Identification of Babesia bovis MSA-1 functionally constraint regions capable of binding to bovine erythrocytes

Merozoite surface antigen-1 is a glycoprotein expressed by Babesia bovis and is considered a vaccine candidate given that antibodies against it are able to partially block in vitro invasion of bovine erythrocytes. Despite this, no study to date has confirmed the target cell binding properties of the...

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Autores:
Cuy Chaparro, Laura
Ricaurte-Contreras, Laura Alejandra
Bohórquez, Michel David
Arevalo-Pinzon, Gabriela
Barreto-Santamaría, Adriana
Pabón, Laura
Reyes, César
Moreno Pérez, Darwin Andrés
Patarroyo, Manuel-Alfonso
Tipo de recurso:
Article of investigation
Fecha de publicación:
2022
Institución:
Universidad de Ciencias Aplicadas y Ambientales U.D.C.A
Repositorio:
Repositorio Institucional UDCA
Idioma:
eng
OAI Identifier:
oai:repository.udca.edu.co:11158/5016
Acceso en línea:
https://repository.udca.edu.co/handle/11158/5016
https://doi.org/10.1016/j.vetpar.2022.109834
https://repository.udca.edu.co/
Palabra clave:
Babesia bovis
Babesiosis
Parásitos
Péptidos
Proteínas Protozoarias
Adhesión de parásitos
Enfermedades del ganado
Rights
openAccess
License
https://creativecommons.org/licenses/by-nc-sa/4.0/legalcode.es
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dc.title.spa.fl_str_mv Identification of Babesia bovis MSA-1 functionally constraint regions capable of binding to bovine erythrocytes
title Identification of Babesia bovis MSA-1 functionally constraint regions capable of binding to bovine erythrocytes
spellingShingle Identification of Babesia bovis MSA-1 functionally constraint regions capable of binding to bovine erythrocytes
Babesia bovis
Babesiosis
Parásitos
Péptidos
Proteínas Protozoarias
Adhesión de parásitos
Enfermedades del ganado
title_short Identification of Babesia bovis MSA-1 functionally constraint regions capable of binding to bovine erythrocytes
title_full Identification of Babesia bovis MSA-1 functionally constraint regions capable of binding to bovine erythrocytes
title_fullStr Identification of Babesia bovis MSA-1 functionally constraint regions capable of binding to bovine erythrocytes
title_full_unstemmed Identification of Babesia bovis MSA-1 functionally constraint regions capable of binding to bovine erythrocytes
title_sort Identification of Babesia bovis MSA-1 functionally constraint regions capable of binding to bovine erythrocytes
dc.creator.fl_str_mv Cuy Chaparro, Laura
Ricaurte-Contreras, Laura Alejandra
Bohórquez, Michel David
Arevalo-Pinzon, Gabriela
Barreto-Santamaría, Adriana
Pabón, Laura
Reyes, César
Moreno Pérez, Darwin Andrés
Patarroyo, Manuel-Alfonso
dc.contributor.author.none.fl_str_mv Cuy Chaparro, Laura
Ricaurte-Contreras, Laura Alejandra
Bohórquez, Michel David
Arevalo-Pinzon, Gabriela
Barreto-Santamaría, Adriana
Pabón, Laura
Reyes, César
Moreno Pérez, Darwin Andrés
Patarroyo, Manuel-Alfonso
dc.subject.decs.none.fl_str_mv Babesia bovis
Babesiosis
Parásitos
Péptidos
Proteínas Protozoarias
topic Babesia bovis
Babesiosis
Parásitos
Péptidos
Proteínas Protozoarias
Adhesión de parásitos
Enfermedades del ganado
dc.subject.proposal.spa.fl_str_mv Adhesión de parásitos
dc.subject.agrovoc.none.fl_str_mv Enfermedades del ganado
