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      Compositional and expression analyses of the glideosome during the Plasmodium life cycle reveal an additional myosin light chain required for maximum motility

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          Abstract

          Myosin A (MyoA) is a Class XIV myosin implicated in gliding motility and host cell and tissue invasion by malaria parasites. MyoA is part of a membrane-associated protein complex called the glideosome, which is essential for parasite motility and includes the MyoA light chain myosin tail domain–interacting protein (MTIP) and several glideosome-associated proteins (GAPs). However, most studies of MyoA have focused on single stages of the parasite life cycle. We examined MyoA expression throughout the Plasmodium berghei life cycle in both mammalian and insect hosts. In extracellular ookinetes, sporozoites, and merozoites, MyoA was located at the parasite periphery. In the sexual stages, zygote formation and initial ookinete differentiation precede MyoA synthesis and deposition, which occurred only in the developing protuberance. In developing intracellular asexual blood stages, MyoA was synthesized in mature schizonts and was located at the periphery of segmenting merozoites, where it remained throughout maturation, merozoite egress, and host cell invasion. Besides the known GAPs in the malaria parasite, the complex included GAP40, an additional myosin light chain designated essential light chain (ELC), and several other candidate components. This ELC bound the MyoA neck region adjacent to the MTIP-binding site, and both myosin light chains co-located to the glideosome. Co-expression of MyoA with its two light chains revealed that the presence of both light chains enhances MyoA-dependent actin motility. In conclusion, we have established a system to study the interplay and function of the three glideosome components, enabling the assessment of inhibitors that target this motor complex to block host cell invasion.

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          Human malaria parasites in continuous culture.

          Plasmodium falciparum can now be maintained in continuous culture in human erythrocytes incubated at 38 degrees C in RPMI 1640 medium with human serum under an atmosphere with 7 percent carbon dioxide and low oxygen (1 or 5 percent). The original parasite material, derived from an infected Aotus trivirgatus monkey, was diluted more than 100 million times by the addition of human erythrocytes at 3- or 4-day intervals. The parasites continued to reproduce in their normal asexual cycle of approximately 48 hours but were no longer highly synchronous. The have remained infective to Aotus.
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            Functional Profiling of a Plasmodium Genome Reveals an Abundance of Essential Genes

            Summary The 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.
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              High efficiency transfection of Plasmodium berghei facilitates novel selection procedures.

              The use of transfection in the study of the biology of malaria parasites has been limited due to poor transfection efficiencies (frequency of 10(-6) to 10(-9)) and a paucity of selection markers. Here, a new method of transfection, using non-viral Nucleofector technology, is described for the rodent parasite Plasmodium berghei. The transfection efficiency obtained (episomal and targeted integration into the genome) is in the range of 10(-2) to 10(-3). Such high transfection efficiency strongly reduces the time, number of laboratory animals and amount of materials required to generate transfected parasites. Moreover, it allows different experimental strategies for reverse genetics to be developed and we demonstrate direct selection of stably and non-reversibly transformed, fluorescent protein (FP)-expressing parasites using FACS. Since there is no need to use a drug-selectable marker, this method increases the (low) number of selectable markers available for transformation of P. berghei and can in principle be extended to utilise additional FP. Furthermore the FACS-selected, FP-expressing parasites may serve as easily visualized reference lines that may still be genetically manipulated with the existing drug-selectable markers. The combination of enhanced transfection efficiency and a versatile rodent model provides a basis for the further development of novel tools for high throughput genome manipulation.
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                Author and article information

                Journal
                J Biol Chem
                J. Biol. Chem
                jbc
                jbc
                JBC
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology (11200 Rockville Pike, Suite 302, Rockville, MD 20852-3110, U.S.A. )
                0021-9258
                1083-351X
                27 October 2017
                11 September 2017
                11 September 2017
                : 292
                : 43
                : 17857-17875
                Affiliations
                From the []Malaria Parasitology Laboratory,
                [** ]Structural Biology and
                [‡‡ ]Mass Spectrometry Science Technology Platforms, and
                [¶¶ ]Single Molecule Enzymology Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, United Kingdom,
                the [§ ]School of Life Sciences, Queens Medical Centre, University of Nottingham, Nottingham NG7 2UH, United Kingdom,
                the []Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway,
                the []Institute of Cell Biology, University of Bern, Bern, Switzerland, and
                the [§§ ]Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu, Aapistie 7, 90220 Oulu, Finland
                Author notes
                [1 ] To whom correspondence may be addressed: Malaria Parasitology Laboratory, The Francis Crick Institute, 1 Midland Rd., London NW1 1AT, United Kingdom. E-mail: judith.green@ 123456crick.ac.uk .
                [7 ] To whom correspondence may be addressed: Malaria Parasitology Laboratory, The Francis Crick Institute, 1 Midland Rd., London NW1 1AT, United Kingdom. E-mail: tony.holder@ 123456crick.ac.uk .
                [2]

                Present address: Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.

                [3]

                Supported by a research grant 2015/13383-EVR and a postdoctoral fellowship from the Faculty of Medicine and Dentistry, University of Bergen.

                [4]

                Present address: Institute for Medical Research, Jalan Pahang, 50588 Kuala Lumpur, Malaysia.

                [5]

                Present address: Research Institute of Molecular Pathology, Campus-Vienna-Biocenter 1, 1030 Vienna, Austria.

                [6]

                Present address: Dept. of Immunology and Infection, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom.

                Edited by Velia M. Fowler

                Author information
                http://orcid.org/0000-0002-8490-6058
                Article
                M117.802769
                10.1074/jbc.M117.802769
                5663884
                28893907
                b86317a7-b7b8-44d1-887e-21c13643236e
                © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

                Author's Choice—Final version free via Creative Commons CC-BY license.

                History
                : 19 June 2017
                : 4 September 2017
                Funding
                Funded by: Francis Crick Institute , open-funder-registry 10.13039/100010438;
                Award ID: FC001097
                Award ID: FC001119
                Funded by: Wellcome Trust , open-funder-registry 10.13039/100004440;
                Award ID: FC001097
                Award ID: FC001119
                Funded by: Cancer Research UK , open-funder-registry 10.13039/501100000289;
                Award ID: FC001097
                Award ID: FC0001119
                Funded by: Medical Research Council , open-funder-registry 10.13039/501100000265;
                Award ID: FC001097
                Award ID: FC001119
                Award ID: G0900278 MR/K011782/1
                Funded by: School of Life Sciences, University of Nottingham
                Funded by: Universitetet i Bergen , open-funder-registry 10.13039/501100005036;
                Award ID: 2015/13383-EVR
                Categories
                Cell Biology

                Biochemistry
                cell motility,invasion,malaria,myosin,plasmodium,glideosome,myosin light chain
                Biochemistry
                cell motility, invasion, malaria, myosin, plasmodium, glideosome, myosin light chain

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