Evaluación de la unión de la proteína secretada con dominio de repetición de trombospondina alterado (SPATR) de Plasmodium vivax a células hospederas.

La malaria en Colombia representa una enfermedad importante a nivel nacional debido a que nuestro país presenta las condiciones climáticas, geográficas y epidemiológicas aptas para la transmisión de esta enfermedad. Particularmente la especie Plasmodium vivax es una de las más prevalentes en nuestro...

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Autores:
Abril Loaiza, Juan Sebastián
Tipo de recurso:
Trabajo de grado de pregrado
Fecha de publicación:
2021
Institución:
Colegio Mayor de Cundinamarca
Repositorio:
Repositorio Colegio Mayor de Cundinamarca
Idioma:
spa
OAI Identifier:
oai:repositorio.unicolmayor.edu.co:unicolmayor/5545
Acceso en línea:
https://repositorio.unicolmayor.edu.co/handle/unicolmayor/5545
Palabra clave:
Plasmodium vivax
pre-eritrocitica
receptor-ligando
eritrocítica
malaria
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network_name_str Repositorio Colegio Mayor de Cundinamarca
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dc.title.spa.fl_str_mv Evaluación de la unión de la proteína secretada con dominio de repetición de trombospondina alterado (SPATR) de Plasmodium vivax a células hospederas.
title Evaluación de la unión de la proteína secretada con dominio de repetición de trombospondina alterado (SPATR) de Plasmodium vivax a células hospederas.
spellingShingle Evaluación de la unión de la proteína secretada con dominio de repetición de trombospondina alterado (SPATR) de Plasmodium vivax a células hospederas.
Plasmodium vivax
pre-eritrocitica
receptor-ligando
eritrocítica
malaria
title_short Evaluación de la unión de la proteína secretada con dominio de repetición de trombospondina alterado (SPATR) de Plasmodium vivax a células hospederas.
title_full Evaluación de la unión de la proteína secretada con dominio de repetición de trombospondina alterado (SPATR) de Plasmodium vivax a células hospederas.
title_fullStr Evaluación de la unión de la proteína secretada con dominio de repetición de trombospondina alterado (SPATR) de Plasmodium vivax a células hospederas.
title_full_unstemmed Evaluación de la unión de la proteína secretada con dominio de repetición de trombospondina alterado (SPATR) de Plasmodium vivax a células hospederas.
title_sort Evaluación de la unión de la proteína secretada con dominio de repetición de trombospondina alterado (SPATR) de Plasmodium vivax a células hospederas.
dc.creator.fl_str_mv Abril Loaiza, Juan Sebastián
dc.contributor.advisor.none.fl_str_mv Sánchez Mora, Ruth Mélida
Arévalo Pinzón, Gabriela
dc.contributor.author.none.fl_str_mv Abril Loaiza, Juan Sebastián
dc.subject.lemb.none.fl_str_mv Plasmodium vivax
pre-eritrocitica
receptor-ligando
topic Plasmodium vivax
pre-eritrocitica
receptor-ligando
eritrocítica
malaria
dc.subject.proposal.spa.fl_str_mv eritrocítica
malaria
description La malaria en Colombia representa una enfermedad importante a nivel nacional debido a que nuestro país presenta las condiciones climáticas, geográficas y epidemiológicas aptas para la transmisión de esta enfermedad. Particularmente la especie Plasmodium vivax es una de las más prevalentes en nuestro territorio lo que refleja entonces, la necesidad de implementar estudios sobre la biología básica de este parásito que involucren la búsqueda de blancos terapéuticos de acción. Durante el complejo ciclo de invasión de Plasmodium, se ha observado que el parásito expresa distintos antígenos para interactuar con la célula hospedera, entre estas, la proteína secretada con dominio de repetición de trombospondina alterado (SPATR). Esta proteína se reconoce como una proteína multietapa ya que en especies diferentes a Plasmodium vivax se ha evidenciado la presencia de esta en estadio sexual y asexual, además de tener capacidad para inducir una respuesta de anticuerpos. Sin embargo, en P. vivax no se ha establecido si esta proteína puede estar participando en eventos de adhesión a la célula hospedera (hepatocitos y reticulocitos), lo que supone un gran reto para la investigación de esta proteína y su importancia en el proceso de invasión. En este trabajo se evaluó la unión de la proteína SPATR a células hepáticas y reticulocitos humanos. Para cumplir con el objetivo se utilizó plásmido que contenía la secuencia de nucleótidos del gen spatr para transformar bacterias E. coli y obtener en estas células la proteína PvSPATR recombinante. La proteína fue purificada mediante procesos cromatográficos y fue incubada con células hospederas. Los resultados mostraron que, a pesar de la importancia de esta proteína en otras especies parasitarias, la proteína PvSPATR no interactúa directamente con reticulocitos y hepatocitos humanos. Esto sugiere que SPATR en P. vivax puede estar cumpliendo otras funciones durante el proceso de invasión del parásito que no involucran interacciones del tipo receptor-ligando o puede estar mediando interacciones macromoleculares con la membrana de la célula hospedera indirectamente. Esta investigación muestra que si bien, los parásitos de la malaria que infectan al humano comparten varias proteínas durante su ciclo de invasión, cada una de ellas puede estar cumpliendo distintas funciones como es el caso de la proteína PvSPATR.
