Characterisation of the eukaryotic initiation factor 2alpha kinases of Plasmodium falciparum

Malaria remains a devastating disease with respect to both mortality and the constraints it places on the economic development of the countries in which it is endemic. Our laboratory is seeking new antimalarial targets, by characterising the protein kinases of the most lethal human malaria parasite, Plasmodium falciparum. As central components of many diverse signalling pathways, protein kinases are crucial for the control of proliferation and differentiation in other eukaryotes; we hypothesise that they play similar roles in P. falciparum. The life cycle of P. falciparum is complex, consisting of a series of tightly controlled stages of division and differentiation. In the related apicomplexan parasite Toxoplasma gondii, stress stimuli have been implicated in an important differentiation step, from rapidly dividing tachyzoites, to quiescent bradyzoites (which enable immune evasion). Evidence suggests that stress may also contribute to an essential differentiation stage, gametocytogenesis, in P. falciparum. In yeast and metazoans, part of the stress response is mediated through phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha), which results in selective translation of mRNAs encoding stress response proteins. Post-transcriptional control of gene expression is suspected to play an important role in P. falciparum. Importantly, the Goldberg laboratory recently demonstrated that similarly, in P. falciparum the eIF2alpha orthologue is phosphorylated in response to starvation. Here we identify the P. falciparum orthologue of the translation initiation factor eIF2alpha and provide bioinformatic evidence for the presence of three eIF2alpha kinases in P. falciparum; PfeIK1, PfeIK2 and PfPK4, only one of which (PfPK4) has been described previously (Mohrle et al., 1997). We show that one of the novel eIF2alpha kinases, PfeIK1, is able to phosphorylate P. falciparum eIF2alpha in vitro. In addition, initial experiments support previous observations that PfPK4 is indeed an active protein kinase (Mohrle et al., 1997). We present evidence that PfPK4 is essential for asexual growth, which precludes straightforward reverse genetics studies aiming to determine its possible role in gametocytogenesis. In contrast, transgenic parasites allowed us to show that neither PfeIK1 nor PfeIK2 are required for asexual growth, or sexual development of the parasite in the mosquito vector. However, preliminary evidence (requiring confirmation) may indicate that parasites lacking PfeIK1 over-express PfPK4, which would suggest that PfeIK1 may play an important function in the parasite. This study strongly suggests that a mechanism for versatile regulation of translation by several kinases with a similar catalytic domain, but distinct regulatory domains, is conserved in P. falciparum

PfeIK1, a eukaryotic initiation factor 2α kinase of the human malaria parasite Plasmodium falciparum, regulates stress-response to amino-acid starvation

<p>Abstract</p> <p>Background</p> <p>Post-transcriptional control of gene expression is suspected to play an important role in malaria parasites. In yeast and metazoans, part of the stress response is mediated through phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), which results in the selective translation of mRNAs encoding stress-response proteins.</p> <p>Methods</p> <p>The impact of starvation on the phosphorylation state of PfeIF2α was examined. Bioinformatic methods were used to identify plasmodial eIF2α kinases. The activity of one of these, PfeIK1, was investigated using recombinant protein with non-physiological substrates and recombinant PfeIF2α. Reverse genetic techniques were used to disrupt the <it>pfeik1 </it>gene.</p> <p>Results</p> <p>The data demonstrate that the <it>Plasmodium falciparum </it>eIF2α orthologue is phosphorylated in response to starvation, and provide bioinformatic evidence for the presence of three eIF2α kinases in <it>P. falciparum</it>, only one of which (PfPK4) had been described previously. Evidence is provided that one of the novel eIF2α kinases, PfeIK1, is able to phosphorylate the <it>P. falciparum </it>eIF2α orthologue <it>in vitro</it>. PfeIK1 is not required for asexual or sexual development of the parasite, as shown by the ability of <it>pfeik1</it>^-parasites to develop into sporozoites. However, eIF2α phosphorylation in response to starvation is abolished in <it>pfeik1</it>^-asexual parasites</p> <p>Conclusion</p> <p>This study strongly suggests that a mechanism for versatile regulation of translation by several kinases with a similar catalytic domain but distinct regulatory domains, is conserved in <it>P. falciparum</it>.</p

The <i>Plasmodium</i> eukaryotic initiation factor-2α kinase IK2 controls the latency of sporozoites in the mosquito salivary glands

Sporozoites, the invasive form of malaria parasites transmitted by mosquitoes, are quiescent while in the insect salivary glands. Sporozoites only differentiate inside of the hepatocytes of the mammalian host. We show that sporozoite latency is an active process controlled by a eukaryotic initiation factor-2α (eIF2α) kinase (IK2) and a phosphatase. IK2 activity is dominant in salivary gland sporozoites, leading to an inhibition of translation and accumulation of stalled mRNAs into granules. When sporozoites are injected into the mammalian host, an eIF2α phosphatase removes the PO4 from eIF2α-P, and the repression of translation is alleviated to permit their transformation into liver stages. In IK2 knockout sporozoites, eIF2α is not phosphorylated and the parasites transform prematurely into liver stages and lose their infectivity. Thus, to complete their life cycle, Plasmodium sporozoites exploit the mechanism that regulates stress responses in eukaryotic cells

Induction of Strain-Transcending Antibodies Against Group A PfEMP1 Surface Antigens from Virulent Malaria Parasites

