Estudio de la interacción molecular huésped-patógeno utilizando el modelo insecto-hongo gallería mellonella-fusarium oxysporum, mediante la caracterización de genes, proteínas y péptidos de defensa provenientes de la respuesta humoral innata y del ataque fúngico

• Autores: Amalia Muñoz Gomez
• Directores de la Tesis: Carlos Alberto Peláez Jaramillo (dir. tes.)
• Lectura: En la Universidad de Antioquia ( Colombia ) en 2015
• Idioma: español
• Enlaces
  • Tesis en acceso abierto en: hdl.handle.net
• Resumen
  • español

    Se estudiaron aspectos involucrados en el ataque del hongo Fusarium oxysporum a larvas de Galleria mellonella (Lepidóptera), y el proceso de defensa que se desencadena en el sistema inmune innato del huésped. Los insectos responden al desafío de agentes microbianos o a lesiones, mediante la rápida y transitoria síntesis de potentes compuestos antimicrobianos. Numerosas proteínas del sistema inmune provenientes de insectos que inhiben microorganismos han sido identificadas caracterizadas y clonadas in vitro, la mayor parte de las cuales exhiben actividad antibacteriana, mientras que en otras se demostró que afectaban a hongos. Los genes más estudiados de F. oxysporum son aquellos que producen toxinas, y en el caso de G. mellonella, son muy pocos los genes identificados, provenientes de su respuesta inmune innata. Para ambas especies es aún muy somera la caracterización de sus respectivos proteomas. Para la comprensión de la relación huésped patógeno (insecto-hongo), las estrategias metodológicas seguidas incluyeron, la estandarización de la colonia de G. mellonella y del proceso de inoculación del hongo en larvas del insecto; la identificación de temperaturas óptimas de mantenimiento de las larvas y las concentraciones sub-letales de microconidias del hongo donde se produjo la mayor supervivencia de las larvas; el empleo de la tecnología iTRAQ (Isobaric tags for relative and absolute quantitation). Esta última permitió identificar y cuantificar las proteínas expresadas, además, mediante la utilización de ARN mensajero se identificaron genes expresados en condiciones deseadas. Finalmente, se llevó a cabo la identificación in vivo de células activas de las larvas atacadas por F. oxysporum, mediante microscopía láser confocal...

  • English

    In this work it was investigated different molecular and physiological aspects related with attack of pathogen Fusarium oxysporum (fungus) and the immune defense of Galleria mellonella (Lepidoptera). The challenge of insect is to reply the attack of microbial pathogens or make the improvements to the lesions, producing rapidly and transitory a large number of proteins and antimicrobial compounds. Many proteins and peptides of innate immune system from insect that inhibit microorganisms, were previously in vitro identified, characterized and cloned, most of them has antibacterial activity, while another ones are related with fungi defense. The most studied genes in F. oxysporum are the responsible of toxins. For the case of G. mellonella not so much genes were identified and the immune system genes are essentially poorly studied. Both organisms are poorly studied to the proteomics level.

    For the best comprehension of the relationship host-pathogen (insect-fungus), the methodological strategy was follow the next steps: G. mellonella colony culture standardization, the right procedure of injection of fungus microconidia into insect, the identification of the best temperature for the maintenance of larvae, the best sublethal microconidia concentration for producing more larvae survivors and the usefulness of iTRAQ technology (Isobaric Tags for Relative and Absolute Quantitation). This last technique allowed identify and quantify a large number of expressed proteins (up or down regulated), furthermore, isolating mRNA those proteins identified were correlated with gene expression under suitable conditions. Finally, It was carry out the in vivo identification of hemocoel active cells from larvae challenged F. oxysporum, by confocal microscopy.

    It was stablished by fungus radial grown, where F. oxysporum grows in correlation with temperature, exhibiting low grow at 37 that at 30°C. The larvae survive in best proportion to the challenge of microconidia from 104 to 106 microconidia/mL, when were incubated at 37°C, during 48 h post-infection, and present best surviving when were pre-inoculated with Latobacillus platarum. Also, it was observed cellular differentiation into hemolymph larvae with fungus- challenge. Using iTRAQ 8-plex were identify 59 proteins, which 20 belong to G. mellonella, 20 were identified using other lepidotera species and the remained were identified using other species from bacteria, protozoan and fungus.

    In conclusion, the standardization of breeding system, the handling and the injection of G. mellonella larvae with F. oxysporum microconidia were reliable and consistent, as well as the maintenance and culture of F. oxysporum was meticulous. The larvae survived with high microconidia/mL concentration, when the temperature incubation was 37°C, especially when the larvae were pre-inoculated with L. plantarum, before the fungus challenge. iTRAQ was a reliable method and consistent for detecting proteins related to innate immune system from G. mellonella in response to the pathogen F. oxysporum. In fact, iTRAQ as a regard of quantitative proteomics analytic method allowed quantify protein and and peptides, therefore allow to study their differential expression and improvement of the immune system from G. mellonella through the treatmet from the low to high of microconidia concentration and from the 25 to 37°C. Our proteomic study allowed following the improvement of immune response in G. mellonella against to fungus challenge at 37°C. In conclusion it was found that using 104 microconidia/mL at 37°C, over-expressed much more proteins than other treatments.