Malaria Parasite-Infected Erythrocytes Secrete PfCK1, the Plasmodium Homologue of the Pleiotropic Protein Kinase Casein Kinase 1

Casein kinase 1 (CK1) is a pleiotropic protein kinase implicated in several fundamental processes of eukaryotic cell biology. Plasmodium falciparum encodes a single CK1 isoform, PfCK1, that is expressed at all stages of the parasite’s life cycle. We have previously shown that the pfck1 gene cannot be disrupted, but that the locus can be modified if no loss-of-function is incurred, suggesting an important role for this kinase in intra-erythrocytic asexual proliferation. Here, we report on the use of parasite lines expressing GFP- or His-tagged PfCK1 from the endogenous locus to investigate (i) the dynamics of PfCK1 localisation during the asexual cycle in red blood cells, and (ii) potential interactors of PfCK1, so as to gain insight into the involvement of the enzyme in specific cellular processes. Immunofluorescence analysis reveals a dynamic localisation of PfCK1, with evidence for a pool of the enzyme being directed to the membrane of the host erythrocyte in the early stages of infection, followed by a predominantly intra-parasite localisation in trophozoites and schizonts and association with micronemes in merozoites. Furthermore, we present strong evidence that a pool of enzymatically active PfCK1 is secreted into the culture supernatant, demonstrating that PfCK1 is an ectokinase. Our interactome experiments and ensuing kinase assays using recombinant PfCK1 to phosphorylate putative interactors in vitro suggest an involvement of PfCK1 in many cellular processes such as mRNA splicing, protein trafficking, ribosomal, and host cell invasion.     

Validation of the theoretical domains framework for use in behaviour change and implementation research

Background

An integrative theoretical framework, developed for cross-disciplinary implementation and other behaviour change research, has been applied across a wide range of clinical situations. This study tests the validity of this framework.

Methods

Validity was investigated by behavioural experts sorting 112 unique theoretical constructs using closed and open sort tasks. The extent of replication was tested by Discriminant Content Validation and Fuzzy Cluster Analysis.

Results

There was good support for a refinement of the framework comprising 14 domains of theoretical constructs (average silhouette value 0.29): ??Knowledge??, ??Skills??, ??Social/Professional Role and Identity??, ??Beliefs about Capabilities??, ??Optimism??, ??Beliefs about Consequences??, ??Reinforcement??, ??Intentions??, ??Goals??, ??Memory, Attention and Decision Processes??, ??Environmental Context and Resources??, ??Social Influences??, ??Emotions??, and ??Behavioural Regulation??.

Conclusions

The refined Theoretical Domains Framework has a strengthened empirical base and provides a method for theoretically assessing implementation problems, as well as professional and other health-related behaviours as a basis for intervention development.     

“Shaping” of cell signaling via AKAP-tethered PDE4D: Probing with AKAR2-AKAP5 biosensor

Background

PKA, a key regulator of cell signaling, phosphorylates a diverse and important array of target molecules and is spatially docked to members of the A-kinase Anchoring Protein (AKAP) family. AKAR2 is a biosensor which yields a FRET signal in vivo, when phosphorylated by PKA. AKAP5, a prominent member of the AKAP family, docks several signaling molecules including PKA, PDE4D, as well as GPCRs, and is obligate for the propagation of the activation of the mitogen-activated protein kinase cascade from GPCRs to ERK1,2.

Results

Using an AKAR2-AKAP5 fusion ??biosensor??, we investigated the spatial-temporal activation of AKAP5 undergoing phosphorylation by PKA in response to ??-adrenergic stimulation. The pattern of PKA activation reported by AKAR2-AKAP5 is a more rapid and spatially distinct from those ??sensed?? by AKAR2-AKAP12. Spatial-temporal restriction of activated PKA by AKAP5 was found to ??shape?? the signaling response. Phosphatase PDE4D tethered to AKAP5 also later reverses within 60?s elevated intracellular cyclic AMP levels stimulated by ??-adrenergic agonist. AKAP12, however, fails to attenuate the rise in cyclic AMP over this time. Fusion of the AKAP5 PDE4D-binding-domain to AKAP12 was found to accelerate a reversal of accumulation of intracellular cyclic AMP.