publishDate 2021
dc.date.issued.none.fl_str_mv 2021-09
dc.date.accessioned.none.fl_str_mv 2022-05-06T01:39:48Z
dc.date.available.none.fl_str_mv 2022-05-06T01:39:48Z
dc.type.spa.fl_str_mv Trabajo de grado - Pregrado
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url https://repositorio.unicolmayor.edu.co/handle/unicolmayor/5545
dc.language.iso.spa.fl_str_mv spa
language spa
dc.relation.references.spa.fl_str_mv Organización Mundial de la Salud, Sección paludismo. Informe mundial 2020 sobre el paludismo. 2020. [Internet] [Citado 14 Octubre 2020] Disponible en: https://cdn.who.int/media/docs/default-source/malaria/world-malaria-reports/world-malaria-report-2020-briefing-kit-sp.pdf?sfvrsn=a6de03a5_11
Ferro C, Informe del evento de malaria en Colombia, Periodo epidemiológico VI Colombia 2021. Grupo enfermedades transmisibles, 2021 [Internet] [Citado 23 de Mayo 2021] Disponible en: https://www.ins.gov.co/buscador-eventos/Informesdeevento/MALARIA%20PE%20VI%202021.pdf
Kanjee U, Rangel G, Clarck M, Duraisingh M. Molecular and cellular interactions defining the tropism of Plasmodium vivax for reticulocytes. Curr Opin Microbiol 2019 [Internet] [Cited 14 October 2020] Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688184/
Johns F, Bass C. The cultivation of malaria plasmodia (Plasmodium vivax and Plasmodium falciparum) in vitro. Journal of Experimental Medicine. 2016 [Internet] [Cited 14 October 2020] Available in:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2124976/
Hertig E. Distribution of Anopheles vectors and potential malaria transmission stability in Europe and the Mediterranean area under future climate change. Parasites & Vectors. 2019 [Internet] [Cited 14 October 2020] Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6325871/
Saliba K, Luque R, Obaldia N, Nuñez M, Dutary S, Lim C, et al. Insights into an Optimization of Plasmodium vivax Sal-1In vitro culture: The Aotus Primate model. 2016 [Internet] [Cited 14 October 2020] Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4963040/
Bourgard C, Albrecht L, Kayano A, Sunnerhagen P, Costa F. Plasmodium vivax Biology: Insights Provided by Genomics, Transcriptomics and Proteomics. Front Cell Infect Microbiol. 2018. [Internet] [Cited 14 October 2020] Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809496/#B41
Mongui A, Angel D, Moreno D, Gonzalez S, Almonacid H, Vanegas M, et al. Identification and characterization of the Plasmodium vivax thrombospondin-related apical merozoite protein. Malaria Journal. 2010. [Internet][Cited 14 October 2020]Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3020679/
Aparicio F. Fenómenos de citoadherencia asociados al paludismo falciparum. Universidad Complutense de Madrid. Facultad de farmacia. 2015. [Internet] [Citado 14 Octubre 2020] Disponible en: http://147.96.70.122/Web/TFG/TFG/Memoria/FEDERICO%20MIGUEL%20BECERRA%20APARICIO.pdf
Luque R, Adams J, Kocken C, Pasini E. From marginal to essential: the golden thread between nutrient sensing, medium composition and Plasmodium vivax maturation in in vitro culture. Malaria Journal. 2019. [Internet] [Cited 14 October 2020] Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6785855/
Bèrmudez M, Pèrez D, Arèvalo G, Curtidor H, Patarroyo M. Plasmodium vivax in vitro continuous culture: the spoke in the wheel. Malaria Journal. 2018. [Internet] [Cited 14 October 2020] Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102941/#CR53
Brockelman C, Laovanitch R, Kaewkes S. Supportive effects of magnesium chloride on viability of Plasmodium vivax in vitro. J Sci Soc Thailand. 1984; [Internet] [Cited 14 October 2020] Available in:http://www.scienceasia.org/1984.10.n2/109.php
Zunping L, Sullivan S, Carlton J. The biology of Plasmodium vivax explored through genomics. Ann N Y Acad Sci. 2015.[Internet] [Cited 14 October 2020] Available in:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4405435/
Palaeya V, Lau Y, Mahmud R, Chen Y, Fong M. Cloning, expression, and immunocharacterization of surface protein containing an altered thrombospondin repeat domain (SPATR) from Plasmodium knowlesi. Malaria Journal. [Internet] [Cited 14 October 2020] Available in: https://malariajournal.biomedcentral.com/articles/10.1186/1475-2875-12-182
Mahajan B, Jani D, Chattopadhyay R, Nagarkatti R, Zheng H, Majam V, et al. Identification, Cloning, Expression, and Characterization of the Gene for Plasmodium knowlesi Surface Protein Containing an Altered Thrombospondin Repeat Domain. Infect Immun.2005. [Internet] [Cited 14 October 2020] Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1231135/
Gupta R, Mishra A, Choudhary H, Narwal S, Nayak B, Srivastava P. et. al. Secreted protein with altered thrombospondin repeat (SPATR) is essential for asexual blood stages but not required for hepatocyte invasion by the malaria parasite Plasmodium berghei.Molecular microbiology. 2019. [Internet] [Cited 14 October 2020] Available in: https://onlinelibrary.wiley.com/doi/abs/10.1111/mmi.14432
