Análisis proteico del patógeno acuícola flavobacterium psychrophilum cultivado en condiciones restrictivas de hierro
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2014
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Profesor/a Guía
Idioma
es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
Flavobacterium psychrophilum es una bacteria patógena de peces cultivados en agua
dulce. En su proceso de infección, la bacteria se ve enfrentada al crecimiento en un medio
restrictivo de hierro, bioelemento que resulta ser imprescindible para su desarrollo y óptimo crecimiento. Las proteínas presentes en la superficie de la bacteria y los
mecanismos en que éstas participan (como la captación de hierro), le podría permitir el
inicio del proceso infeccioso. El objetivo de esta tesis fue identificar y seleccionar
componentes proteicos de la célula completa de aislados chilenos de F. psychrophilum
cultivados en condiciones con y sin restricción de hierro para determinar su implicancia en
la respuesta inmune humoral de trucha arcoíris. Los análisis de SDS-PAGE y 20-PAGE
establecen que cuando las cepas son cultivadas en condiciones normales de crecimiento,
se observa un patrón muy conservado entre los 7 aislados chilenos de F. psychrophilum y las otras 2 cepas americanas estudiadas. Sin embargo, al crecer estos aislados en
condiciones restrictivas de hierro, se observó la expresión de siete bandas diferenciales
(p107, p103, p92, p87, p62, p53 y p46), las cuales sólo se expresan en ésta condición de
crecimiento. Utilizando la técnica de MALDI-TOF, se identificó un total de doce proteínas,
de las cuales seis fueron proteínas inmunoreactivas al suero de trucha arcoíris. Éstas se
identificaron como la enzima fumarato hidratasa, el factor de elongación Tu, la proteína
valina deshidrogenasa, dos proteínas reconocidas como peroxiredoxinas y la proteína
difosfatasa inorgánica. De las otras cinco proteínas identificadas, dos corresponden a
proteínas con aumento en su expresión en condiciones normales de crecimiento (la
proteína Succinii-CoA sintetasa y el Factor de elongación Tu) y otras tres corresponden a
proteínas diferenciales expresadas en condiciones limitantes de hierro (proteína de
membrana externa OmpA (2) y la enzima Adhesina SprC). En conclusión, todos los
aislados de F. psychrophilum estudiados mostraron la expresión de bandas y proteínas
que fueron diferenciales para cada condición de crecimiento, estableciendo que no existe
un patrón específico de expresión al crecer en condiciones limitantes de hierro. Los
componentes de la célula completa que generan una respuesta inmune del tipo humoral
en trucha arcoíris, tampoco parecen estar directamente relacionados a la restricción de
hierro, ya que la mayoría de las proteínas inmunoreactivas que se identificaron,
corresponden a proteínas del aislado cultivado en condiciones normales de crecimiento.
Flavobacterium psychrophilum is a pathogenic bacteria in freshwater cultured fish. In process of infection, the bacteria growth is confronted to an iron restrictive medium, bioelement that result to be essential for optimal growth and development. The proteins present on the surface of the bacteria and the mechanisms in which they participate (as iron uptake ), it could enable them to initiate the infectious process. The aim of this thesis was to identify and select components of the whole cell protein of Chilean isolates of F. psychrophilum grown in conditions with and without iron restriction to determine their implication in the humoral immune response of rainbow trout. The SDS-PAGE and 20- PAGE analysis establish that when strains are grown in normal growth conditions, there is a conserved pattern between the 7 F. psychrophilum Chilean isolates and the other 2 American strains studied. However, when the isolates grow in iron restrictive conditions, we observed differential expression of seven bands (p107, p103, p92, p87, p62, p53 and p46), which are expressed only in this condition . Using the MALDI -TOF technique, we identified a total of eleven proteins, of which six were immunoreactive proteins with rainbow trout serum. They were identified as the enzyme fumarate hydratase, elongation factor Tu, valine dehydrogenase protein, two proteins known as protein peroxiredoxins and an inorganic diphosphatase. For the others five identified proteins, two of them are proteins that show increased protein expression in normal growth conditions (protein succinyl -CoA synthetase and Elongation Factor Tu ) and three other differential proteins are expressed in iron limiting conditions (outer membrane protein OmpA (2) and Andhesine SprC). In conclusion, all isolates of F. psychrophilum studied showed the expression of differential bands and proteins for each grow condition, establishing that there is not an specific pattern of expression by growing in iron limiting conditions. The complete cell components that generate an humoral immune response in rainbow trout, doesn 't seem to be directly related to iron restriction, as most immunoreactive proteins that were identified, were proteins from the isolate grown under normal growth conditions.
Flavobacterium psychrophilum is a pathogenic bacteria in freshwater cultured fish. In process of infection, the bacteria growth is confronted to an iron restrictive medium, bioelement that result to be essential for optimal growth and development. The proteins present on the surface of the bacteria and the mechanisms in which they participate (as iron uptake ), it could enable them to initiate the infectious process. The aim of this thesis was to identify and select components of the whole cell protein of Chilean isolates of F. psychrophilum grown in conditions with and without iron restriction to determine their implication in the humoral immune response of rainbow trout. The SDS-PAGE and 20- PAGE analysis establish that when strains are grown in normal growth conditions, there is a conserved pattern between the 7 F. psychrophilum Chilean isolates and the other 2 American strains studied. However, when the isolates grow in iron restrictive conditions, we observed differential expression of seven bands (p107, p103, p92, p87, p62, p53 and p46), which are expressed only in this condition . Using the MALDI -TOF technique, we identified a total of eleven proteins, of which six were immunoreactive proteins with rainbow trout serum. They were identified as the enzyme fumarate hydratase, elongation factor Tu, valine dehydrogenase protein, two proteins known as protein peroxiredoxins and an inorganic diphosphatase. For the others five identified proteins, two of them are proteins that show increased protein expression in normal growth conditions (protein succinyl -CoA synthetase and Elongation Factor Tu ) and three other differential proteins are expressed in iron limiting conditions (outer membrane protein OmpA (2) and Andhesine SprC). In conclusion, all isolates of F. psychrophilum studied showed the expression of differential bands and proteins for each grow condition, establishing that there is not an specific pattern of expression by growing in iron limiting conditions. The complete cell components that generate an humoral immune response in rainbow trout, doesn 't seem to be directly related to iron restriction, as most immunoreactive proteins that were identified, were proteins from the isolate grown under normal growth conditions.
Notas
Tesis (Magíster en Biotecnología) Proyecto FONDECYT 1110219
Palabras clave
Piscicultura, Chile, Bacterias|, Aislamiento y Purificación