| Congresso Brasileiro de Microbiologia 2023 | Resumo: 415-1 | ||||
Resumo:Histoplasma capsulatum (Hc) is a dimorphic fungus with a global distribution and the etiological agent of histoplasmosis. The disease occurs in immunocompetent and immunocompromised patients, with a high mortality rate for individuals in the latter group, especially those who do not receive treatment. Our group has been investigating the involvement of fungal extracellular vesicles (EVs) in fungal pathogenesis as EVs are an alternative secretion mechanism used by fungal organisms to release a broad variety of biomolecules, some of which are related do virulence. One of the major knowledge gaps in the biological role of EVs is our lack of understanding how these compartments traverse the cell wall, as well as our inability to inhibit the secretion of EVs, as there are currently no known molecules that inhibit fungal EV secretion directly. Previous works of our group demonstrated that there are enzymes associated with the hydrolysis of cell wall structures among the molecules transported by these compartments, such as glucanases, mannosidases, proteases and chitinases. The latter were found in high quantities in EVs produce by Hc and seem to be intimately involved in the cell wall remodeling process, which is essential for fungal growth and survival. The presence of enzymes in the EVs cargo, especially chitinases, suggests that this vesicular content could be involved in the mechanism by which EVs pass through the fungal cell wall. Our research aims to investigate this phenomenon using caffeine, a methylxanthine, which inhibits chitinases. Using a combination of fluorescence microscopy and a fluorogenic substrate to chitinase (4-MU-(GlcNAc)3) we were able to confirm the enhanced concentration of chitinase in Hc EVs compared to other fungal species. We were also able to demonstrate caffeine chitinolytic inhibition as well as to determine the optimal parameters for EV isolation and subsequent comparison. In parallel, caffeine antifungal activity was assessed with Bioscreen growth curves and scanning electron microscopy to define fungistatic and -cidal concentrations. Hc were cultivated without (control) or with sub-inhibitory concentrations of caffeine. EV isolation was performed followed by Nanotracking analysis and transmission electron microscopy. EVs from the treated conditions were similar morphologically to control EVs; however, there was a ~50% reduction of EVs produced by Hc treated with caffeine compared to control EVs, corroborating the hypothesis that chitinase plays a role in EV liberation. Finally, taking advantage of what it is the first confirmed pharmacological fungal EV release inhibitor, we aimed to further assess the role Hc EVs plays in pathogenesis using a G. mellonella infection model. Our results demonstrated that caffeine treatment significantly reduced the virulence of Hc, but the addition of EVs isolated from the control group restored virulence. Taken together, our results demonstrate that inhibition of chitinase by caffeine has a direct impact on the release of Hc EVs and, although more research in this area is needed, our in vivo model suggests that the ability to secrete EVs is directly correlated to virulence in histoplasmosis and that EV release inhibition can be explored to further investigate the biological role of fungal EVs in pathogenesis. Palavras-chave: Caffeine, Extracellular Vesicles, H. capsulatum, Histoplasmosis Agência de fomento:CAPES, CNPq, FAPERJ, NIH | |||||