Chattopadhyay R, Rathore D, Fujioka H, Kumar S, de la Vega P, Haynes D. et al PfSPATR, a Plasmodium falciparum Protein Containing an Altered Thrombospondin Type I Repeat Domain Is Expressed at Several Stages of the Parasite Life Cycle and Is the Target of Inhibitory Antibodies. Journal of Biological Chemistry. 2019. [Internet] [Cited 14 October 2020] Available in: https://www.jbc.org/content/278/28/25977.long
Patarroyo ME, Franky J, Gómez M, Arévalo G, Patarroyo MA. Hotspots in Plasmodium and RBC Receptor-Ligand Interactions: Key Pieces for Inhibiting Malarial Parasite Invasion. Int J Mol Sci. 2020. [Internet] [Cited 14 October 2020] Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7370042
Crosnier C, Bustamante L, Barthlodson S, Bei A, Theron M, Uchikawa M. et al. BASIGIN is a receptor essential for erythrocyte invasion by Plasmodium falciparum. Nature. 2011 [Internet] [Cited 14 October 2020] Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3245779/
Markus M. Biological concepts in recurrent Plasmodium vivax malaria. Epub. 2018. [Internet] [Cited 14 October 2020] Available in: https://pubmed.ncbi.nlm.nih.gov/29564998/
Howick V, Russell A, Andrews T, Heaton H, Reid A, Natarajan K. et al. The Malaria Cell Atlas: Single parasite transcriptomes across the complete Plasmodium life cycle. Science. 2019.[Internet] [Cited 14 October 2020] Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7056351/
Ansari HR, Templeton TJ, Subudhi AK, Ramaprasad A, Tang J, Lu F, et al. Genome-scale comparison of expanded gene families in Plasmodium ovale wallikeri and Plasmodium ovale curtisi with Plasmodium malariae and with other Plasmodium species.International Jorurnal of Parasitology 2016. 46 1-2016 [Internet] [Cited 14 October 2020] Avaliable in: https://www.ncbi.nlm.nih.gov/pubmed/27392654
Kumar H and Tolia N. Getting in: The structural biology of malaria invasion, PLoS Pathog. 2019 [Internet] [Cited 14 October 2020] Avaliable in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6728024/
Kumar H, Tolia N. Getting in: The structural biology of malaria invasion. Plos pathogens. 2019.[Internet] [Cited 14 October 2020] Avaliable in: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1007943
XD Fang , DC Kaslow , JH Adams , LH Miller. Clonación del receptor Duffy de Plasmodium vivax. Mol Biochem Parasitol. 1990. [Internet] [Cited 14 October 2020] Avaliable in: https://pubmed.ncbi.nlm.nih.gov/1849231/
Langhorne J, Duffy P. Expanding the antimalarial toolkit: Targeting host–parasite interactions. J Exp Med. 2016 [Internet] [Cited 14 October 2020] Avaliable in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4749928/#bib2
Ministerio de Justicia. Ley 100 de 1993. Dairio oficial- página 1-1993 [Internet] [Citado 14 Octubre 2020] Disponible en:http://www.suinjuriscol.gov.co/viewDocument.asp?ruta=Leyes/1635955
Hietanen J, Chim A, Chiramanewong T, Gruszczyk J, Roobsoong W, Tham W. et al Gene Models, Expression Repertoire, and Immune Response of Plasmodium vivax Reticulocyte Binding Proteins. Infect Immun. 2016. [Internet] [Cited 14 October 2020] Avaliable in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4771344/
Srivastava A, Creek D, Evans K, Souza D, Schofield L, Müller S. et al. Host Reticulocytes Provide Metabolic Reservoirs That Can Be Exploited by Malaria Parasites. PLoS Pathog. 2015 [Internet] [Cited 14 October 2020] Avaliable in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4456406/
Spencer M, Gomez A, Collovini A. Mecanismos de invasion del esporozoíto y merozoíto de Plasmodium. Revista bionatura. 2016. [Internet] [Citado 14 Octubre 2020] Disponible en: https://www.revistabionatura.com/files/Mecanismos-de-invasion-del-esporozoito-y-merozoito.pdf.
Lynne S, García MS. Malaria. Revista Elsevier. 2010. [Internet] [Citado 10 Enero 2021] Disponible en: https://www.sciencedirect.com/science/article/abs/pii/S0272271209001188?via%3Dihub
Danny A. Milner Jr. Malaria Pathogenesis. Advance May. 2017 [Internet] [Cited 19 April 2021] Avaliable in: http://perspectivesinmedicine.cshlp.org/content/8/1/a025569.long
Lover A, Baird J, Gosling R. Malaria Elimination: Time to Target All Species The American Journal of Tropical Medicine and Hygiene. 2018. [Internet] [Cited 19 April 2021] Avaliable in: https://www.ajtmh.org/view/journals/tpmd/99/1/article-p17.xml
Muñoz J, Marcos G, Ramírez G, Salas J, Treviño B, Perez J.L. et. al. Diagnosis and treatment of imported malaria in Spain: Recommendations from the Malaria Working Group of the Spanish Society of Tropical Medicine and International Health (SEMTSI). Elsevier. 2015. [Internet] [Cited 19 April 2021] Avaliable in: https://www.sciencedirect.com/science/article/abs/pii/S0213005X14000196?via%3Dihub
Iborra M, García E, Carrilero B, Segovia M. Malaria diagnosis and treatment: analysis of a cohort of hospitalised patients at a tertiary level hospital (1998-2010). Rev Esp Quimioter. 2013. [Internet] [Cited 19 April 2021] Avaliable in: https://seq.es/seq/0214-3429/26/1/iborra.pdf
Santa P, Vázquez MC, Latorre E, Mairal P, et al. First autochthonous malaria case due to Plasmodium vivax since eradication, Spain, October 2010. Euro Surveill. 2010;15(41):19684. [Internet][Cited 02 apr 2020]. Available in: https://pubmed.ncbi.nlm.nih.gov/20961517/