Conclusion

AKAPs, which are scaffolds with tethered enzymes, can ??shape?? the temporal and spatial aspects of cell signaling.     

Inhibition of γ-secretase worsens memory deficits in a genetically congruous mouse model of Danish dementia

Background

A mutation in the BRI2/ITM2b gene causes familial Danish dementia (FDD). BRI2 is an inhibitor of amyloid-?? precursor protein (APP) processing, which is genetically linked to Alzheimer??s disease (AD) pathogenesis. The FDD mutation leads to a loss of BRI2 protein and to increased APP processing. APP haplodeficiency and inhibition of APP cleavage by ??-secretase rescue synaptic/memory deficits of a genetically congruous mouse model of FDD (FDD_KI). ??-cleavage of APP yields the ??-carboxyl-terminal (??-CTF) and the amino-terminal-soluble APP?? (sAPP??) fragments. ??-secretase processing of ??-CTF generates A??, which is considered the main cause of AD. However, inhibiting A?? production did not rescue the deficits of FDD_KI mice, suggesting that sAPP??/??-CTF, and not A??, are the toxic species causing memory loss.

Results

Here, we have further analyzed the effect of ??-secretase inhibition. We show that treatment with a ??-secretase inhibitor (GSI) results in a worsening of the memory deficits of FDD_KI mice. This deleterious effect on memory correlates with increased levels of the ??/??-CTFs APP fragments in synaptic fractions isolated from hippocampi of FDD_KI mice, which is consistent with inhibition of ??-secretase activity.

Conclusion

This harmful effect of the GSI is in sharp contrast with a pathogenic role for A??, and suggests that the worsening of memory deficits may be due to accumulation of synaptic-toxic ??/??-CTFs caused by GSI treatment. However, ??-secretase cleaves more than 40 proteins; thus, the noxious effect of GSI on memory may be dependent on inhibition of cleavage of one or more of these other ??-secretase substrates. These two possibilities do not need to be mutually exclusive. Our results are consistent with the outcome of a clinical trial with the GSI Semagacestat, which caused a worsening of cognition, and advise against targeting ??-secretase in the therapy of AD. Overall, the data also indicate that FDD_KI is a valuable mouse model to study AD pathogenesis and predict the clinical outcome of therapeutic agents for AD.     

Sympatric Plasmodium falciparum isolates from Venezuela have structured var gene repertoires

Background

Plasmodium falciparum antigenic diversity and polymorphism confuses the issue of antimalarial vaccine development. Merozoite surface protein (MSP)-1 and -2 are two highly polymorphic vaccine candidates. Characterisation of their precise polymorphism in endemic regions may facilitate the design of an effective vaccine.

Methods

Isolates obtained in 52 Gabonese children presenting with uncomplicated malaria were genotyped by nested-PCR of msp -1 block 2, and msp -2 block 3, to analyze both parasite population polymorphism and clone fluctuations.

Results

Twenty-five and 19 different alleles were respectively obtained for msp-1 and msp-2 loci, the RO33 family of msp-1 being poorly polymorphic. Four cases of non-random distribution of alleles were reported of the FC27, and/or 3D7 families of msp-2. All but two isolates were composed of more than one genotype, and the multiplicity of infection (MOI) was 4.0. Neither parasite density nor age was related to MOI. Clone fluctuations were studied for ten subjects who were sampled again at reappearance of parasites in blood. Disappearance and reappearance of alleles were observed following treatment, suggesting difficulties in assessing polymorphism and in distinguishing reinfection from recrudescence.

Conclusion

P. falciparum polymorphism is extensive in Southeast Gabon, and most of infections are composed of multiple clones. The fluctuation of clones contributes to parasite diversity.     