description Merozoite surface antigen-1 is a glycoprotein expressed by Babesia bovis and is considered a vaccine candidate given that antibodies against it are able to partially block in vitro invasion of bovine erythrocytes. Despite this, no study to date has confirmed the target cell binding properties of the full MSA-1 or its fragments. This research has thus been focused on identifying protein regions playing a role in erythrocyte attachment, based on genetic diversity and natural selection analysis. Two regions under functional constraint (nucleotides 134–428 and 464–629) having a preponderance of negatively-selected signals were identified in silico. Three non-overlapping peptides derived from functionally constraint regions (42422 (39PEGSFYDDMSKFYGAVGSFD58), 42424 (91NALIKNNPMIRPDLFNATIV110) and 42426 (150TDIVEEDREKAVEYFKKHVY169)) were able to specifically bind to a sialoglycoprotein located on the bovine erythrocyte surface as confirmed by sensitive and specific peptide-cell interaction competition assays using both enzymatically treated and untreated red blood cells. Interestingly, it was predicted that peptides 42422 and 42426 have a helical structure and conserved motifs in all strain/isolates. These findings provide evidence, for the first time, related to B. bovis MSA-1 short regions used by the parasite in erythrocyte binding which could be predicted using natural selection analysis. Future work focused on evaluating these peptides’ antigenic ability during natural infection, and their ability to induce protection in immunisation assays are needed to confirm their usefulness as synthetic vaccine candidates.
publishDate 2022
dc.date.issued.none.fl_str_mv 2022
dc.date.accessioned.none.fl_str_mv 2023-01-20T17:36:21Z
dc.date.available.none.fl_str_mv 2023-01-20T17:36:21Z
dc.type.spa.fl_str_mv Artículo de revista
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dc.identifier.citation.spa.fl_str_mv Cuy-Chaparro, L., Ricaurte-Contreras, L. A., Bohórquez, M. D., Arévalo-Pinzón, G., Barreto-Santamaria, A., Pabón, L., . . . Patarroyo, M. A. (2022). Identification of babesia bovis MSA-1 functionally constraint regions capable of binding to bovine erythrocytes. Veterinary Parasitology, 312 doi:10.1016/j.vetpar.2022.109834
dc.identifier.issn.spa.fl_str_mv 0304-4017
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dc.identifier.doi.none.fl_str_mv https://doi.org/10.1016/j.vetpar.2022.109834
dc.identifier.eissn.spa.fl_str_mv 1873-2550
dc.identifier.instname.spa.fl_str_mv Universidad de Ciencias Aplicadas y Ambientales
dc.identifier.reponame.spa.fl_str_mv UDCA
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identifier_str_mv Cuy-Chaparro, L., Ricaurte-Contreras, L. A., Bohórquez, M. D., Arévalo-Pinzón, G., Barreto-Santamaria, A., Pabón, L., . . . Patarroyo, M. A. (2022). Identification of babesia bovis MSA-1 functionally constraint regions capable of binding to bovine erythrocytes. Veterinary Parasitology, 312 doi:10.1016/j.vetpar.2022.109834
0304-4017
1873-2550
Universidad de Ciencias Aplicadas y Ambientales
UDCA
url https://repository.udca.edu.co/handle/11158/5016
https://doi.org/10.1016/j.vetpar.2022.109834
https://repository.udca.edu.co/
dc.language.iso.spa.fl_str_mv eng
language eng
dc.relation.indexed.spa.fl_str_mv Agrosavia
dc.relation.citationedition.spa.fl_str_mv (Dic., 2022) Artículo número 109834
dc.relation.citationendpage.spa.fl_str_mv 8
dc.relation.citationstartpage.spa.fl_str_mv 1
dc.relation.citationvolume.spa.fl_str_mv 312
dc.relation.ispartofjournal.spa.fl_str_mv Veterinary Parasitology
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spelling Cuy Chaparro, LauraRicaurte-Contreras, Laura AlejandraBohórquez, Michel DavidArevalo-Pinzon, GabrielaBarreto-Santamaría, AdrianaPabón, LauraReyes, CésarMoreno Pérez, Darwin AndrésPatarroyo, Manuel-Alfonso2023-01-20T17:36:21Z2023-01-20T17:36:21Z2022Cuy-Chaparro, L., Ricaurte-Contreras, L. A., Bohórquez, M. D., Arévalo-Pinzón, G., Barreto-Santamaria, A., Pabón, L., . . . Patarroyo, M. A. (2022). Identification of babesia bovis MSA-1 functionally constraint regions capable of binding to bovine erythrocytes. Veterinary Parasitology, 312 doi:10.1016/j.vetpar.2022.1098340304-4017https://repository.udca.edu.co/handle/11158/5016https://doi.org/10.1016/j.vetpar.2022.1098341873-2550Universidad