Markus M. Biological concepts in recurrent Plasmodium vivax malaria. Cambridge University Press. 2018. [Internet][Cited 02 apr 2020]. Available in: https://pubmed.ncbi.nlm.nih.gov/20961517/
Howes R, Battle K, Mendis K, Smith D, Cibulskis R, Baird J. et al. Global Epidemiology of Plasmodium vivax. The American Journal of Tropical Medicine and Hygiene. Vol 95. 2016 [Internet][Cited 02 apr 2020]. Available in: https://www.ajtmh.org/view/journals/tpmd/95/6_Suppl/article-p15.xml
Flannery E, Markus M, Vaughan M. Plasmodium vivax. Trends in parasitology. 2019. [Internet][Cited 02 apr 2020]. Available in: https://www.cell.com/trends/parasitology/fulltext/S1471-4922(19)30081-9?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS1471492219300819%3Fshowall%3Dtrue
Curtidor H, García J, Vanegas M, Puentes F, Forero M, Patarroyo M. Identification of peptides with high red blood cell and hepatocyte binding activity in the Plasmodium falciparum multi-stage invasion proteins: PfSPATR and MCP-. Elsevier. 2008 [Internet][Cited 02 apr 2020]. Available in: https://www.sciencedirect.com/science/article/abs/pii/S0300908408002514
Gant S, Clavijo P, Bai X, Esko J, Sinnis P. Cell Adhesion to a Motif Shared by the Malaria Circumsporozoite Protein and Thrombospondin Is Mediated by Its Glycosaminoglycan-binding Region and Not by CSVTCG*. Journal Biological Chemistry. 1997. [Internet][Cited 02 apr 2020]. Available in: https://www.jbc.org/article/S0021-9258(18)38944-0/fulltext?keytype2=tf_ipsecsha&ijkey=3d6583af49adac59ba8e5f69942a200172d836f3
Chan Li, Dietrich M, Nguitragool W, Tham W. Plasmodium vivax Reticulocyte Binding Proteins for invasion into reticulocytes. Cellular Microbiology. 2019[Internet][Cited 02 apr 2020]. Available in: https://onlinelibrary.wiley.com/doi/full/10.1111/cmi.13110
Pereira A, Pérez M. Epidemiología y tratamiento del paludismo. Elsevier 2002. [Internet][Citado 02 Abril 2021]. Disponible en: https://www.elsevier.es/es-revista-offarm-4-articulo-epidemiologia-tratamiento-del-paludismo-13033516
Adams H, Mueller I. The Biology of Plasmodium vivax. CSH perspectives. 2017 [Internet][Citado 02 Abril 2021]. Disponible en: http://perspectivesinmedicine.cshlp.org/content/7/9/a025585.long.
OMS. Paludismo. 2020 [Internet][Citado 02 Abril 2021]. Disponible en: https://www.who.int/es/news-room/fact-sheets/detail/malaria
OMS. Control y eliminación del paludismo por Plasmodium vivax Informe técnico. 2020 [Internet][Citado 02 Abril 2021]. Disponible en: http://apps.who.int/iris/bitstream/handle/10665/204494/9789243509242_spa.pdf?sequence=1&isAllowed=y
Jia Baolei, Jeon Che. High-throughput recombinant protein expression in Escherichia coli: current status and future perspectives. [Internet] ][Cited 22 August 2021]. Available in: https://royalsocietypublishing.org/doi/10.1098/rsob.160196
Wandrey G, Bier C, Binder D, Hoffman K, Jaeger K, Pietruszka J, et al Light-induced gene expression with photocaged IPTG for induction profiling in a high-throughput screening system [Internet] ][Cited 22 August 2021]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4842301/
Farewell A, Neidhardt F.Effect of Temperature on In Vivo Protein Synthetic Capacity in Escherichia coli. [Internet] ][Cited 22 August 2021]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC107486/
Kaur J, Reinhardt D. Immobilized Metal Affinity Chromatography Co-Purifies TGF-β1 with Histidine-Tagged Recombinant Extracellular Proteins [Internet] ][Cited 22 August 2021]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3485342/
Spencer L, Gómez A, Collivini E. Mecanismos de invasion del esporozoíto y merozoíto de Plasmodium. Revista Bionatura. [Internet] ][Citado 22 Agosto 2021]. Disponible en: http://revistabionatura.com/files/Mecanismos-de-invasion-del-esporozoito-y-merozoito.pdf
Bourgard C, Albrecht L, Kayano A, Sunnerhagen P, Costa F, Plasmodium vivax Biology: Insights Provided by Genomics, Transcriptomics and Proteomics. 2018. Frontiers in. [Internet] ][Cited 22 August 2021]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809496/
Vaughan A, Kappe S. Malaria Parasite Liver Infection and Exoerythrocytic Biology. 2017. Cold Spring Harb Perspect Med. [Internet] ][Cited 22 August 2021]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5453383/
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spelling Sánchez Mora, Ruth Mélidaefe8f5fbfdf7ec7c24ba850634bfbb9aArévalo Pinzón, Gabriela3dd83e7b546fe22342d6cc31cc2cbc79Abril Loaiza, Juan Sebastián33a45338c06ea1d7a4df9513f78a84a52022-05-06T01:39:48Z2022-05-06T01:39:48Z2021-09https://repositorio.unicolmayor.edu.co/handle/unicolmayor/5545La malaria en Colombia representa una enfermedad importante a nivel nacional debido a que nuestro país presenta las condiciones climáticas, geográficas y epidemiológicas aptas para la transmisión de esta enfermedad. Particularmente la especie Plasmodium vivax es una de las más prevalentes en nuestro territorio lo que refleja entonces, la necesidad de implementar estudios sobre la biología básica de este parásito que involucren la búsqueda de blancos terapéuticos de acción. Durante el complejo ciclo de invasión de Plasmodium, se ha observado que el parásito expresa