and with the bovine and human coronin homologues recently cloned. This homology lies not only within the N-terminus containing the five WD repeats that characterize coronin but also extends in the C-terminal part. Furthermore, using an affinity-purified mouse monoclonal antibody against D. discoideum coronin, we have detected in extracts of P. falciparum young and mature schizonts a 42-kDa polypeptide which binds this antibody and is present in a Triton insoluble fraction that also contains parasite actin filaments. In addition, the recombinant protein encoded by the homologue nucleotidic sequence of P. falciparum coronin is indeed recognized by the antibody against D. discoideum coronin. Finally, the cross-reactive polypeptide displays the ability to cosediment with exogenous F-actin, a property which fits with its involvement in actin dynamics.

The use of nocodazole in cell cycle analysis and parasite purification from Theileria parva-infected B cells.

Theileria parasites transform bovine leukocytes and induce uncontrolled lymphoproliferation only in the macroschizont stage of their life cycle. The isolation of highly purified stage-specific parasite RNA and proteins is an essential prerequisite when studying the Theileria-host relationship. We therefore improved a protocol based on the cytolytic bacterial toxin aerolysin by taking advantage of the microtubule inhibitor nocodazole. In this report we describe that nocodazole-mediated separation of the parasite from the host cell microtubule network was used with success to improve quantity and quality of purified parasites. We furthermore show that nocodazole is a useful tool to study cell cycle checkpoints due to its capacity to induce reversible cell cycle arrest in Theileria-infected B cells.

A Plasmodium falciparum blood stage antigen highly homologous to the glycophorin binding protein GBP.

We have isolated a gene coding for a protein highly homologous to an antigen known as the glycophorin binding protein (GBP) which was therefore called GBPH. The gene consists of 2 exons interrupted by an intron located at a position corresponding to that of the GBP gene. The deduced amino acid sequence of GBPH comprises 427 residues and is characterized by a signal sequence and by an extended repeat region consisting of 8 units of 40 amino acid residues. The comparison of the amino acid sequences of GBPH and GBP reveals an identity of 69%. Antisera raised against a GBPH fragment that carries part of the repetitive region cross-react with GBP (105 kDa) and additionally detect some bands between 40 and 70 kDa, one of which may correspond to GBPH. The genes coding for GBP and GBPH are located on chromosomes 10 and 14, respectively. The GBP gene is transcribed as a highly abundant 6.5 kb mRNA in the blood-stage form, whereas Northern blot analysis using a GBPH specific probe detects 2 less abundant mRNAs of 2.3 kb and 2.7 kb. Southern blot analysis of P. falciparum DNA identifies a third member of the GBP gene family.

A Theileria parva type 1 protein phosphatase activity.

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.

Molecular characterization of the heat shock protein 90 gene of the human malaria parasite Plasmodium falciparum.

We report here the nucleotide sequence of hsp90 (heat shock protein 90) of Plasmodium falciparum. Computer analysis of the deduced protein sequence revealed an unusually large region of charged amino acids when compared to hsp90 from other species. This region shows striking homology to the calcium binding domain of calreticulin, the major calcium binding protein of endoplasmic reticulum. Phylogenetic tree analysis indicates that P. falciparum hsp90 is more closely related to hsp90 from plants than to hsp90 from vertebrates or other parasites. The malaria hsp90 is an ATP binding protein encoded by a single gene constitutively expressed in both asexual (trophozoite) and sexual (gametocyte) stage parasites. The hsp90 protein is homologous to a previously identified 90-kDa antigen strongly recognised by both sera from vaccinated monkeys and monoclonal antibody XIV/7.

Loss of matrix metalloproteinase 9 activity in Theileria annulata-attenuated cells is at the transcriptional level and is associated with differentially expressed AP-1 species.