de Ciencias Aplicadas y AmbientalesUDCAhttps://repository.udca.edu.co/Merozoite surface antigen-1 is a glycoprotein expressed by Babesia bovis and is considered a vaccine candidate given that antibodies against it are able to partially block in vitro invasion of bovine erythrocytes. Despite this, no study to date has confirmed the target cell binding properties of the full MSA-1 or its fragments. This research has thus been focused on identifying protein regions playing a role in erythrocyte attachment, based on genetic diversity and natural selection analysis. Two regions under functional constraint (nucleotides 134–428 and 464–629) having a preponderance of negatively-selected signals were identified in silico. Three non-overlapping peptides derived from functionally constraint regions (42422 (39PEGSFYDDMSKFYGAVGSFD58), 42424 (91NALIKNNPMIRPDLFNATIV110) and 42426 (150TDIVEEDREKAVEYFKKHVY169)) were able to specifically bind to a sialoglycoprotein located on the bovine erythrocyte surface as confirmed by sensitive and specific peptide-cell interaction competition assays using both enzymatically treated and untreated red blood cells. Interestingly, it was predicted that peptides 42422 and 42426 have a helical structure and conserved motifs in all strain/isolates. These findings provide evidence, for the first time, related to B. bovis MSA-1 short regions used by the parasite in erythrocyte binding which could be predicted using natural selection analysis. Future work focused on evaluating these peptides’ antigenic ability during natural infection, and their ability to induce protection in immunisation assays are needed to confirm their usefulness as synthetic vaccine candidates.Incluye referencias bibliográficasapplication/pdfenghttps://creativecommons.org/licenses/by-nc-sa/4.0/legalcode.eshttps://creativecommons.org/licenses/by-nc-sa/4.0/info:eu-repo/semantics/openAccessAtribución-NoComercial-CompartirIgual 4.0 Internacional (CC BY-NC-SA 4.0)http://purl.org/coar/access_right/c_abf2https://www.sciencedirect.com/science/article/pii/S0304401722001881Identification of Babesia bovis MSA-1 functionally constraint regions capable of binding to bovine erythrocytesArtículo de revistahttp://purl.org/coar/resource_type/c_2df8fbb1info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionTexthttp://purl.org/redcol/resource_type/ARThttp://purl.org/coar/version/c_970fb48d4fbd8a85Babesia bovisBabesiosisParásitosPéptidosProteínas ProtozoariasAdhesión de parásitosEnfermedades del ganadoAgrosavia(Dic., 2022) Artículo número 10983481312Veterinary ParasitologyPublicationORIGINAL1-s2.0-S0304401722001881-main.pdf1-s2.0-S0304401722001881-main.pdfapplication/pdf2688297https://repository.udca.edu.co/bitstreams/660d8c5c-228e-48b0-8568-ba485d8bd918/downloadf623609716f348f030174578d760b74bMD51LICENSElicense.txtlicense.txttext/plain; charset=utf-814775https://repository.udca.edu.co/bitstreams/96aa81d4-0714-4a30-b6e2-fcebe4f930e3/downloadf661acf14bedbf9f5d13897a0387e751MD52TEXT1-s2.0-S0304401722001881-main.pdf.txt1-s2.0-S0304401722001881-main.pdf.txtExtracted texttext/plain51804https://repository.udca.edu.co/bitstreams/b44f1753-10fb-4ec6-b71e-02382f639ce0/download354a7001dacd48b5859f4df37d47419cMD53THUMBNAIL1-s2.0-S0304401722001881-main.pdf.jpg1-s2.0-S0304401722001881-main.pdf.jpgGenerated 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en las Obras Colectivas;
b.	Distribuir copias o fonogramas de las Obras, exhibirlas públicamente, ejecutarlas públicamente y/o ponerlas a disposición pública, incluyéndolas como incorporadas en Obras Colectivas, según corresponda;
c.	Distribuir copias de las Obras Derivadas que se generen, exhibirlas públicamente, ejecutarlas públicamente y/o ponerlas a disposición pública.