distintos antígenos para interactuar con la célula hospedera, entre estas, la proteína secretada con dominio de repetición de trombospondina alterado (SPATR). Esta proteína se reconoce como una proteína multietapa ya que en especies diferentes a Plasmodium vivax se ha evidenciado la presencia de esta en estadio sexual y asexual, además de tener capacidad para inducir una respuesta de anticuerpos. Sin embargo, en P. vivax no se ha establecido si esta proteína puede estar participando en eventos de adhesión a la célula hospedera (hepatocitos y reticulocitos), lo que supone un gran reto para la investigación de esta proteína y su importancia en el proceso de invasión. En este trabajo se evaluó la unión de la proteína SPATR a células hepáticas y reticulocitos humanos. Para cumplir con el objetivo se utilizó plásmido que contenía la secuencia de nucleótidos del gen spatr para transformar bacterias E. coli y obtener en estas células la proteína PvSPATR recombinante. La proteína fue purificada mediante procesos cromatográficos y fue incubada con células hospederas. Los resultados mostraron que, a pesar de la importancia de esta proteína en otras especies parasitarias, la proteína PvSPATR no interactúa directamente con reticulocitos y hepatocitos humanos. Esto sugiere que SPATR en P. vivax puede estar cumpliendo otras funciones durante el proceso de invasión del parásito que no involucran interacciones del tipo receptor-ligando o puede estar mediando interacciones macromoleculares con la membrana de la célula hospedera indirectamente. Esta investigación muestra que si bien, los parásitos de la malaria que infectan al humano comparten varias proteínas durante su ciclo de invasión, cada una de ellas puede estar cumpliendo distintas funciones como es el caso de la proteína PvSPATR.Malaria in Colombia represents an important disease at the national level because our country presents the climatic, geographical and epidemiological conditions suitable for the transmission of this disease. Particularly the Plasmodium vivax species is one of the most prevalent in our territory, which then reflects the need to implement studies on the basic biology of this parasite that involve the search for therapeutic targets of action. During the complex invasion cycle of Plasmodium, it has been observed that the parasite expresses different antigens to interact with the host cell, including the secreted protein with altered thrombospondin repeat domain (SPATR). This protein is recognized as a multistage protein since in species other than Plasmodium vivax, its presence in sexual and asexual stages has been evidenced, in addition to having the capacity to induce an antibody response. However, in P. vivax it has not been established whether this protein may be participating in events of adhesion to the host cell (hepatocytes and reticulocytes), which represents a great challenge for the investigation of this protein and its importance in the process of invasion. In this work, the binding of the SPATR protein to liver cells and human reticulocytes was evaluated. To meet the objective, a plasmid containing the nucleotide sequence of the spatr gene was used to transform E. coli bacteria and obtain the recombinant PvSPATR protein in these cells. The protein was purified by chromatographic processes and incubated with host cells. The results show that, despite the importance of this protein in other parasitic species, the PvSPATR protein does not interact directly with human reticulocytes and hepatocytes. This suggests that SPATR in P. vivax may be fulfilling other functions during the parasite invasion process that do not involve receptor-ligand interactions or may be indirectly mediating macromolecular interactions with the host cell membrane. This research shows that although the malaria parasites that infect humans share several proteins during their invasion cycle, each of them may be fulfilling different functions, such as the PvSPATR protein.Tabla de contenido Pág. 1. Resumen 1 2. Introducción 2 3. Marco teórico 3 3.1 Antecedentes 3 3.2 Malaria 7 3.3 Epidemiología de la malaria 7 3.4 Presentación clínica 9 3.5 Ciclo de vida y etiología 10 3.6 Plasmodium vivax 12 3.7 Proceso de invasión a los reticulocitos 13 3.8 Proceso de invasión a los hepatocitos 14 4. Diseño metodológico 15 4.1 Objetivo general 15 4.2 Objetivos específicos 16 4.3 Tipo de investigación 16 4.4 Enfoque de estudio 16 4.5 Materiales y métodos 16 4.5.1 Preparación del vector 17 4.5.2 Transformación bacteriana 18 4.5.3 Expresión de la proteína PvSPATR 18 4.5.4 Tratamiento de la proteína recombinante 19 4.5.5 Purificación de PvSPATR por cromatografía de afinidad 20 4.5.6 Diálisis de PvSPATR 21 4.5.7 SDS-PAGE 21 4.5.8 Western-blot 21 4.5.9 Tinción azul de Coomasie 22 4.5.10 Dot Blot 22 4.5.11 Cuantificación de PvSPATR recombinante 22 4.5.12 Citometría de flujo con células HepG2 23 4.5.13 Radiomarcaje de PvSPATR recombinante 23 4.5.14 Ensayo de unión con PvSPATR radiomarcada a células HepG2 23 4.5.15 Obtención y purificación de reticulocitos 24 4.5.16 Citometría de flujo con reticulocitos 25 5. Resultados 25 5.1 Obtención