| Congresso Brasileiro de Microbiologia 2023 | Resumo: 476-2 | ||||
Resumo:Sporothrix brasiliensis is considered the most pathogenic species, responsible for the Brazilian cat-transmitted sporotrichosis hyperendemics. New data contributing to the understanding of this species interaction with the host immune effectors are needed. The close similarity between the immune response of Galleria mellonella - hemocytes - and the components of the mammalian innate immune response plus the vertebrate use ethical concerns, raised G. mellonella to the post of one of the most recognized biomodel of infection. In parallel, the use of fluorescent nanotechnology, such as labeled Quantum dots (QDs) has been expanding the possibilities of tracking pathogen-host interaction in different scales, facilitating the 3D reconstructions and fluorescence quantification. The aim of this work was to in vitro and in vivo investigate S. brasiliensis infection dynamics in the G. mellonella. To this purpose, histological analyzes was used to assess tissue damage in G. mellonella infected by this fungus. Three larvae infected with 1x107 yeast of S. brasiliensis were formalin-fixed after 24 and 120 hours of infection. Whole larvae were transferred to the same solution and after one week were ethanol dehydrated and paraffin embedded. Tissue sections of 3 μm were stained with hematoxylin-eosin and sections examined with a Leica DM500 light microscope (United States). Nanotechnology was designed for the synthesis of Silicon Quantum Dots (SiQD) to allow the internalization and marking of S. brasiliensis by fluorescent nanoparticles by using organosilane compounds [3-(2-aminoethylamino)propyl]trimethoxysilane (DAMO) as a silicon source, further stabilized with mercaptoacetic acid. Concanvalin A was conjugated with SiQDs to optimize internalization by the yeasts. The in vitro assay of SiQDs internalization began with five days of S. brasiliensis growth in Yeast Dextrose Peptone (YPD). Yeast suspensions (1x107) were incubated with diluted blue light emitting SiQD nanocrystals in distinct concentrations 1000, 500, 250, 125, 62,5 μg/mL, at 37°C. Toxicity was evaluated by daily cell counts in Neubauer chamber and later by colony forming units (CFU). Fluorescence detection was analyzed with 900x magnification fluorescent microscopy (Motic, China) after 24 and 120 hours of incubation. Whole insect inoculated with nanoparticles at the same concentrations were analyzed by micro-CT Scan (Skyscan 1275, Bruker, Belgium). G. mellonella sagittal sections allowed the visualization of external cuticle, internal organs, fat body, muscle fibers and numerous foci of melanization. S. brasiliensis triggered a rapid biomodel response, with hemocytes recruitment forming melanized nodules-trapping yeast cells. SiQDs at 1000 μg/mL and 500 μg/mL effectively labelled S. brasiliensis yeasts while did not affect yeast integrity demonstrating that this nanoparticle can act as physiological fluorescent markers. Contrarily, yeast labelling was undetected by microtomography, a poor tool in G. mellonella-S. brasiliensis infection dynamics. The combination of routine methods such as histopathology with the innovative ones such as the use of fluorescent nanoparticles can add relevant data for the investigation of fungus-host dynamics in sporotrichosis. The unprecedented promissing application of nanotechnology on future imaging of G. mellonella-fungus interaction was demonstrated. Palavras-chave: Host-Parasite Interactions, Invertebrates, Sporotrichosis, Quantum dots, Zoonosis Agência de fomento:Conselho Nacional de Desenvolvimento Científico e Tecnológico (PIBIC-CNPq-UFF, Brazil); Fundação de Amparo à Pesquisa no Estado do Rio de Janeiro, Brasil (FAPERJ E-26/211.502/2021), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brasil | |||||