SummaryThe genomes of malaria parasites contain many genes of unknown function. To assist drug development through the identification of essential genes and pathways, we have measured competitive growth rates in mice of 2,578 barcoded Plasmodium berghei knockout mutants, representing >50% of the genome, and created a phenotype database. At a single stage of its complex life cycle, P. berghei requires two-thirds of genes for optimal growth, the highest proportion reported from any organism and a probable consequence of functional optimization necessitated by genomic reductions during the evolution of parasitism. In contrast, extreme functional redundancy has evolved among expanded gene families operating at the parasite-host interface. The level of genetic redundancy in a single-celled organism may thus reflect the degree of environmental variation it experiences. In the case of Plasmodium parasites, this helps rationalize both the relative successes of drugs and the greater difficulty of making an effective vaccine.
SummaryThe genome-wide identification of gene functions in malaria parasites is hampered by a lack of reverse genetic screening methods. We present a large-scale resource of barcoded vectors with long homology arms for effective modification of the Plasmodium berghei genome. Cotransfecting dozens of vectors into the haploid blood stages creates complex pools of barcoded mutants, whose competitive fitness can be measured during infection of a single mouse using barcode sequencing (barseq). To validate the utility of this resource, we rescreen the P. berghei kinome, using published kinome screens for comparison. We find that several protein kinases function redundantly in asexual blood stages and confirm the targetability of kinases cdpk1, gsk3, tkl3, and PBANKA_082960 by genotyping cloned mutants. Thus, parallel phenotyping of barcoded mutants unlocks the power of reverse genetic screening for a malaria parasite and will enable the systematic identification of genes essential for in vivo parasite growth and transmission.
The Plasmodium Genetic Modification (PlasmoGEM) database (http://plasmogem.sanger.ac.uk) provides access to a resource of modular, versatile and adaptable vectors for genome modification of Plasmodium spp. parasites. PlasmoGEM currently consists of >2000 plasmids designed to modify the genome of Plasmodium berghei, a malaria parasite of rodents, which can be requested by non-profit research organisations free of charge. PlasmoGEM vectors are designed with long homology arms for efficient genome integration and carry gene specific barcodes to identify individual mutants. They can be used for a wide array of applications, including protein localisation, gene interaction studies and high-throughput genetic screens. The vector production pipeline is supported by a custom software suite that automates both the vector design process and quality control by full-length sequencing of the finished vectors. The PlasmoGEM web interface allows users to search a database of finished knock-out and gene tagging vectors, view details of their designs, download vector sequence in different formats and view available quality control data as well as suggested genotyping strategies. We also make gDNA library clones and intermediate vectors available for researchers to produce vectors for themselves.
Human parechoviruses (HPeVs) are frequent pathogens with a seroprevalance of over 90 % in adults. Recent studies on these viruses have increased the number of HPeV types to eight. Here we analyse the complete genome of one clinical isolate, PicoBank/HPeV1/a, and VP1 and 3D protein sequences of PicoBank/HPeV6/a, isolated from the same individual 13 months later. PicoBank/HPeV1/a is closely related to other recent HPeV1 isolates but is distinct from the HPeV1 Harris prototype isolated 50 years ago. The availability of an increasing number of HPeV sequences has allowed a detailed analysis of these viruses. The results add weight to the observations that recombination plays a role in the generation of HPeV diversity. An important finding is the presence of unexpected conservation of codons utilized in part of the 3D-encoding region, some of which can be explained by the presence of a phylogenetically conserved predicted secondary structure domain. This suggests that in addition to the cis-acting replication element, RNA secondary structure domains in coding regions play a key role in picornavirus replication. INTRODUCTIONPicornaviruses are non-enveloped, positive-sense RNA viruses with icosahedral capsids, composed of 60 copies of three or four virus-encoded proteins (VP1-4 or VP0, VP1 and VP3) (Stanway et al., 2002(Stanway et al., , 2005. They have a genome of around 7000-8000 nt encoding one polyprotein which is cleaved by virus proteases to give the structural and non-structural (2A-C and 3A-D) proteins. Picornaviruses consist of economically and socially very important human and animal viruses such as polioviruses, other enteroviruses, rhinoviruses, hepatitis A virus, footand-mouth disease virus and parechoviruses. Currently, parechoviruses consist of two species: human parechovirus (HPeV) and Ljungan virus (LV) (Johansson et al., 2002;Joki-Korpela & Hyypiä, 2001;Stanway & Hyypiä, 1999;Stanway et al., 2000). HPeVs are frequent infectious agents and although they usually cause mild gastroenteritis and respiratory disease in young children, more serious cases, such as flaccid paralysis, encephalitis and myocarditis, have also been reported, particularly associated with HPeV3 infection (Baumgarte et al., 2008;Benschop et al., 2006a Benschop et al., , 2008bEhrnst & Eriksson, 1993;Figueroa et al., 1989;Harvala et al., 2008Harvala et al., , 2009Joki-Korpela & Hyypiä, 2001). Recently, HPeV1 has also been linked to otitis media (Tauriainen et al., 2008). With the isolation of several HPeV types in recent studies AlSunaidi et al., 2007;Baumgarte et al., 2008;Benschop et al., 2006b; Drexler et al., 2009;Ito et al., 2004;Li et al., 2009), eight types (1-8) of HPeVs are currently known.HPeVs have several distinctive features compared with other picornaviruses (Ghazi et al., 1998;Hyypiä et al., 1992;Stanway et al., 1994Stanway et al., , 2000. Post-translational cleavage of the polyprotein results in only three structural proteins (VP0, VP3 and VP1), as the cleavage of VP0 to VP4 and VP2 seen in other picornaviruses does n...
SignificanceMalaria is caused by a parasite that is deposited in the skin through the bite of an infected mosquito. From the skin, parasites navigate through host tissues where they must locate and invade liver cells. We know that a parasite surface protein called TRAP is important for this process, making it a leading vaccine candidate. TRAP is thought to work by specifically binding a defined host cell surface protein, but its identity has remained a long-standing mystery. Our research has identified an integrin—a class of host cell surface proteins—as a TRAP receptor. This finding provides an important piece of the puzzle relating to TRAP function and may help improve the development of an effective malaria vaccine.
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