Los derechos mencionados anteriormente pueden ser ejercidos en todos los medios y formatos, actualmente conocidos o que se inventen en el futuro. Los derechos antes mencionados incluyen el derecho a realizar dichas modificaciones en la medida que sean técnicamente necesarias para ejercer los derechos en otro medio o formatos, pero de otra manera usted no está autorizado para realizar obras derivadas.Todos los derechos no otorgados expresamente por el Licenciante quedan por este medio reservados, incluyendo pero sin limitarse a aquellos que se mencionan en las secciones 4(d) y 4(e).
4. Restricciones.
La licencia otorgada en la anterior Sección 3 está expresamente sujeta y limitada por las siguientes restricciones:
a.	Usted puede distribuir, exhibir públicamente, ejecutar públicamente, o poner a disposición pública la Obra sólo bajo las condiciones de esta Licencia, y Usted debe incluir una copia de esta licencia o del Identificador Universal de Recursos de la misma con cada copia de la Obra que distribuya, exhiba públicamente, ejecute públicamente o ponga a disposición pública. No es posible ofrecer o imponer ninguna condición sobre la Obra que altere o limite las condiciones de esta Licencia o el ejercicio de los derechos de los destinatarios otorgados en este documento. No es posible sublicenciar la Obra. Usted debe mantener intactos todos los avisos que hagan referencia a esta Licencia y a la cláusula de limitación de garantías. Usted no puede distribuir, exhibir públicamente, ejecutar públicamente, o poner a disposición pública la Obra con alguna medida tecnológica que controle el acceso o la utilización de ella de una forma que sea inconsistente con las condiciones de esta Licencia. Lo anterior se aplica a la Obra incorporada a una Obra Colectiva, pero esto no exige que la Obra Colectiva aparte de la obra misma quede sujeta a las condiciones de esta Licencia. Si Usted crea una Obra Colectiva, previo aviso de cualquier Licenciante debe, en la medida de lo posible, eliminar de la Obra Colectiva cualquier referencia a dicho Licenciante o al Autor Original, según lo solicitado por el Licenciante y conforme lo exige la cláusula 4(c).
b.	Usted no puede ejercer ninguno de los derechos que le han sido otorgados en la Sección 3 precedente de modo que estén principalmente destinados o directamente dirigidos a conseguir un provecho comercial o una compensación monetaria privada. El intercambio de la Obra por otras obras protegidas por derechos de autor, ya sea a través de un sistema para compartir archivos digitales (digital file-sharing) o de cualquier otra manera no será considerado como estar destinado principalmente o dirigido directamente a conseguir un provecho comercial o una compensación monetaria privada, siempre que no se realice un pago mediante una compensación monetaria en relación con el intercambio de obras protegidas por el derecho de autor.
