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      CRISPR/Cas9 in Leishmania mexicana: A case study of LmxBTN1

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          Abstract

          Leishmania parasites cause human cutaneous, mucocutaneous and visceral leishmaniasis. Several studies proposed involvement of certain genes in infectivity of these parasites based on differential mRNA expression data. Due to unusual gene expression mechanism, functions of such genes must be further validated experimentally. Here, we investigated a role of one of the putative virulence factors, LmxM. 22. 0010-encoded BTN1 (a protein involved in Batten disease in humans), in L. mexicana infectivity. Due to the incredible plasticity of the L. mexicana genome, we failed to obtain a complete knock-out of LmxM. 22. 0010 using conventional recombination-based approach even after ablating four alleles of this gene. To overcome this, we established a modified CRISPR-Cas9 system with genomic expression of Cas9 nuclease and gRNA. Application of this system allowed us to establish a complete BTN1 KO strain of L. mexicana. The mutant strain did not show any difference in growth kinetics and differentiation in vitro, as well as in the infectivity for insect vectors and mice hosts. Based on the whole-transcriptome profiling, LmxM. 22. 0010-encoded BTN1 was considered a putative factor of virulence in Leishmania. Our study suggests that ablation of LmxM. 22. 0010 does not influence L. mexicana infectivity and further illustrates importance of experimental validation of in silico-predicted virulence factors. Here we also describe the whole genome sequencing of the widely used model isolate L. mexicana M379 and report a modified CRISPR/Cas9 system suitable for complete KO of multi-copy genes in organisms with flexible genomes.

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          A Historical Overview of the Classification, Evolution, and Dispersion of Leishmania Parasites and Sandflies

          Background The aim of this study is to describe the major evolutionary historical events among Leishmania, sandflies, and the associated animal reservoirs in detail, in accordance with the geographical evolution of the Earth, which has not been previously discussed on a large scale. Methodology and Principal Findings Leishmania and sandfly classification has always been a controversial matter, and the increasing number of species currently described further complicates this issue. Despite several hypotheses on the origin, evolution, and distribution of Leishmania and sandflies in the Old and New World, no consistent agreement exists regarding dissemination of the actors that play roles in leishmaniasis. For this purpose, we present here three centuries of research on sandflies and Leishmania descriptions, as well as a complete description of Leishmania and sandfly fossils and the emergence date of each Leishmania and sandfly group during different geographical periods, from 550 million years ago until now. We discuss critically the different approaches that were used for Leishmana and sandfly classification and their synonymies, proposing an updated classification for each species of Leishmania and sandfly. We update information on the current distribution and dispersion of different species of Leishmania (53), sandflies (more than 800 at genus or subgenus level), and animal reservoirs in each of the following geographical ecozones: Palearctic, Nearctic, Neotropic, Afrotropical, Oriental, Malagasy, and Australian. We propose an updated list of the potential and proven sandfly vectors for each Leishmania species in the Old and New World. Finally, we address a classical question about digenetic Leishmania evolution: which was the first host, a vertebrate or an invertebrate? Conclusions and Significance We propose an updated view of events that have played important roles in the geographical dispersion of sandflies, in relation to both the Leishmania species they transmit and the animal reservoirs of the parasites.
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            Phlebotomine sandflies and the spreading of leishmaniases and other diseases of public health concern.

            Phlebotomine sandflies transmit pathogens that affect humans and animals worldwide. We review the roles of phlebotomines in the spreading of leishmaniases, sandfly fever, summer meningitis, vesicular stomatitis, Chandipura virus encephalitis and Carrión's disease. Among over 800 species of sandfly recorded, 98 are proven or suspected vectors of human leishmaniases; these include 42 Phlebotomus species in the Old World and 56 Lutzomyia species in the New World (all: Diptera: Psychodidae). Based on incrimination criteria, we provide an updated list of proven or suspected vector species by endemic country where data are available. Increases in sandfly diffusion and density resulting from increases in breeding sites and blood sources, and the interruption of vector control activities contribute to the spreading of leishmaniasis in the settings of human migration, deforestation, urbanization and conflict. In addition, climatic changes can be expected to affect the density and dispersion of sandflies. Phlebovirus infections and diseases are present in large areas of the Old World, especially in the Mediterranean subregion, in which virus diversity has proven to be higher than initially suspected. Vesiculovirus diseases are important to livestock and humans in the southeastern U.S.A. and Latin America, and represent emerging human threats in parts of India. Carrión's disease, formerly restricted to regions of elevated altitude in Peru, Ecuador and Colombia, has shown recent expansion to non-endemic areas of the Amazon basin. © 2012 The Authors. Medical and Veterinary Entomology © 2012 The Royal Entomological Society.
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              Leishmania development in sand flies: parasite-vector interactions overview

