Induction, Isolation and Biological function of Excreted/Secreted Products released by entomopathogenic nematode Heterorhabditis bacteriophora.

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Entomopathogenic nematodes (EPNs) are a type of parasitic nematode characterized by the development in insect hosts and symbiotic relationship with specific bacterial species. The EPN Heterorhabditis bacteriophora has a symbiotic association with bacterium Photorhabdus luminesces. During the infection of the host, the EPNs produce a variety of molecules known as Excreted/Secreted Products (ESPs) that have been described with immunomodulatory functions. Although EPNs only infect insects, humans and insects share analogous immune reactions of the innate immune system. Characterized ESPs from H. bacteriophora have the capacity to down-regulate the production of antimicrobial peptides (AMPs), induced by insect host hemolymph during infection. In addition, P. luminesces produces lectins that decrease the production of reactive oxygen species (ROS) in human blood phagocytes. In this study, we optimize the production process of H. bacteriophora ESPs in infective juveniles (IJs), by getting rid of contamination and using a variety of materials that induce their production. The different activating materials used come from a variety of compounds from insect host Galleria mellonella, which we name as homogenate. Furthermore, we isolated these ESPs and analyzed their immunomodulatory functions in insects and humans. We confirmed that the contamination found in the production of ESPs was due to Gram-positive and Gram-negative bacteria, and that this contamination could be eliminated with kanamycin (50 mg/ml) and streptomycin/penicillin (10,000 U/ml). Moreover, the use of these antibiotics showed an increase in the protein concentration of ESPs. We did not detect any increase in the protein concentration of ESPs, by the addition or removal of 0,01 % NaClO, nor with activation or secretion times selected in this study. An increase in the protein concentration of ESPs was observed when using as activating material concentrated heat-inactivated homogenate. We analysed the effect of ESPs over the antimicrobial activity of hemolymph from G. mellonella, by viability of bioluminescent Escherichia coli. We could not conclude if ESPs impede the antimicrobial activity of hemolymph or served as nutrients for the bacteria, due to lack of statistical significance. Lastly, we decided to measure the effect of ESPs over the production of ROS in human blood, concluding that collected ESPs do not significantly affect this type of immune response. This study benefits the ongoing research of bioactive ESPs over the areas of pharmacology, biocontrol and human medicine.

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