de células E. coli transformadas por el vector Pet24-PvSPATR 25 5.2 Expresión de la proteína PvSPATR 26 5.3 Obtención de PvSPATR purificada 26 5.4 Unión total de la proteína PvSPATR a células HepG2 por citometría de flujo 27 5.5 Unión de la proteína PvSPATR radiomarcada a células HepG2 30 5.6 Obtención y purificación de reticulocitos 31 5.7 Unión total de PvSPATR a reticulocitos por citometría de flujo 32 6. Discusión 33 7. Conclusiones 37 8. Bibliografía 38PregradoBacteriólogo(a) y Laboratorista Clínico52p.application/pdfspaUniversidad Colegio Mayor de CundinamarcaFacultad de Ciencias de la SaludBogotáBacteriología y Laboratorio ClínicoDerechos Reservados - Universidad Colegio Mayor de Cundinamarca, 2021https://creativecommons.org/licenses/by-nc-sa/4.0/info:eu-repo/semantics/closedAccessAtribución-NoComercial-CompartirIgual 4.0 Internacional (CC BY-NC-SA 4.0)http://purl.org/coar/access_right/c_14cbEvaluación de la unión de la proteína secretada con dominio de repetición de trombospondina alterado (SPATR) de Plasmodium vivax a células hospederas.Trabajo de grado - Pregradohttp://purl.org/coar/resource_type/c_7a1fhttp://purl.org/coar/version/c_970fb48d4fbd8a85Textinfo:eu-repo/semantics/bachelorThesishttps://purl.org/redcol/resource_type/TPinfo:eu-repo/semantics/publishedVersionOrganización Mundial de la Salud, Sección paludismo. Informe mundial 2020 sobre el paludismo. 2020. [Internet] [Citado 14 Octubre 2020] Disponible en: https://cdn.who.int/media/docs/default-source/malaria/world-malaria-reports/world-malaria-report-2020-briefing-kit-sp.pdf?sfvrsn=a6de03a5_11Ferro C, Informe del evento de malaria en Colombia, Periodo epidemiológico VI Colombia 2021. Grupo enfermedades transmisibles, 2021 [Internet] [Citado 23 de Mayo 2021] Disponible en: https://www.ins.gov.co/buscador-eventos/Informesdeevento/MALARIA%20PE%20VI%202021.pdfKanjee U, Rangel G, Clarck M, Duraisingh M. Molecular and cellular interactions defining the tropism of Plasmodium vivax for reticulocytes. Curr Opin Microbiol 2019 [Internet] [Cited 14 October 2020] Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688184/Johns F, Bass C. The cultivation of malaria plasmodia (Plasmodium vivax and Plasmodium falciparum) in vitro. Journal of Experimental Medicine. 2016 [Internet] [Cited 14 October 2020] Available in:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2124976/Hertig E. Distribution of Anopheles vectors and potential malaria transmission stability in Europe and the Mediterranean area under future climate change. Parasites & Vectors. 2019 [Internet] [Cited 14 October 2020] Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6325871/Saliba K, Luque R, Obaldia N, Nuñez M, Dutary S, Lim C, et al. Insights into an Optimization of Plasmodium vivax Sal-1In vitro culture: The Aotus Primate model. 2016 [Internet] [Cited 14 October 2020] Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4963040/Bourgard C, Albrecht L, Kayano A, Sunnerhagen P, Costa F. Plasmodium vivax Biology: Insights Provided by Genomics, Transcriptomics and Proteomics. Front Cell Infect Microbiol. 2018. [Internet] [Cited 14 October 2020] Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809496/#B41Mongui A, Angel D, Moreno D, Gonzalez S, Almonacid H, Vanegas M, et al. Identification and characterization of the Plasmodium vivax thrombospondin-related apical merozoite protein. Malaria Journal. 2010. [Internet][Cited 14 October 2020]Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3020679/Aparicio F. Fenómenos de citoadherencia asociados al paludismo falciparum. Universidad Complutense de Madrid. Facultad de farmacia. 2015. [Internet] [Citado 14 Octubre 2020] Disponible en: http://147.96.70.122/Web/TFG/TFG/Memoria/FEDERICO%20MIGUEL%20BECERRA%20APARICIO.pdfLuque R, Adams J, Kocken C, Pasini E. From marginal to essential: the golden thread between nutrient sensing, medium composition and Plasmodium vivax maturation in in vitro culture. Malaria Journal. 2019. [Internet] [Cited 14 October 2020] Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6785855/Bèrmudez M, Pèrez D, Arèvalo G, Curtidor H, Patarroyo M. Plasmodium vivax in vitro continuous culture: the spoke in the wheel. Malaria Journal. 2018. [Internet] [Cited 14 October 2020] Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102941/#CR53Brockelman C, Laovanitch R, Kaewkes S. Supportive effects of magnesium chloride on viability of Plasmodium vivax in vitro. J Sci Soc Thailand. 1984; [Internet] [Cited 14 October 2020] Available in:http://www.scienceasia.org/1984.10.n2/109.phpZunping L, Sullivan S, Carlton J. The biology of Plasmodium vivax explored through genomics. Ann N Y Acad Sci. 2015.