c.	Si usted distribuye, exhibe públicamente, ejecuta públicamente o ejecuta públicamente en forma digital la Obra o cualquier Obra Derivada u Obra Colectiva, Usted debe mantener intacta toda la información de derecho de autor de la Obra y proporcionar, de forma razonable según el medio o manera que Usted esté utilizando: (i) el nombre del Autor Original si está provisto (o seudónimo, si fuere aplicable), y/o (ii) el nombre de la parte o las partes que el Autor Original y/o el Licenciante hubieren designado para la atribución (v.g., un instituto patrocinador, editorial, publicación) en la información de los derechos de autor del Licenciante, términos de servicios o de otras formas razonables; el título de la Obra si está provisto; en la medida de lo razonablemente factible y, si está provisto, el Identificador Uniforme de Recursos (Uniform Resource Identifier) que el Licenciante especifica para ser asociado con la Obra, salvo que tal URI no se refiera a la nota sobre los derechos de autor o a la información sobre el licenciamiento de la Obra; y en el caso de una Obra Derivada, atribuir el crédito identificando el uso de la Obra en la Obra Derivada (v.g., "Traducción Francesa de la Obra del Autor Original," o "Guión Cinematográfico basado en la Obra original del Autor Original"). Tal crédito puede ser implementado de cualquier forma razonable; en el caso, sin embargo, de Obras Derivadas u Obras Colectivas, tal crédito aparecerá, como mínimo, donde aparece el crédito de cualquier otro autor comparable y de una manera, al menos, tan destacada como el crédito de otro autor comparable.
d.	Para evitar toda confusión, el Licenciante aclara que, cuando la obra es una composición musical:
i.	Regalías por interpretación y ejecución bajo licencias generales. El Licenciante se reserva el derecho exclusivo de autorizar la ejecución pública o la ejecución pública digital de la obra y de recolectar, sea individualmente o a través de una sociedad de gestión colectiva de derechos de autor y derechos conexos (por ejemplo, SAYCO), las regalías por la ejecución pública o por la ejecución pública digital de la obra (por ejemplo Webcast) licenciada bajo licencias generales, si la interpretación o ejecución de la obra está primordialmente orientada por o dirigida a la obtención de una ventaja comercial o una compensación monetaria privada.
ii.	Regalías por Fonogramas. El Licenciante se reserva el derecho exclusivo de recolectar, individualmente o a través de una sociedad de gestión colectiva de derechos de autor y derechos conexos (por ejemplo, SAYCO), una agencia de derechos musicales o algún agente designado, las regalías por cualquier fonograma que Usted cree a partir de la obra (“versión cover”) y distribuya, en los términos del régimen de derechos de autor, si la creación o distribución de esa versión cover está primordialmente destinada o dirigida a obtener una ventaja comercial o una compensación monetaria privada.
e.	Gestión de Derechos de Autor sobre Interpretaciones y Ejecuciones Digitales (WebCasting). Para evitar toda confusión, el Licenciante aclara que, cuando la obra sea un fonograma, el Licenciante se reserva el derecho exclusivo de autorizar la ejecución pública digital de la obra (por ejemplo, webcast) y de recolectar, individualmente o a través de una sociedad de gestión colectiva de derechos de autor y derechos conexos (por ejemplo, Acinpro), las regalías por la ejecución pública digital de la obra (por ejemplo, webcast), sujeta a las disposiciones aplicables del régimen de Derecho de Autor, si esta ejecución pública digital está primordialmente dirigida a obtener una ventaja comercial o una compensación monetaria privada.
5. Representaciones, Garantías y Limitaciones de Responsabilidad.
A MENOS QUE LAS PARTES LO ACORDARAN DE OTRA FORMA POR ESCRITO, EL LICENCIANTE OFRECE LA OBRA (EN EL ESTADO EN EL QUE SE ENCUENTRA) “TAL CUAL”, SIN BRINDAR GARANTÍAS DE CLASE ALGUNA RESPECTO DE LA OBRA, YA SEA EXPRESA, IMPLÍCITA, LEGAL O CUALQUIERA OTRA, INCLUYENDO, SIN LIMITARSE A ELLAS, GARANTÍAS DE TITULARIDAD, COMERCIABILIDAD, ADAPTABILIDAD O ADECUACIÓN A PROPÓSITO DETERMINADO, AUSENCIA DE INFRACCIÓN, DE AUSENCIA DE DEFECTOS LATENTES O DE OTRO TIPO, O LA PRESENCIA O AUSENCIA DE ERRORES, SEAN O NO DESCUBRIBLES (PUEDAN O NO SER ESTOS DESCUBIERTOS). ALGUNAS JURISDICCIONES NO PERMITEN LA EXCLUSIÓN DE GARANTÍAS IMPLÍCITAS, EN CUYO CASO ESTA EXCLUSIÓN PUEDE NO APLICARSE A USTED.
