Purification of Trypanosoma cruzi metacyclic trypomastigotes by ion exchange chromatography in sepharose-DEAE, a novel methodology for host-pathogen interaction studies

Lissa Cruz-Saavedra, Marina Muñoz, Cielo León, Manuel Alfonso Patarroyo, Gabriela Arevalo, Paula Pavia, Gustavo Vallejo, Julio César Carranza, Juan David Ramírez

Resultado de la investigación: Contribución a RevistaArtículo

3 Citas (Scopus)

Resumen

Las tripomastigotes metacíclicas son esenciales para la comprensión de la biología de Trypanosoma cruzi, el agente de la enfermedad.
Enfermedad de Chagas. Sin embargo, la obtención de estas etapas biológicas en medio axénico es difícil. Técnicas basadas en
de carga y densidad del parásito durante las diferentes etapas, sin que se haya observado un alto grado de
eficacia en la purificación de tripomastigotes metacíclicos. Hasta ahora, no se ha implementado ningún protocolo en el que
sefarosa - DEAE se utiliza como resina. Por lo tanto, aquí probamos su capacidad para purificar tripomastigotes metacíclicos en
Infusión hepática Triptosa (LIT) en cultivos medianos. Un protocolo simple, fácil de ejecutar y eficaz basado en la tecnología de iones.
Se describe la cromatografía de intercambio en resina de Sefarado-DEAE para la purificación de tripomastigotes de T. cruzi.
Se utilizaron cepas de T. cruzi de las unidades de tipificación discreta (DTU) I y II. Las cepas fueron cosechadas en LIT
medio a una concentración de 1 × 107 epimastigotas/mL. Calculamos el tiempo de incremento de tripomastigotes
(TTI). A partir de los datos obtenidos, se realizó cromatografía de intercambio iónico con resina de sífilis DEAE. Para
verificar la pureza y viabilidad de las tripomastigotas, se realizó un cultivo en medio LIT con el consiguiente
verificación con tinción de giemsa. Evaluar si la técnica afectó la infectividad de los tripomastigotes, in vitro
se realizaron ensayos en células Vero e in vivo en ratones ICR-CD1. La técnica permitió la purificación de
tripomastigotes metacíclicos de otros estadios de T. cruzi en un porcentaje del 100%, se observó una mayor recuperación.
en cultivos de 12 días. Hubo diferencias con respecto a la recuperación de los tripomastigotes metacíclicos para ambos.
DTUs, siendo DTU TcI la que recuperó una mayor cantidad de estas formas. La técnica no afectó
infecciosidad del parásito in vitro y/o in vivo.
Idioma originalEnglish (US)
Páginas (desde-hasta)27-32
Número de páginas6
PublicaciónJournal of Microbiological Methods
Volumen142
DOI
EstadoE-pub ahead of print - sep 1 2017

Huella dactilar

Host-Pathogen Interactions
Trypanosoma cruzi
Ion Exchange Chromatography
Sepharose
Liver
Parasites
Inbred ICR Mouse
Vero Cells
Chagas Disease
Culture Media
Staining and Labeling
2-diethylaminoethanol
In Vitro Techniques

Citar esto

@article{f7f409587fe84104ba70bf0d09c981c5,
title = "Purification of Trypanosoma cruzi metacyclic trypomastigotes by ion exchange chromatography in sepharose-DEAE, a novel methodology for host-pathogen interaction studies",
abstract = "Metacyclic trypomastigotes are essential for the understanding of the biology of Trypanosoma cruzi, the agent of Chagas disease. However, obtaining these biological stages in axenic medium is difficult. Techniques based on charge and density of the parasite during different stages have been implemented, without showing a high efficiency in the purification of metacyclic trypomastigotes. So far, there is no protocol implemented where sepharose-DEAE is used as a resin. Therefore, herein we tested its ability to purify metacyclic trypomastigotes in Liver Infusion Triptose (LIT) medium cultures. A simple, easy-to-execute and effective protocol based on ion exchange chromatography on Sepharose-DEAE resin for the purification of T. cruzi trypomastigotes is described. T. cruzi strains from the Discrete Typing Units (DTUs) I and II were used. The strains were harvested in LIT medium at a concentration of 1×10(7)epimastigotes/mL. We calculated the time of trypomastigotes increment (TTI). Based on the data obtained, Ion exchange chromatography was performed with DEAE-sepharose resin. To verify the purity and viability of the trypomastigotes, a culture was carried out in LIT medium with subsequent verification with giemsa staining. To evaluate if the technique affected the infectivity of trypomastigotes, in vitro assays were performed in Vero cells and in vivo in ICR-CD1 mice. The technique allowed the purification of metacyclic trypomastigotes of other stages of T. cruzi in a percentage of 100{\%}, a greater recovery was observed in cultures of 12days. There were differences regarding the recovery of metacyclic trypomastigotes for both DTUs, being DTU TcI the one that recovered a greater amount of these forms. The technique did not affect parasite infectivity in vitro or/and in vivo.",
author = "Lissa Cruz-Saavedra and Marina Mu{\~n}oz and Cielo Le{\'o}n and Patarroyo, {Manuel Alfonso} and Gabriela Arevalo and Paula Pavia and Gustavo Vallejo and Carranza, {Julio C{\'e}sar} and Ram{\'i}rez, {Juan David}",
note = "Copyright {\circledC} 2017 Elsevier B.V. All rights reserved.",
year = "2017",
month = "9",
day = "1",
doi = "10.1016/j.mimet.2017.08.021",
language = "English (US)",
volume = "142",
pages = "27--32",
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Purification of Trypanosoma cruzi metacyclic trypomastigotes by ion exchange chromatography in sepharose-DEAE, a novel methodology for host-pathogen interaction studies. / Cruz-Saavedra, Lissa; Muñoz, Marina; León, Cielo; Patarroyo, Manuel Alfonso; Arevalo, Gabriela; Pavia, Paula; Vallejo, Gustavo; Carranza, Julio César; Ramírez, Juan David.