Sequence diversity in pathogen antigens is an obstacle to the development of interventions against many infectious diseases. In malaria caused by Plasmodium falciparum, the PfEMP1 family of variant surface antigens encoded by var genes are adhesion molecules that play a pivotal role in malaria pathogenesis and clinical disease. PfEMP1 is a major target of protective immunity, however, development of drugs or vaccines based on PfEMP1 is problematic due to extensive sequence diversity within the PfEMP1 family. Here we identified the PfEMP1 variants transcribed by P. falciparum strains selected for a virulence-associated adhesion phenotype (IgM-positive rosetting). The parasites transcribed a subset of Group A PfEMP1 variants characterised by an unusual PfEMP1 architecture and a distinct N-terminal domain (either DBLα1.5 or DBLα1.8 type). Antibodies raised in rabbits against the N-terminal domains showed functional activity (surface reactivity with live infected erythrocytes (IEs), rosette inhibition and induction of phagocytosis of IEs) down to low concentrations (<10 µg/ml of total IgG) against homologous parasites. Furthermore, the antibodies showed broad cross-reactivity against heterologous parasite strains with the same rosetting phenotype, including clinical isolates from four sub-Saharan African countries that showed surface reactivity with either DBLα1.5 antibodies (variant HB3var6) or DBLα1.8 antibodies (variant TM284var1). These data show that parasites with a virulence-associated adhesion phenotype share IE surface epitopes that can be targeted by strain-transcending antibodies to PfEMP1. The existence of shared surface epitopes amongst functionally similar disease-associated P. falciparum parasite isolates suggests that development of therapeutic interventions to prevent severe malaria is a realistic goal

Examination of the Accuracy and Applicability of Information in Popular Books on Dog Training

There is a wealth of popular literature available on dog behavior and training; sourcing reliable and trustworthy advice is important to achieving successful training. The aim of this study was to select five best-selling (at that time) dog training books, and review their general content and references to basic learning theory and human communicative cues. An Internet search was performed on three online bookstores’ websites for “best selling” “dog training” books. The books were by Millan and Peltier (2006), Fennell (2002), Stilwell (2005), Pryor (1999), and Monks of New Skete (2002). The results showed marked differences across all books, including inconsistencies in the depth of information provided, and some starkly contrasting training methods were advocated. Overall, these books were not all considered to function as instructional manuals. The persistent popularity of these books suggests that they have likely contributed appreciably to the type of information accessed by dog guardians

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

The evolution of lung cancer and impact of subclonal selection in TRACERx

Lung cancer is the leading cause of cancer-associated mortality worldwide. Here we analysed 1,644 tumour regions sampled at surgery or during follow-up from the first 421 patients with non-small cell lung cancer prospectively enrolled into the TRACERx study. This project aims to decipher lung cancer evolution and address the primary study endpoint: determining the relationship between intratumour heterogeneity and clinical outcome. In lung adenocarcinoma, mutations in 22 out of 40 common cancer genes were under significant subclonal selection, including classical tumour initiators such as TP53 and KRAS. We defined evolutionary dependencies between drivers, mutational processes and whole genome doubling (WGD) events. Despite patients having a history of smoking, 8% of lung adenocarcinomas lacked evidence of tobacco-induced mutagenesis. These tumours also had similar detection rates for EGFR mutations and for RET, ROS1, ALK and MET oncogenic isoforms compared with tumours in never-smokers, which suggests that they have a similar aetiology and pathogenesis. Large subclonal expansions were associated with positive subclonal selection. Patients with tumours harbouring recent subclonal expansions, on the terminus of a phylogenetic branch, had significantly shorter disease-free survival. Subclonal WGD was detected in 19% of tumours, and 10% of tumours harboured multiple subclonal WGDs in parallel. Subclonal, but not truncal, WGD was associated with shorter disease-free survival. Copy number heterogeneity was associated with extrathoracic relapse within 1 year after surgery. These data demonstrate the importance of clonal expansion, WGD and copy number instability in determining the timing and patterns of relapse in non-small cell lung cancer and provide a comprehensive clinical cancer evolutionary data resource

The evolution of non-small cell lung cancer metastases in TRACERx

Metastatic disease is responsible for the majority of cancer-related deaths. We report the longitudinal evolutionary analysis of 126 non-small cell lung cancer (NSCLC) tumours from 421 prospectively recruited patients in TRACERx who developed metastatic disease, compared with a control cohort of 144 non-metastatic tumours. In 25% of cases, metastases diverged early, before the last clonal sweep in the primary tumour, and early divergence was enriched for patients who were smokers at the time of initial diagnosis. Simulations suggested that early metastatic divergence more frequently occurred at smaller tumour diameters (less than 8 mm). Single-region primary tumour sampling resulted in 83% of late divergence cases being misclassified as early, highlighting the importance of extensive primary tumour sampling. Polyclonal dissemination, which was associated with extrathoracic disease recurrence, was found in 32% of cases. Primary lymph node disease contributed to metastatic relapse in less than 20% of cases, representing a hallmark of metastatic potential rather than a route to subsequent recurrences/disease progression. Metastasis-seeding subclones exhibited subclonal expansions within primary tumours, probably reflecting positive selection. Our findings highlight the importance of selection in metastatic clone evolution within untreated primary tumours, the distinction between monoclonal versus polyclonal seeding in dictating site of recurrence, the limitations of current radiological screening approaches for early diverging tumours and the need to develop strategies to target metastasis-seeding subclones before relapse