[Internet] [Cited 14 October 2020] Available in:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4405435/Palaeya V, Lau Y, Mahmud R, Chen Y, Fong M. Cloning, expression, and immunocharacterization of surface protein containing an altered thrombospondin repeat domain (SPATR) from Plasmodium knowlesi. Malaria Journal. [Internet] [Cited 14 October 2020] Available in: https://malariajournal.biomedcentral.com/articles/10.1186/1475-2875-12-182Mahajan B, Jani D, Chattopadhyay R, Nagarkatti R, Zheng H, Majam V, et al. Identification, Cloning, Expression, and Characterization of the Gene for Plasmodium knowlesi Surface Protein Containing an Altered Thrombospondin Repeat Domain. Infect Immun.2005. [Internet] [Cited 14 October 2020] Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1231135/Gupta R, Mishra A, Choudhary H, Narwal S, Nayak B, Srivastava P. et. al. Secreted protein with altered thrombospondin repeat (SPATR) is essential for asexual blood stages but not required for hepatocyte invasion by the malaria parasite Plasmodium berghei.Molecular microbiology. 2019. [Internet] [Cited 14 October 2020] Available in: https://onlinelibrary.wiley.com/doi/abs/10.1111/mmi.14432Chattopadhyay R, Rathore D, Fujioka H, Kumar S, de la Vega P, Haynes D. et al PfSPATR, a Plasmodium falciparum Protein Containing an Altered Thrombospondin Type I Repeat Domain Is Expressed at Several Stages of the Parasite Life Cycle and Is the Target of Inhibitory Antibodies. Journal of Biological Chemistry. 2019. [Internet] [Cited 14 October 2020] Available in: https://www.jbc.org/content/278/28/25977.longPatarroyo ME, Franky J, Gómez M, Arévalo G, Patarroyo MA. Hotspots in Plasmodium and RBC Receptor-Ligand Interactions: Key Pieces for Inhibiting Malarial Parasite Invasion. Int J Mol Sci. 2020. [Internet] [Cited 14 October 2020] Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7370042Crosnier C, Bustamante L, Barthlodson S, Bei A, Theron M, Uchikawa M. et al. BASIGIN is a receptor essential for erythrocyte invasion by Plasmodium falciparum. Nature. 2011 [Internet] [Cited 14 October 2020] Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3245779/Markus M. Biological concepts in recurrent Plasmodium vivax malaria. Epub. 2018. [Internet] [Cited 14 October 2020] Available in: https://pubmed.ncbi.nlm.nih.gov/29564998/Howick V, Russell A, Andrews T, Heaton H, Reid A, Natarajan K. et al. The Malaria Cell Atlas: Single parasite transcriptomes across the complete Plasmodium life cycle. Science. 2019.[Internet] [Cited 14 October 2020] Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7056351/Ansari HR, Templeton TJ, Subudhi AK, Ramaprasad A, Tang J, Lu F, et al. Genome-scale comparison of expanded gene families in Plasmodium ovale wallikeri and Plasmodium ovale curtisi with Plasmodium malariae and with other Plasmodium species.International Jorurnal of Parasitology 2016. 46 1-2016 [Internet] [Cited 14 October 2020] Avaliable in: https://www.ncbi.nlm.nih.gov/pubmed/27392654Kumar H and Tolia N. Getting in: The structural biology of malaria invasion, PLoS Pathog. 2019 [Internet] [Cited 14 October 2020] Avaliable in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6728024/Kumar H, Tolia N. Getting in: The structural biology of malaria invasion. Plos pathogens. 2019.[Internet] [Cited 14 October 2020] Avaliable in: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1007943XD Fang , DC Kaslow , JH Adams , LH Miller. Clonación del receptor Duffy de Plasmodium vivax. Mol Biochem Parasitol. 1990. [Internet] [Cited 14 October 2020] Avaliable in: https://pubmed.ncbi.nlm.nih.gov/1849231/Langhorne J, Duffy P. Expanding the antimalarial toolkit: Targeting host–parasite interactions. J Exp Med. 2016 [Internet] [Cited 14 October 2020] Avaliable in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4749928/#bib2Ministerio de Justicia. Ley 100 de 1993. Dairio oficial- página 1-1993 [Internet] [Citado 14 Octubre 2020] Disponible en:http://www.suinjuriscol.gov.co/viewDocument.asp?ruta=Leyes/1635955Hietanen J, Chim A, Chiramanewong T, Gruszczyk J, Roobsoong W, Tham W. et al Gene Models, Expression Repertoire, and Immune Response of Plasmodium vivax Reticulocyte Binding Proteins. Infect Immun. 2016. [Internet] [Cited 14 October 2020] Avaliable in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4771344/Srivastava A, Creek D, Evans K, Souza D, Schofield L, Müller S. et al. Host Reticulocytes Provide Metabolic Reservoirs That Can Be Exploited by Malaria Parasites. PLoS Pathog. 2015 [Internet] [Cited 14 October 2020] Avaliable in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4456406/Spencer M, Gomez A, Collovini A. Mecanismos de invasion del esporozoíto y merozoíto de Plasmodium. Revista bionatura. 2016. [Internet] [Citado 14 Octubre 2020] Disponible en: https://www.revistabionatura.com/files/Mecanismos-de-invasion-del-esporozoito-y-merozoito.pdf.Lynne S, García MS. Malaria. Revista Elsevier. 2010. [Internet] [Citado 10 Enero 2021] Disponible en: https://www.sciencedirect.com/science/article/abs/pii/S0272271209001188?via%3DihubDanny A. Milner Jr. Malaria Pathogenesis. Advance May. 2017 [Internet] [Cited 19 April 2021] Avaliable in: http://perspectivesinmedicine.cshlp.org/content/8/1/a025569.longLover A, Baird J, Gosling R. Malaria Elimination: Time to Target All Species The American Journal of Tropical Medicine and Hygiene. 2018. [Internet] [Cited 19 April 2021] Avaliable in: https://www.ajtmh.org/view/journals/tpmd/99/1/article-p17.xmlMuñoz J, Marcos G, Ramírez G, Salas J, Treviño B, Perez J.L. et. al. Diagnosis and treatment of imported malaria in Spain: Recommendations from the Malaria Working Group of the Spanish Society of Tropical Medicine and International Health (SEMTSI). Elsevier. 