Reduced glycerol incorporation into phospholipids contributes to impaired intra-erythrocytic growth of glycerol kinase knockout Plasmodium falciparum parasites

Background

Malaria is a devastating disease and Plasmodium falciparum is the most lethal parasite infecting humans. Understanding the biology of this parasite is vital in identifying potential novel drug targets. During every 48-hour intra-erythrocytic asexual replication cycle, a single parasite can produce up to 32 progeny. This extensive proliferation implies that parasites require substantial amounts of lipid precursors for membrane biogenesis. Glycerol kinase is a highly conserved enzyme that functions at the interface of lipid synthesis and carbohydrate metabolism. P. falciparum glycerol kinase catalyzes the ATP-dependent phosphorylation of glycerol to glycerol-3-phosphate, a major phospholipid precursor.

Methods

The P. falciparum glycerol kinase gene was disrupted using double crossover homologous DNA recombination to generate a knockout parasite line. Southern hybridization and mRNA analysis were used to verify gene disruption. Parasite growth rates were monitored by flow cytometry. Radiolabelling studies were used to assess incorporation of glycerol into parasite phospholipids.

Results

Disruption of the P. falciparum glycerol kinase gene produced viable parasites, but their growth was significantly reduced to 56.5 ± 1.8% when compared to wild type parasites. ¹⁴C-glycerol incorporation into the major phospholipids of the parasite membrane, phosphatidylcholine and phosphatidylethanolamine, was 48.4 ± 10.8% and 53.1 ± 5.7% relative to an equivalent number of wild type parasites.

Conclusions

P. falciparum glycerol kinase is required for optimal intra-erythrocytic asexual parasite development. Exogenous glycerol may be used as an alternative carbon source for P. falciparum phospholipid biogenesis, despite the lack of glycerol kinase to generate glycerol-3-phosphate.

General significance

These studies provide new insight into glycerolipid metabolism in P. falciparum.     

Regulation of Toll-like receptor 4-mediated immune responses through Pasteurella multocida toxin-induced G protein signalling

Background

Lipopolysaccharide (LPS)-triggered Toll-like receptor (TLR) 4-signalling belongs to the key innate defence mechanisms upon infection with Gram-negative bacteria and triggers the subsequent activation of adaptive immunity. There is an active crosstalk between TLR4-mediated and other signalling cascades to secure an effective immune response, but also to prevent excessive inflammation. Many pathogens induce signalling cascades via secreted factors that interfere with TLR signalling to modify and presumably escape the host response. In this context heterotrimeric G proteins and their coupled receptors have been recognized as major cellular targets. Toxigenic strains of Gram-negative Pasteurella multocida produce a toxin (PMT) that constitutively activates the heterotrimeric G proteins G??_q, G??₁₃ and G??_i independently of G protein-coupled receptors through deamidation. PMT is known to induce signalling events involved in cell proliferation, cell survival and cytoskeleton rearrangement.

Results

Here we show that the activation of heterotrimeric G proteins through PMT suppresses LPS-stimulated IL-12p40 production and eventually impairs the T cell-activating ability of LPS-treated monocytes. This inhibition of TLR4-induced IL-12p40 expression is mediated by G??_i-triggered signalling as well as by G?|?-dependent activation of PI3kinase and JNK. Taken together we propose the following model: LPS stimulates TLR4-mediated activation of the NF?B-pathway and thereby the production of TNF-??, IL-6 and IL-12p40. PMT inhibits the production of IL-12p40 by G??_i-mediated inhibition of adenylate cyclase and cAMP accumulation and by G?|?-mediated activation of PI3kinase and JNK activation.

Conclusions

On the basis of the experiments with PMT this study gives an example of a pathogen-induced interaction between G protein-mediated and TLR4-triggered signalling and illustrates how a bacterial toxin is able to interfere with the host??s immune response.     

