Análise da localização, da expressão gênica e predição estrutural de adesinas hipotéticas no patógeno emergente Trichosporon asahii

Abstract

Trichosporon spp. belong to the Basidiomycota phylum and currently possess 12 species, being Trichosporon asahii the most clinically relevant species, causing invasive infections, with mortality rate ranging up to 80%, depending on the patient's immune status, the ability of the fungus to grow in different morphologies and the limited susceptibility to antifungal agents, as well as its ability to form biofilms. Biofilm formation depends on the expression of adhesins that mediate cell-environment interactions in the different morphologies of the fungus.The aim of this work was to verify if proteins with predictive function of adhesins are expressed in yeast and hyphae of T. asahii and if they are found in the cellular surface of this fungus, as well as to analyze the three-dimensional models of these adhesins. For this, isogenic 129 / Sv mice were inoculated with total yeast and hyphae protein extracts for the generation of polyclonal antisera that were evaluated for titration by enzyme-linked immunosorbent assay (ELISA), showing that there was a difference in the amount of antibodies raised against the total proteins of yeasts and hyphae and that they differentially recognized the opposite protein extracts to which they were produced in significantly way (p <0.05), and that the antibodies generated against hyphae proteins have greater binding strength. Polyclonal antisera were used in immunofluorescence microscopy assays, both against yeasts and hyphae, showing recognition of the different morphologies in a similar way and independent of the cells having undergone permeabilization treatment or not. In addition, four proteins previously characterized in silico by our group as potential adhesins (Restin-like, CFL1-like, Mar-like and Beta-like) had their tertiary protein structure modeled on the I-TASSER server. Among five 3D probabilistic models generated for each protein, the one that presented the highest confidence value (C-score) was chosen. Thereafter, the amino acid sequences of the proteins were analyzed in the TepiTool and NetMHCII softwares to identify peptides recognized by human class II MHC and Murine H2 alleles. The peptides were analyzed in the VaxiJen v.2.0 software to verify their immunogenicity and it was used the cutoff value > 0.5. Thus, the peptides of each protein were located in the 3D structure of each protein using the PyMol 3.1 software. Finally, those that were on the surface of each protein model and close to the N-terminus region were selected. These peptides were synthesized by commercial company and inoculated in mice for production of specific antiserum against each peptide. Through the polyclonal antibodies generated against each peptide, surface proteins were detected on the surface of yeast and hyphae of T. asahii. The Western blot protein recognition pilot assay did not provide definitive conclusions regarding the efficacy of adhesins recognition for antibodies raised against peptides. Gene expression analysis of the four potential adhesins in yeast and hyphae showed that all are synthesized to a greater degree in the hyphae of T. asahii. From the 3D structures of the four adhesins an in silico molecular docking assay was performed, showing that all four adhesins bind to human proteins that are the target of cellular adhesion of pathogens. This work contributed to the understanding of the molecular interaction between the emerging pathogen T. asahii and the tissues of its human host.