              Leishmaniases are vector-borne parasitic diseases with 0.9 – 1.4 million new human cases each year worldwide. In the vectorial part of the life-cycle, Leishmania development is confined to the digestive tract. During the first few days after blood feeding, natural barriers to Leishmania development include secreted proteolytic enzymes, the peritrophic matrix surrounding the ingested blood meal and sand fly immune reactions. As the blood digestion proceeds, parasites need to bind to the midgut epithelium to avoid being excreted with the blood remnant. This binding is strictly stage-dependent as it is a property of nectomonad and leptomonad forms only. While the attachment in specific vectors (P. papatasi, P. duboscqi and P. sergenti) involves lipophosphoglycan (LPG), this Leishmania molecule is not required for parasite attachment in other sand fly species experimentally permissive for various Leishmania. During late-stage infections, large numbers of parasites accumulate in the anterior midgut and produce filamentous proteophosphoglycan creating a gel-like plug physically obstructing the gut. The parasites attached to the stomodeal valve cause damage to the chitin lining and epithelial cells of the valve, interfering with its function and facilitating reflux of parasites from the midgut. Transformation to metacyclic stages highly infective for the vertebrate host is the other prerequisite for effective transmission. Here, we review the current state of knowledge of molecular interactions occurring in all these distinct phases of parasite colonization of the sand fly gut, highlighting recent discoveries in the field.
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                Author and article information

                Contributors
                Role: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: Writing – original draft
                Role: Data curationRole: Investigation
                Role: Formal analysisRole: Investigation
                Role: Data curationRole: Formal analysisRole: Investigation
                Role: Data curationRole: Funding acquisitionRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: Project administrationRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: Project administrationRole: ResourcesRole: SupervisionRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                13 February 2018
                2018
                : 13
                : 2
                : e0192723
                Affiliations
                [1 ] Life Science Research Centre, Faculty of Science, University of Ostrava, Ostrava, Czech Republic
                [2 ] Biology Centre, Institute of Parasitology, Czech Academy of Sciences, České Budejovice (Budweis), Czech Republic
                [3 ] Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic
                [4 ] University of South Bohemia, Faculty of Sciences, České Budejovice (Budweis), Czech Republic
                [5 ] Institute of Environmental Technologies, Faculty of Science, University of Ostrava, Ostrava, Czech Republic
                University of Liverpool, UNITED KINGDOM
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0001-7701-5778
                http://orcid.org/0000-0003-4765-3263
                Article
                PONE-D-18-02690
                10.1371/journal.pone.0192723
                5811015
                29438445
                d40efb54-84be-4de9-a056-f4ac1a7af190
                © 2018 Ishemgulova 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
                : 31 October 2017
                : 29 January 2018
                Page count
                Figures: 3, Tables: 0, Pages: 17
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100001824, Grantová Agentura České Republiky;
                Award ID: 17-10656S
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001824, Grantová Agentura České Republiky;
                Award ID: 17-10656S
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001824, Grantová Agentura České Republiky;
                Award ID: 16-18699S
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001824, Grantová Agentura České Republiky;
                Award ID: 16-18699S
                Award Recipient :
                Funded by: European Cooperation in Science and Technology ()
                Award ID: CM1307
                Award Recipient :
                Funded by: European Research Council ()
                Award ID: CZ LL1601
                Award Recipient :
                Funded by: Ministerstvo Školství, Mládeže a Tělovýchovy (CZ)
                Award ID: LO1208 “TEWEP”
                Funded by: Charles University Research Center
                Award ID: UNCE project 204072
                Funded by: University of Ostrava
                Award ID: SGS/PrF/2018
                Support from the Czech Grant Agency ( www.gacr.cz) awards 17-10656S to VY and JV, 16-18699S to JL and VY; ERC CZ ( www.msmt.cz) grant LL1601 to JL, Charles University Research Center UNCE ( https://www.cuni.cz) project 204072 to JH, KM, JV, PV and the COST action ( www.cost.eu) CM1307 to JL is kindly acknowledged. This work was also financially supported by the Ministry of Education, Youth and Sports of the Czech Republic ( www.msmt.cz) in the “National Feasibility Program I”, project LO1208 “TEWEP” and University of Ostrava ( www.osu.cz) SGS/PrF/2018. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Medicine and Health Sciences
                Parasitic Diseases
                Biology and Life Sciences
                Organisms
                Eukaryota
                Protozoans
                Parasitic Protozoans
                Leishmania
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Biology and Life Sciences
                Developmental Biology
                Life Cycles
                Protozoan Life Cycles
                Promastigotes
                Biology and Life Sciences
                Microbiology
                Protozoology
                Protozoan Life Cycles
                Promastigotes
                Medicine and Health Sciences
                Infectious Diseases
                Disease Vectors
                Insect Vectors
                Sand Flies
                Biology and Life Sciences
                Species Interactions
                Disease Vectors
                Insect Vectors
                Sand Flies
                Biology and Life Sciences
                Genetics
                Genomics
                Animal Genomics
                Mammalian Genomics
                Medicine and Health Sciences
                Otorhinolaryngology
                Otology
                Ear Infections
                Biology and Life Sciences
                Genetics
                Genomics
                Animal Genomics
                Invertebrate Genomics
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                All relevant data are within the paper and its Supporting Information files.

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