Bridging the gap to nontoxic fungal control

Doutoramento em Biologia - Instituto Superior de AgronomiaBlad is an edible, Lupinus cotyledon-derived 173 amino acid residue polypeptide which occurs naturally and undissociated as part of a soluble 210 kDa oligomer, hereby termed Bladcontaining oligomer (BCO). BCO is isolated during growth of Lupinus seedlings and is a breakdown product of β-conglutin catabolism. In this work, BCO inherent, potent and wide spectrum antifungal activity was demonstrated under in vitro and greenhouse conditions. Several field trials showed that BCO exhibits an equal or better performance than the conventional chemicals over a large variety of crops/fungal pathogens. BCO antifungal activity was also showed to extend to human pathogens, including both yeasts and filamentous fungi. In vitro tests demonstrated a higher efficacy on a molar basis when compared to the best chemical fungicides tested, with an absence of mammal toxicity or genotoxicity. In addition to the lectin and catalytic activities which allow BCO to target the fungal cell wall, this work revealed other highly complex and multitarget mode(s) of action. BCO enters fungal cells causing several morphological alterations and seems to essentially act through inhibition of metal ion homeostasis resulting in an apoptotic cell death. In an attempt to provide a structural characterization of Blad, data from electronic microscopy, dynamic light scattering, SAXS and X-ray crystallography were obtained from heterologous expressed Blad, however the results were not conclusive. Last but not least, this work also aimed at the development of a “super- Blad”: a fusion protein consisting of Blad and two selected antibacterial peptides, SP10-5 and Sub5. The resulting chimeras not only maintained a strong antibacterial activity but were also able to retain the ability to inhibit the growth of both yeast and filamentous fungi. In summary, the overall approach of this thesis considered phytopathogens and human pathogens as belonging to the same scientific area and therefore aimed at producing a uniform natural multitarget, step-stone antifungal solutionN/