En: Journal of Microbiological Methods, Vol. 142, 01.09.2017, p. 27-32.

Resultado de la investigación: Contribución a RevistaArtículo

TY - JOUR

T1 - Purification of Trypanosoma cruzi metacyclic trypomastigotes by ion exchange chromatography in sepharose-DEAE, a novel methodology for host-pathogen interaction studies

AU - Cruz-Saavedra, Lissa

AU - Muñoz, Marina

AU - León, Cielo

AU - Patarroyo, Manuel Alfonso

AU - Arevalo, Gabriela

AU - Pavia, Paula

AU - Vallejo, Gustavo

AU - Carranza, Julio César

AU - Ramírez, Juan David

N1 - Copyright © 2017 Elsevier B.V. All rights reserved.

PY - 2017/9/1

Y1 - 2017/9/1

N2 - Metacyclic trypomastigotes are essential for the understanding of the biology of Trypanosoma cruzi, the agent of Chagas disease. However, obtaining these biological stages in axenic medium is difficult. Techniques based on charge and density of the parasite during different stages have been implemented, without showing a high efficiency in the purification of metacyclic trypomastigotes. So far, there is no protocol implemented where sepharose-DEAE is used as a resin. Therefore, herein we tested its ability to purify metacyclic trypomastigotes in Liver Infusion Triptose (LIT) medium cultures. A simple, easy-to-execute and effective protocol based on ion exchange chromatography on Sepharose-DEAE resin for the purification of T. cruzi trypomastigotes is described. T. cruzi strains from the Discrete Typing Units (DTUs) I and II were used. The strains were harvested in LIT medium at a concentration of 1×10(7)epimastigotes/mL. We calculated the time of trypomastigotes increment (TTI). Based on the data obtained, Ion exchange chromatography was performed with DEAE-sepharose resin. To verify the purity and viability of the trypomastigotes, a culture was carried out in LIT medium with subsequent verification with giemsa staining. To evaluate if the technique affected the infectivity of trypomastigotes, in vitro assays were performed in Vero cells and in vivo in ICR-CD1 mice. The technique allowed the purification of metacyclic trypomastigotes of other stages of T. cruzi in a percentage of 100%, a greater recovery was observed in cultures of 12days. There were differences regarding the recovery of metacyclic trypomastigotes for both DTUs, being DTU TcI the one that recovered a greater amount of these forms. The technique did not affect parasite infectivity in vitro or/and in vivo.

AB - Metacyclic trypomastigotes are essential for the understanding of the biology of Trypanosoma cruzi, the agent of Chagas disease. However, obtaining these biological stages in axenic medium is difficult. Techniques based on charge and density of the parasite during different stages have been implemented, without showing a high efficiency in the purification of metacyclic trypomastigotes. So far, there is no protocol implemented where sepharose-DEAE is used as a resin. Therefore, herein we tested its ability to purify metacyclic trypomastigotes in Liver Infusion Triptose (LIT) medium cultures. A simple, easy-to-execute and effective protocol based on ion exchange chromatography on Sepharose-DEAE resin for the purification of T. cruzi trypomastigotes is described. T. cruzi strains from the Discrete Typing Units (DTUs) I and II were used. The strains were harvested in LIT medium at a concentration of 1×10(7)epimastigotes/mL. We calculated the time of trypomastigotes increment (TTI). Based on the data obtained, Ion exchange chromatography was performed with DEAE-sepharose resin. To verify the purity and viability of the trypomastigotes, a culture was carried out in LIT medium with subsequent verification with giemsa staining. To evaluate if the technique affected the infectivity of trypomastigotes, in vitro assays were performed in Vero cells and in vivo in ICR-CD1 mice. The technique allowed the purification of metacyclic trypomastigotes of other stages of T. cruzi in a percentage of 100%, a greater recovery was observed in cultures of 12days. There were differences regarding the recovery of metacyclic trypomastigotes for both DTUs, being DTU TcI the one that recovered a greater amount of these forms. The technique did not affect parasite infectivity in vitro or/and in vivo.

U2 - 10.1016/j.mimet.2017.08.021

DO - 10.1016/j.mimet.2017.08.021

M3 - Article

C2 - 28865682

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JO - Journal of Microbiological Methods

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