6. Limitación de responsabilidad.
A MENOS QUE LO EXIJA EXPRESAMENTE LA LEY APLICABLE, EL LICENCIANTE NO SERÁ RESPONSABLE ANTE USTED POR DAÑO ALGUNO, SEA POR RESPONSABILIDAD EXTRACONTRACTUAL, PRECONTRACTUAL O CONTRACTUAL, OBJETIVA O SUBJETIVA, SE TRATE DE DAÑOS MORALES O PATRIMONIALES, DIRECTOS O INDIRECTOS, PREVISTOS O IMPREVISTOS PRODUCIDOS POR EL USO DE ESTA LICENCIA O DE LA OBRA, AUN CUANDO EL LICENCIANTE HAYA SIDO ADVERTIDO DE LA POSIBILIDAD DE DICHOS DAÑOS. ALGUNAS LEYES NO PERMITEN LA EXCLUSIÓN DE CIERTA RESPONSABILIDAD, EN CUYO CASO ESTA EXCLUSIÓN PUEDE NO APLICARSE A USTED.
7. Término.
a.	Esta Licencia y los derechos otorgados en virtud de ella terminarán automáticamente si Usted infringe alguna condición establecida en ella. Sin embargo, los individuos o entidades que han recibido Obras Derivadas o Colectivas de Usted de conformidad con esta Licencia, no verán terminadas sus licencias, siempre que estos individuos o entidades sigan cumpliendo íntegramente las condiciones de estas licencias. Las Secciones 1, 2, 5, 6, 7, y 8 subsistirán a cualquier terminación de esta Licencia.
b.	Sujeta a las condiciones y términos anteriores, la licencia otorgada aquí es perpetua (durante el período de vigencia de los derechos de autor de la obra). No obstante lo anterior, el Licenciante se reserva el derecho a publicar y/o estrenar la Obra bajo condiciones de licencia diferentes o a dejar de distribuirla en los términos de esta Licencia en cualquier momento; en el entendido, sin embargo, que esa elección no servirá para revocar esta licencia o que deba ser otorgada , bajo los términos de esta licencia), y esta licencia continuará en pleno vigor y efecto a menos que sea terminada como se expresa atrás. La Licencia revocada continuará siendo plenamente vigente y efectiva si no se le da término en las condiciones indicadas anteriormente.
8. Varios.
a.	Cada vez que Usted distribuya o ponga a disposición pública la Obra o una Obra Colectiva, el Licenciante ofrecerá al destinatario una licencia en los mismos términos y condiciones que la licencia otorgada a Usted bajo esta Licencia.
b.	Si alguna disposición de esta Licencia resulta invalidada o no exigible, según la legislación vigente, esto no afectará ni la validez ni la aplicabilidad del resto de condiciones de esta Licencia y, sin acción adicional por parte de los sujetos de este acuerdo, aquélla se entenderá reformada lo mínimo necesario para hacer que dicha disposición sea válida y exigible.
c.	Ningún término o disposición de esta Licencia se estimará renunciada y ninguna violación de ella será consentida a menos que esa renuncia o consentimiento sea otorgado por escrito y firmado por la parte que renuncie o consienta.
d.	Esta Licencia refleja el acuerdo pleno entre las partes respecto a la Obra aquí licenciada. No hay arreglos, acuerdos o declaraciones respecto a la Obra que no estén especificados en este documento. El Licenciante no se verá limitado por ninguna disposición adicional que pueda surgir en alguna comunicación emanada de Usted. Esta Licencia no puede ser modificada sin el consentimiento mutuo por escrito del Licenciante y Usted.