The schizont stage of the protozoan parasite Theileria annulata reversibly transforms bovine monocytes into an immortalised and metastatic state. We have been studying T. annulata induction of host matrix metalloproteinases (MMP) which are involved in parasite dissemination and pathogenesis. We have observed that prolonged in vitro culture of T. annulata-infected cell lines results in their attenuation and this process is associated with alterations in both host and parasite gene expression. In particular, a loss in bovine MMP expression in later passage cultures suggests that these parasite-induced MMPs are virulence factors. As a means to further our understanding of the attenuation process we examine in detail the parasite-induced differential expression of one particular bovine proteinase, MMP9, in non-attenuated (p58) and attenuated (p158) passage levels of the Ode vaccine line. We show here that MMP9 expression is regulated at the transcriptional level and we suggest that a particular parasite-induced AP-1 recognition transcription factor present in the Ode non-attenuated line may have a role to play in the expression of this host gene.

Gametocytogenesis and ribosomal rRNA gene organisation in the rodent malarias Plasmodium chabaudi and Plasmodium berghei.

A cloned Plasmodium berghei (ANKA) isolate was syringe passaged repeatedly to generate a line that was non-infective to Anopheles stephensi. Ribosomal gene organisation of this non-infective line was then compared to its infective ancestor. DNA was also prepared from asexual parasites and gametocytes of P. chabaudi and the arrangement of the rRNA genes of this species was studied. Although macrogametocytes have many more ribosomes than microgametocytes, this increase does not appear to stem from an amplification of the rRNA genes, as no differences either in the quantity or the arrangement of the rDNA could be detected. Furthermore, the loss of infectivity of the P. berghei gametocytes does not seem to be due to a reduction or rearrangement of sequences coding for the rRNA genes. P. chabaudi and P. berghei DNA failed to show any homology to a repetitive DNA sequence cloned from P. falciparum. We conclude that this probe, PFH8rep20, is specific for P. falciparum.

Characterization and complete nucleotide sequence of a 5.8S ribosomal RNA gene from Plasmodium falciparum.

The 5.8S and 5S rRNA components from the FCR-3/The Gambia strain of Plasmodium falciparum have been identified and the complete nucleotide sequence of a 5.8S ribosomal RNA gene determined. Unlike the 5S rRNA species, the 5.8S is a single homogeneous population of molecules of 157 nucleotides. Comparison of its nucleotide sequence with previously reported 5.8S rRNA sequences indicates that it is homologous to these molecules, but distantly related to them. The sequence of the 5.8S rRNA coding region from the pfrib-2 recombinant of the HG13 Gambian isolate of P. falciparum is identical.

The S-antigen of Plasmodium falciparum Palo Alto represents a new S-antigen serotype.

The S-antigen from the Palo Alto isolate of Plasmodium falciparum has been characterized. The partial sequence for the gene coding for this antigen (clone 281) reveals the presence of tandem repeats of eight amino acids which defines a new S-antigen serotype. Antibodies raised against the 281 recombinant clone reacted with a 140 kDa antigen by immunoblotting with parasite extracts and culture supernatants. The 140 kDa peptide was also identified by immunoprecipitation of metabolic labelled parasites. The 281 mouse antiserum was used to localize the antigen on parasite smears by indirect immunofluorescence assay and more precisely by immunoelectron microscopy. The S-antigen is localized within the parasitophorous vacuole. Furthermore, different isolates were examined for the presence of the Palo Alto S-antigen specificity.

Characterisation of a repetitive DNA sequence from the malaria parasite, Plasmodium falciparum.

A repetitive DNA fragment cloned from the malaria parasite, Plasmodium falciparum, has been analysed. It contains a 21 base pair sequence which occurs in multiple tandem repeats. Two clusters of the same repeat are found in opposite orientations on the same DNA fragment. The repetitive DNA provides an additional way to distinguish between different strains of parasite by hybridisation to genomic blots and may serve as a species-specific probe for diagnosis.

Chromosome size variation in the malaria parasite of rodents, Plasmodium chabaudi.

Pulsed field gradient gel electrophoresis has been used to identify at least 10 large DNA fragments in the genome of the rodent malaria species Plasmodium chabaudi. The fragments range in size from approximately 650 to 5000 kb. All the fragments contain sequences homologous to a P. berghei telomere probe, suggesting that they represent intact chromosomes. Ribosomal RNA genes and P. chabaudi cDNA sequences have been mapped to specific fragments. The fragments vary in size in different cloned isolates of the parasite. In a cross between two cloned parasites differing in the sizes of chromosomes 4 and 5, independent segregation of each chromosome occurred during meiosis.