Highly syntenic and yet divergent: a tale of two Theilerias

The published genomic sequences of the two major host-transforming Theileria species of cattle represent a rich resource of information that has allowed novel bioinformatic and experimental studies into these important apicomplexan parasites. Since their publication in 2005, the genomes of T. annulata and T. parva have been utilised for a diverse range of applications, ranging from candidate antigen discovery to the identification of genetic markers for population analysis. This has led to advancements in the quest for a sub-unit vaccine, while providing a greater understanding of variation among parasite populations in the field. The unique ability of these Theileria species to induce host cell transformation is the subject of considerable scientific interest and the availability of full genomic sequences has provided new insights into this area of research. This article reviews the data underlying published comparative analyses, focussing on the general features of gene expression, the major Tpr/Tar multi-copy gene family and a re-examination of the predicted macroschizont secretome. Codon usage between the Theileria species is reviewed in detail, as this underpins ongoing comparative studies investigating selection at the intra- and inter-species level. The TashAT/TpshAT family of genes, conserved between T. annulata and T. parva, encodes products targeted to the host nucleus and has been implicated in contributing to the transformed bovine phenotype. Species-specific expansion and diversification at this critical locus is discussed with reference to the availability, in the near future, of genomic datasets which are based on non-transforming Theileria species

Extensive differential protein phosphorylation as intraerythrocytic Plasmodium falciparumschizonts develop into extracellular invasive merozoites

Pathology of the most lethal form of malaria is caused by Plasmodium falciparum asexual blood stages and initiated by merozoite invasion of erythrocytes. We present a phosphoproteome analysis of extracellular merozoites revealing 1765 unique phosphorylation sites including 785 sites not previously detected in schizonts. All MS data have been deposited in the ProteomeXchange with identifier PXD001684 (http://proteomecentral.proteomexchange.org/dataset/PXD001684). The observed differential phosphorylation between extra and intraerythrocytic life-cycle stages was confirmed using both phospho-site and phospho-motif specific antibodies and is consistent with the core motif [K/R]xx[pS/pT] being highly represented in merozoite phosphoproteins. Comparative bioinformatic analyses highlighted protein sets and pathways with established roles in invasion. Within the merozoite phosphoprotein interaction network a subnetwork of 119 proteins with potential roles in cellular movement and invasion was identified and suggested that it is coregulated by a further small subnetwork of protein kinase A (PKA), two calcium-dependent protein kinases (CDPKs), a phosphatidyl inositol kinase (PI3K), and a GCN2-like elF2-kinase with a predicted role in translational arrest and associated changes in the ubquitinome. To test this notion experimentally, we examined the overall ubiquitination level in intracellular schizonts versus extracellular merozoites and found it highly upregulated in merozoites. We propose that alterations in the phosphoproteome and ubiquitinome reflect a starvation-induced translational arrest as intracellular schizonts transform into extracellular merozoites

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

Plasmodium falciparum infection induces dynamic changes in the erythrocyte phospho-proteome

The phosphorylation status of red blood cell proteins is strongly altered during the infection by the malaria parasite Plasmodium falciparum. We identify the key phosphorylation events that occur in the erythrocyte membrane and cytoskeleton during infection, by a comparative analysis of global phospho-proteome screens between infected (obtained at schizont stage) and uninfected RBCs. The meta-analysis of reported mass spectrometry studies revealed a novel compendium of 495 phosphorylation sites in 182 human proteins with regulatory roles in red cell morphology and stability, with about 25% of these sites specific to infected cells. A phosphorylation motif analysis detected 7 unique motifs that were largely mapped to kinase consensus sequences of casein kinase II and of protein kinase A/protein kinase C. This analysis highlighted prominent roles for PKA/PKC involving 78 phosphorylation sites. We then compared the phosphorylation status of PKA (PKC) specific sites in adducin, dematin, Band 3 and GLUT-1 in uninfected RBC stimulated or not by cAMP to their phosphorylation status in iRBC. We showed cAMP-induced phosphorylation of adducin S59 by immunoblotting and we were able to demonstrate parasite-induced phosphorylation for adducin S726, Band 3 and GLUT-1, corroborating the protein phosphorylation status in our erythrocyte phosphorylation site compendium

Plasmodium falciparum infection induces dynamic changes in the erythrocyte phospho-proteome.

