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      A Putative Small Solute Transporter Is Responsible for the Secretion of G377 and TRAP-Containing Secretory Vesicles during Plasmodium Gamete Egress and Sporozoite Motility

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          Abstract

          Regulated protein secretion is required for malaria parasite life cycle progression and transmission between the mammalian host and mosquito vector. During transmission from the host to the vector, exocytosis of highly specialised secretory vesicles, such as osmiophilic bodies, is key to the dissolution of the red blood cell and parasitophorous vacuole membranes enabling gamete egress. The positioning of adhesins from the TRAP family, from micronemes to the sporozoite surface, is essential for gliding motility of the parasite and transmission from mosquito to mammalian host. Here we identify a conserved role for the putative pantothenate transporter PAT in Plasmodium berghei in vesicle fusion of two distinct classes of vesicles in gametocytes and sporozoites. PAT is a membrane component of osmiophilic bodies in gametocytes and micronemes in sporozoites. Despite normal formation and trafficking of osmiophilic bodies to the cell surface upon activation, PAT-deficient gametes fail to discharge their contents, remain intraerythrocytic and unavailable for fertilisation and further development in the mosquito. Sporozoites lacking PAT fail to secrete TRAP, are immotile and thus unable to infect the subsequent rodent host. Thus, P. berghei PAT appears to regulate exocytosis in two distinct populations of vesicles in two different life cycle forms rather than acting as pantothenic transporter during parasite transmission.

          Author Summary

          Transmission of the malaria parasite between mosquito and host requires two different life cycle stages—the gametocyte and the sporozoite. In both parasite forms, transmission is dependent on exocytosis of stage-specific vesicles. In gametocytes these vesicles release proteins allowing egress from red blood cells and fertilization, and are hence needed to establish an infection in the mosquito. In contrast, proteins are secreted into the membrane of the sporozoite, where they play distinct roles during adhesion and motility, both crucial for transmission back into the mammalian host. Here we show that parasites lacking the putative small solute transporter PAT are still able to form vesicles in both parasite forms but are unable to fuse and secrete their contents. This results in impaired parasite transmission into and from the mosquito. Our work shows that a single protein can regulate the function of functionally distinct classes of vesicles in different life cycle forms of a parasite.

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          Most cited references54

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          Major facilitator superfamily.

          The major facilitator superfamily (MFS) is one of the two largest families of membrane transporters found on Earth. It is present ubiquitously in bacteria, archaea, and eukarya and includes members that can function by solute uniport, solute/cation symport, solute/cation antiport and/or solute/solute antiport with inwardly and/or outwardly directed polarity. All homologous MFS protein sequences in the public databases as of January 1997 were identified on the basis of sequence similarity and shown to be homologous. Phylogenetic analyses revealed the occurrence of 17 distinct families within the MFS, each of which generally transports a single class of compounds. Compounds transported by MFS permeases include simple sugars, oligosaccharides, inositols, drugs, amino acids, nucleosides, organophosphate esters, Krebs cycle metabolites, and a large variety of organic and inorganic anions and cations. Protein members of some MFS families are found exclusively in bacteria or in eukaryotes, but others are found in bacteria, archaea, and eukaryotes. All permeases of the MFS possess either 12 or 14 putative or established transmembrane alpha-helical spanners, and evidence is presented substantiating the proposal that an internal tandem gene duplication event gave rise to a primordial MFS protein prior to divergence of the family members. All 17 families are shown to exhibit the common feature of a well-conserved motif present between transmembrane spanners 2 and 3. The analyses reported serve to characterize one of the largest and most diverse families of transport proteins found in living organisms.
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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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              TRAP is necessary for gliding motility and infectivity of plasmodium sporozoites.

              Many protozoans of the phylum Apicomplexa are invasive parasites that exhibit a substrate-dependent gliding motility. Plasmodium (malaria) sporozoites, the stage of the parasite that invades the salivary glands of the mosquito vector and the liver of the vertebrate host, express a surface protein called thrombospondin-related anonymous protein (TRAP) that has homologs in other Apicomplexa. By gene targeting in a rodent Plasmodium, we demonstrate that TRAP is critical for sporozoite infection of the mosquito salivary glands and the rat liver, and is essential for sporozoite gliding motility in vitro. This suggests that in Plasmodium sporozoites, and likely in other Apicomplexa, gliding locomotion and cell invasion have a common molecular basis.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, CA USA )
                1553-7366
                1553-7374
                18 July 2016
                July 2016
                : 12
                : 7
                : e1005734
                Affiliations
                [1 ]Integrative Parasitology, Center for Infectious Diseases, University of Heidelberg Medical School, Heidelberg, Germany
                [2 ]Instituto Medicina Molecular, Lisbon, Portugal
                Wellcome Trust Sanger Institute, UNITED KINGDOM
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: JK MS FF GRM. Performed the experiments: JK MS LL PAGCS GRM. Analyzed the data: JK GRM. Wrote the paper: JK FF GRM.

                Article
                PPATHOGENS-D-16-00522
                10.1371/journal.ppat.1005734
                4948853
                27427910
                13fd5174-7e56-4405-8e84-0fc832c67500
                © 2016 Kehrer et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 6 March 2016
                : 8 June 2016
                Page count
                Figures: 9, Tables: 0, Pages: 25
                Funding
                Funded by: CellNetworks
                Award ID: EcTOP
                Award Recipient :
                Funded by: European Research Council (BE)
                Award ID: ERC-SG 281719
                Award Recipient :
                Funded by: Seventh Framework Programme (BE)
                Award ID: EviMalar
                Award Recipient :
                Funded by: SFB
                Award ID: SFB 1129
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001871, Fundação para a Ciência e a Tecnologia;
                Award ID: SFRH/BPD/72619/2010
                Award Recipient :
                Funded by: Fundação para a Ciência e a Tecnologia (PT)
                Award ID: PTDC/SAU-MIC/122082/2010
                Award Recipient :
                This work was supported by the CellNetworks research cluster of Heidelberg University through EcTop2 ( http://www.cellnetworks.uni-hd.de), the European Research Council (ERC-SG 281719) and the EU FP7 research network EVIMalaR to FF (erc.europa.eu). FF is a member of the collaborative research center SFB 1129 at Heidelberg University ( http://www.sfb1129.de). PAGCS and GRM were supported by Fundação para a Ciência e a Tecnologia grants SFRH/BPD/72619/2010 and PTDC/SAU-MIC/122082/2010 ( www.fct.pt). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Parasitology
                Parasite Groups
                Apicomplexa
                Plasmodium
                Biology and Life Sciences
                Parasitology
                Parasite Groups
                Apicomplexa
                Sporozoites
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Vesicles
                Medicine and Health Sciences
                Parasitic Diseases
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Cell Membranes
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Germ Cells
                Gametocytes
                Biology and Life Sciences
                Developmental Biology
                Life Cycles
                Parasitic Life Cycles
                Biology and Life Sciences
                Parasitology
                Parasitic Life Cycles
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Cell Membranes
                Membrane Proteins
                Custom metadata
                All relevant data are within the paper and its Supporting Information files.

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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