Molecular characterization and localization of Plasmodium falciparum choline kinase

Generation of phosphocholine by choline kinase is important for phosphatidylcholine biosynthesis via Kennedy pathway and phosphatidylcholine biosynthesis is essential for intraerythrocytic growth of malaria parasite. A putative gene (Gene ID PF14_0020) in chromosome 14, having highest sequence homology with choline kinase, has been identified by BLAST searches from P. falciparum genome sequence database. This gene has been PCR amplified, cloned, over-expressed and characterized. Choline kinase activity of the recombinant protein (PfCK) was validated as it catalyzed the formation of phosphocholine from choline in presence of ATP. The K_m values for choline and ATP are found to be 145 ± 20 μM and 2.5 ± 0.3 mM, respectively. PfCK can phosphorylate choline efficiently but not ethanolamine. Southern blotting indicates that PfCK is a single copy gene and it is a cytosolic protein as evidenced by Western immunoblotting and confocal microscopy. A model structure of PfCK was constructed based on the crystal structure of choline kinase of C. elegans to search the structural homology. Consistent with the homology modeling predictions, CD analysis indicates that the α and β content of PfCK are 33% and 14%, respectively. Since choline kinase plays a vital role for growth and multiplication of P. falciparum during intraerythrocytic stages, we can suggest that this well characterized PfCK may be exploited in the screening of new choline kinase inhibitors to evaluate their antimalarial activity.     

Construction of a Plasmodium falciparum Rab-interactome identifies CK1 and PKA as Rab-effector kinases in malaria parasites

Background information

The pathology causing stages of the human malaria parasite Plasmodium falciparum reside within red blood cells that are devoid of any regulated transport system. The parasite, therefore, is entirely responsible for mediating vesicular transport within itself and in the infected erythrocyte cytoplasm, and it does so in part via its family of 11 Rab GTPases. Putative functions have been ascribed to Plasmodium Rabs due to their homology with Rabs of yeast, particularly with Saccharomyces that has an equivalent number of rab/ypt genes and where analyses of Ypt function is well characterized.

Results

Rabs are important regulators of vesicular traffic due to their capacity to recruit specific effectors. In order to identify P. falciparum Rab (PfRab) effectors, we first built a Ypt‐interactome by exploiting genetic and physical binding data available at the Saccharomyces genome database (SGD). We then constructed a PfRab‐interactome using putative parasite Rab‐effectors identified by homology to Ypt‐effectors. We demonstrate its potential by wet‐bench testing three predictions; that casein kinase‐1 (PfCK1) is a specific Rab5B interacting protein and that the catalytic subunit of cAMP‐dependent protein kinase A (PfPKA‐C) is a PfRab5A and PfRab7 effector.

Conclusions

The establishment of a shared set of physical Ypt/PfRab‐effector proteins sheds light on a core set Plasmodium Rab‐interactants shared with yeast. The PfRab‐interactome should benefit vesicular trafficking studies in malaria parasites. The recruitment of PfCK1 to PfRab5B+ and PfPKA‐C to PfRab5A+ and PfRab7+ vesicles, respectively, suggests that PfRab‐recruited kinases potentially play a role in early and late endosome function in malaria parasites.     

Identification of a highly conserved valine-glycine-phenylalanine amino acid triplet required for HIV-1 Nef function

Background

The Nef protein of HIV facilitates virus replication and disease progression in infected patients. This role as pathogenesis factor depends on several genetically separable Nef functions that are mediated by interactions of highly conserved protein-protein interaction motifs with different host cell proteins. By studying the functionality of a series of nef alleles from clinical isolates, we identified a dysfunctional HIV group O Nef in which a highly conserved valine-glycine-phenylalanine (VGF) region, which links a preceding acidic cluster with the following proline-rich motif into an amphipathic surface was deleted. In this study, we aimed to study the functional importance of this VGF region.

Results

The dysfunctional HIV group O8 nef allele was restored to the consensus sequence, and mutants of canonical (NL4.3, NA-7, SF2) and non-canonical (B2 and C1422) HIV-1 group M nef alleles were generated in which the amino acids of the VGF region were changed into alanines (VGF??AAA) and tested for their capacity to interfere with surface receptor trafficking, signal transduction and enhancement of viral replication and infectivity. We found the VGF motif, and each individual amino acid of this motif, to be critical for downregulation of MHC-I and CXCR4. Moreover, Nef??s association with the cellular p21-activated kinase 2 (PAK2), the resulting deregulation of cofilin and inhibition of host cell actin remodeling, and targeting of Lck kinase to the trans-golgi-network (TGN) were affected as well. Of particular interest, VGF integrity was essential for Nef-mediated enhancement of HIV virion infectivity and HIV replication in peripheral blood lymphocytes. For targeting of Lck kinase to the TGN and viral infectivity, especially the phenylalanine of the triplet was essential. At the molecular level, the VGF motif was required for the physical interaction of the adjacent proline-rich motif with Hck.

