Zebrafish embryo sensitivity test as in vivo platform to anti-Shiga toxin compound screening.

Shiga toxin-producing Escherichia coli (STEC) pathotype secretes two types of AB5 cytotoxins (Stx1 and Stx2), responsible for complications such as hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) in infected patients, which could lead to sequels and death. Currently, there is no effective treatment against the cytotoxic effect of these toxins. However, in order to approve any therapy molecule, an animal experiment is required in order to evaluate the efficacy and safety of therapeutic approaches. The use of alternative small host models is growing among human infectious disease studies, particularly the vertebrate zebrafish model, since relevant results have been described for pathogen-host interaction. In this sense, the present work aimed to analyze the toxic effect of Shiga toxins in zebrafish embryo model in order to standardize this method in the future to be used as a fast, simple, and efficient methodology for the screening of therapeutic molecules. Herein, we demonstrated that the embryos were sensitive in a dose-dependent manner to both Stx toxins, with LD50 of 22 µg/mL for Stx1 and 33 µg/mL for Stx2, and the use of anti-Stx polyclonal antibody abolished the toxic effect. Therefore, this methodology can be a rapid alternative method for selecting promising compounds against Stx toxins, such as recombinant antibodies.

Morphological and molecular aspects of sclerotial development in the phytopathogenic fungus Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum (Lib.) de Bary produces a resistance structure called sclerotium, which guarantees its survival in soil for long periods. Morphological and melanization aspects during sclerotial development were evaluated by microscopy and qRT-PCR techniques. S. sclerotiorum produces sclerotia with different phases of maturation and melanization during growth in PDA medium. Using scanning electron microscopy we observed that there are no structural differences in the three stages of formation of melanized and non-melanized sclerotium. Through histochemical analysis we observed that the melanized sclerotium accumulates more glycogen and produces less protein than non-melanized sclerotia. Melanin was most commonly found in the rind of melanized sclerotia, and the highest concentration of lipofucsins was found in non-melanized sclerotia. These molecules are products of the lipid peroxidation pathway and are associated with oxidative stress during differentiation in fungi. The expression of histidine kinase (shk) and adenylate cyclase (sac) genes in melanized and non-melanized sclerotiawere also evaluated. The higher gene expression of shk and lesser expression of sac in non-melanized sclerotiais an indication of the participation of cell signaling in the development of these structures. The higher expression of polyketide synthase (pks), tyrosinase (tyr) and laccase (lac) in non-melanized sclerotia suggested that S. sclerotiorum can use the DHN and L-dopa pathways to produce melanin. Expression studies of the enzymes chitin synthase and glucan synthase suggest that this process occurs along with the formation of melanin. This is interesting since polysaccharides, such as chitin and ß-1,3-glucan, serve as a scaffold to which the melanin granules are cross-linked.