Papel da galactosaminogalactana da ativação endotelial por Aspergillus fumigatus

Abstract

Aspergillus fumigatus is the main etiological agent of invasive aspergillosis and already known as an angioinvasive fungal pathogen. Upon contact with human umbilical vein endothelial cells (HUVECs), this fungus can induce cellular injury and a pro-thrombotic phenotype, characterized by the inflammatory cytokine release and tissue factor expression. Nevertheless, the possible pathogen molecules as well as endothelial cell pathways involved in these processes are still unknown. A. fumigatus hyphae have been shown to produce, both in vivo and in vitro, an extracellular matrix composed of galactomannan (GM), galactosaminogalactan (GAG) and α-(1,3)-glucan. GM is a polysaccharide composed of galactofuranose (Galf) linked to a mannan chain, while GAG is a polymer composed of galactopyranose (Galp) and N-acetylgalactosamine residues. The disruption of the UGM1 gene in A. fumigatus leads to a mutant strain (∆ugm1) with a phenotype characterized by a higher GAG production, a lack of Galf residues in the cell wall and a hyperadhesive phenotype to epithelial cells and inert surfaces. Galactosaminogalactan is an essential adhesine of A. fumigatus, as well as an immunomodulatory molecule. Hence, the goal of this work was to investigate endothelial pathways involved in HUVEC response to A. fumigatus interaction and the role of GAG in this process. Therefore, we applied a label-free proteomic approach using High Definition Mass Spectrometry (2D nanoUPLC-HDMSE) to investigate the biological processes modulated by wild type strains, the ∆ugm1 mutant and the purified GAG of A. fumigatus in endothelial cells. Our data demonstrated that this pathogen is able to modulate HUVEC pathways related to immune response, angiogenesis, coagulation and homeostasis. In addition, the mutant presented a hyperadhesive phenotype to HUVECs, which was correspondent to an increased TNF-α release and tissue factor expression. In addition, the GAG alone induced a significant higher TNF-α secretion by HUVECs. Applying the 2D nanoUPLC-HDMSE strategy, we saw that the UGM1 mutant differently modulated HUVEC pathways related to immune response, inflammation and cell stress. Nevertheless, some of these pathways were also differentially modulated in HUVEC after interaction with the purified GAG. Finally, the TNF-α was predicted as the major upstream regulator cytokine involved in the HUVEC response to A. fumigatus strains and GAG. In conclusion, our data suggest that GAG has an important role in the modulation of endothelial pathways involved in the immune response, inflammation, blood coagulation and response to cell stress, and that these processes seems to be dependent on TNF-α secretion.