Conclusion

Based on these findings, we propose that this highly conserved three amino acid VGF motif together with the acidic cluster and the proline-rich motif form a previously unrecognized amphipathic surface on Nef. This surface appears to be essential for the majority of Nef functions and thus represents a prime target for the pharmacological inhibition of Nef.     

Spatial and temporal variation in malaria transmission in a low endemicity area in northern Tanzania

Background

Current detection or screening for malaria infection necessitates drawing blood by fingerprick or venipuncture, which poses risks and limitations for repeated measurement. This study presents PCR detection of Plasmodium falciparum in human urine and saliva samples, and illustrates this potential application in genotyping malaria infections.

Methods

Urine and saliva were obtained from 47 thick film positive and 4 negative individuals one day after collection of blood slides and filter paper blood spots. P. falciparum DNA was extracted from blood, urine and saliva, in separate groups, using the Chelex method or Qiagen DNEasy^® kit (urine and saliva only). Blood, urine and saliva extracts were subjected to PCR in separate batches. Amplicons from the various sample types were examined for MSP2 polymorphisms and restriction fragment patterns on DHFR amino acid codon 59.

Results and discussion

Malaria infections exhibited primarily low-grade parasite densities, with a geometric mean of 775 asexual parasites/μl. Regularly matching polymorphic MSP2 genotypes were found between the corresponding urine, saliva and peripheral blood amplicons of each individual, with different inter-individual polymorphic genotypes. Amplicon yields were significantly dependent on DNA extraction method, parasite density and primer set (p < 0.001). A Qiagen^® kit extraction had more than 2× higher amplicon yield than the Chelex method, for both urine and saliva. Amplicon yields were 1.6 fold higher from saliva than urine. For each unit increase in log parasite density, the probability of amplicon enhanced 1.8 fold. Highest amplicon yields were obtained from the primer set with the shortest PCR product.

Conclusion

P. falciparum infection is detectable by PCR on human urine and saliva samples. Subject to further refinement of extraction technique and amplicon yields, large-scale malaria parasite screening and epidemiological surveys could be possible without the need to collect blood and use of needles or sharps.     

Enzymatic antioxidant system in the cestode Hymenolepis diminuta after chronic infection of the rat

Background

The aim of the present paper was to describe the enzymatic antioxidant system in Hymenolepis diminuta collected from rats exposed to chronic cestode invasion.

Methodology

We dissected different tissues of H. diminuta (immature proglottids, genital primordia, hermaphroditic proglottids, early uterus, and gravid uterus) and studied activity of: superoxide dismutases (SOD1 and SOD2), catalase (CAT), glutathione peroxidases (non-Se-dependent GSHPx and Se-dependent GSHPx), glutathione-S-transferase (GST) and glutathione reductase (GSHR), and oxidative stress markers ?? reduced glutathione (GSH), and the lipid peroxidation level (TBARS).

Results

We demonstrated changes in antioxidant enzyme activities and levels of oxidative stress markers in different tissues of the parasite. The levels of TBARS and GSH indicate that oxidative stress occurred in tissues located proximal to the intestine wall. Activity of SOD1 was high in all parts of H. diminuta, but the GST activity was the highest of all studied antioxidant enzymes. SOD2 activity differed significantly in various parts of H. diminuta. Significant differences were observed for nonSeGSHPx and activity of other GSH-dependent enzymes was generally similar in all the tissues.

Conclusions

Our results show that the enzymatic antioxidant system of H. diminuta, allows the parasite to adapt and live under conditions of chronic oxidative stress. It suggests an oxidative-antioxidative balance during interactions between parasite and host.