The phosphorylation status of red blood cell proteins is strongly altered during the infection by the malaria parasite Plasmodium falciparum. We identify the key phosphorylation events that occur in the erythrocyte membrane and cytoskeleton during infection, by a comparative analysis of global phospho-proteome screens between infected (obtained at schizont stage) and uninfected RBCs. The meta-analysis of reported mass spectrometry studies revealed a novel compendium of 495 phosphorylation sites in 182 human proteins with regulatory roles in red cell morphology and stability, with about 25% of these sites specific to infected cells. A phosphorylation motif analysis detected 7 unique motifs that were largely mapped to kinase consensus sequences of casein kinase II and of protein kinase A/protein kinase C. This analysis highlighted prominent roles for PKA/PKC involving 78 phosphorylation sites. We then compared the phosphorylation status of PKA (PKC) specific sites in adducin, dematin, Band 3 and GLUT-1 in uninfected RBC stimulated or not by cAMP to their phosphorylation status in iRBC. We showed cAMP-induced phosphorylation of adducin S59 by immunoblotting and we were able to demonstrate parasite-induced phosphorylation for adducin S726, Band 3 and GLUT-1, corroborating the protein phosphorylation status in our erythrocyte phosphorylation site compendium

TGF-b2 induction regulates invasiveness of theileria-transformed leukocytes and disease susceptibility

Theileria parasites invade and transform bovine leukocytes causing either East Coast fever (T. parva), or tropical theileriosis (T. annulata). Susceptible animals usually die within weeks of infection, but indigenous infected cattle show markedly reduced pathology, suggesting that host genetic factors may cause disease susceptibility. Attenuated live vaccines are widely used to control tropical theileriosis and attenuation is associated with reduced invasiveness of infected macrophages in vitro. Disease pathogenesis is therefore linked to aggressive invasiveness, rather than uncontrolled proliferation of Theileria-infected leukocytes. We show that the invasive potential of Theileria-transformed leukocytes involves TGF-b signalling. Attenuated live vaccine lines express reduced TGF-b2 and their invasiveness can be rescued with exogenous TGF-b. Importantly, infected macrophages from disease susceptible Holstein-Friesian (HF) cows express more TGF-b2 and traverse Matrigel with great efficiency compared to those from disease-resistant Sahiwal cattle. Thus, TGF-b2 levels correlate with disease susceptibility. Using fluorescence and time-lapse video microscopy we show that Theileria-infected, disease-susceptible HF macrophages exhibit increased actin dynamics in their lamellipodia and podosomal adhesion structures and develop more membrane blebs. TGF-b2-associated invasiveness in HF macrophages has a transcription-independent element that relies on cytoskeleton remodelling via activation of Rho kinase (ROCK). We propose that a TGF-b autocrine loop confers an amoeboid-like motility on Theileria-infected leukocytes, which combines with MMP-dependent motility to drive invasiveness and virulence

Virulence attenuation of Theileria annulata-transformed macrophages

Tropical theileriosis is a significant tick-borne disease affecting cattle. For decades an empirical live attenuated vaccine has been the primary method of controlling disease. The vaccine is produced through prolonged culture of Theileria annulata schizont-transformed macrophages, but how loss of virulence occurs remains unclear. Notably attenuated (vaccine) macrophages display dampened dissemination potential compared with their original, virulent counterparts. In addition, parasite schizonts in attenuated macrophages have significantly lost their ability to differentiate into merozoites. This review discusses the changes that occur during long-term passage of T. annulata-transformed bovine macrophages and how they contribute to loss of virulence, defined as heightened dissemination. Finally, we also suggest that a common parasite-dependent pathway is potentially involved in both macrophage dissemination and parasite merogony

cAMP-Signalling Regulates Gametocyte-Infected Erythrocyte Deformability Required for Malaria Parasite Transmission.

Blocking Plasmodium falciparum transmission to mosquitoes has been designated a strategic objective in the global agenda of malaria elimination. Transmission is ensured by gametocyte-infected erythrocytes (GIE) that sequester in the bone marrow and at maturation are released into peripheral blood from where they are taken up during a mosquito blood meal. Release into the blood circulation is accompanied by an increase in GIE deformability that allows them to pass through the spleen. Here, we used a microsphere matrix to mimic splenic filtration and investigated the role of cAMP-signalling in regulating GIE deformability. We demonstrated that mature GIE deformability is dependent on reduced cAMP-signalling and on increased phosphodiesterase expression in stage V gametocytes, and that parasite cAMP-dependent kinase activity contributes to the stiffness of immature gametocytes. Importantly, pharmacological agents that raise cAMP levels in transmissible stage V gametocytes render them less deformable and hence less likely to circulate through the spleen. Therefore, phosphodiesterase inhibitors that raise cAMP levels in P. falciparum infected erythrocytes, such as sildenafil, represent new candidate drugs to block transmission of malaria parasites