2015. [Internet] [Cited 19 April 2021] Avaliable in: https://www.sciencedirect.com/science/article/abs/pii/S0213005X14000196?via%3DihubIborra M, García E, Carrilero B, Segovia M. Malaria diagnosis and treatment: analysis of a cohort of hospitalised patients at a tertiary level hospital (1998-2010). Rev Esp Quimioter. 2013. [Internet] [Cited 19 April 2021] Avaliable in: https://seq.es/seq/0214-3429/26/1/iborra.pdfSanta P, Vázquez MC, Latorre E, Mairal P, et al. First autochthonous malaria case due to Plasmodium vivax since eradication, Spain, October 2010. Euro Surveill. 2010;15(41):19684. [Internet][Cited 02 apr 2020]. Available in: https://pubmed.ncbi.nlm.nih.gov/20961517/Markus M. Biological concepts in recurrent Plasmodium vivax malaria. Cambridge University Press. 2018. [Internet][Cited 02 apr 2020]. Available in: https://pubmed.ncbi.nlm.nih.gov/20961517/Howes R, Battle K, Mendis K, Smith D, Cibulskis R, Baird J. et al. Global Epidemiology of Plasmodium vivax. The American Journal of Tropical Medicine and Hygiene. Vol 95. 2016 [Internet][Cited 02 apr 2020]. Available in: https://www.ajtmh.org/view/journals/tpmd/95/6_Suppl/article-p15.xmlFlannery E, Markus M, Vaughan M. Plasmodium vivax. Trends in parasitology. 2019. [Internet][Cited 02 apr 2020]. Available in: https://www.cell.com/trends/parasitology/fulltext/S1471-4922(19)30081-9?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS1471492219300819%3Fshowall%3DtrueCurtidor H, García J, Vanegas M, Puentes F, Forero M, Patarroyo M. Identification of peptides with high red blood cell and hepatocyte binding activity in the Plasmodium falciparum multi-stage invasion proteins: PfSPATR and MCP-. Elsevier. 2008 [Internet][Cited 02 apr 2020]. Available in: https://www.sciencedirect.com/science/article/abs/pii/S0300908408002514Gant S, Clavijo P, Bai X, Esko J, Sinnis P. Cell Adhesion to a Motif Shared by the Malaria Circumsporozoite Protein and Thrombospondin Is Mediated by Its Glycosaminoglycan-binding Region and Not by CSVTCG*. Journal Biological Chemistry. 1997. [Internet][Cited 02 apr 2020]. Available in: https://www.jbc.org/article/S0021-9258(18)38944-0/fulltext?keytype2=tf_ipsecsha&ijkey=3d6583af49adac59ba8e5f69942a200172d836f3Chan Li, Dietrich M, Nguitragool W, Tham W. Plasmodium vivax Reticulocyte Binding Proteins for invasion into reticulocytes. Cellular Microbiology. 2019[Internet][Cited 02 apr 2020]. Available in: https://onlinelibrary.wiley.com/doi/full/10.1111/cmi.13110Pereira A, Pérez M. Epidemiología y tratamiento del paludismo. Elsevier 2002. [Internet][Citado 02 Abril 2021]. Disponible en: https://www.elsevier.es/es-revista-offarm-4-articulo-epidemiologia-tratamiento-del-paludismo-13033516Adams H, Mueller I. The Biology of Plasmodium vivax. CSH perspectives. 2017 [Internet][Citado 02 Abril 2021]. Disponible en: http://perspectivesinmedicine.cshlp.org/content/7/9/a025585.long.OMS. Paludismo. 2020 [Internet][Citado 02 Abril 2021]. Disponible en: https://www.who.int/es/news-room/fact-sheets/detail/malariaOMS. Control y eliminación del paludismo por Plasmodium vivax Informe técnico. 2020 [Internet][Citado 02 Abril 2021]. Disponible en: http://apps.who.int/iris/bitstream/handle/10665/204494/9789243509242_spa.pdf?sequence=1&isAllowed=yJia Baolei, Jeon Che. High-throughput recombinant protein expression in Escherichia coli: current status and future perspectives. [Internet] ][Cited 22 August 2021]. Available in: https://royalsocietypublishing.org/doi/10.1098/rsob.160196Wandrey G, Bier C, Binder D, Hoffman K, Jaeger K, Pietruszka J, et al Light-induced gene expression with photocaged IPTG for induction profiling in a high-throughput screening system [Internet] ][Cited 22 August 2021]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4842301/Farewell A, Neidhardt F.Effect of Temperature on In Vivo Protein Synthetic Capacity in Escherichia coli. [Internet] ][Cited 22 August 2021]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC107486/Kaur J, Reinhardt D. Immobilized Metal Affinity Chromatography Co-Purifies TGF-β1 with Histidine-Tagged Recombinant Extracellular Proteins [Internet] ][Cited 22 August 2021]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3485342/Spencer L, Gómez A, Collivini E. Mecanismos de invasion del esporozoíto y merozoíto de Plasmodium. Revista Bionatura. [Internet] ][Citado 22 Agosto 2021]. Disponible en: http://revistabionatura.com/files/Mecanismos-de-invasion-del-esporozoito-y-merozoito.pdfBourgard C, Albrecht L, Kayano A, Sunnerhagen P, Costa F, Plasmodium vivax Biology: Insights Provided by Genomics, Transcriptomics and Proteomics. 2018. Frontiers in. [Internet] ][Cited 22 August 2021]. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809496/Vaughan A, Kappe S. Malaria Parasite Liver Infection and Exoerythrocytic Biology. 2017. Cold Spring Harb Perspect Med. [Internet] ][Cited 22 August 2021]. 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Unicolmayorrepositorio@unicolmayor.edu.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