Caracterização molecular e análise da expressão gênica de adesivas na levedura emergente Trichosporon asahii

Abstract

The Trichosporon genus is classified within the Basidiomycota phylum and comprehends 12 species with Trichosporon asahii being the most clinically relevant species, causing invasive infections with mortality 80%. This is due not only to the patient's immune status, but also, to treatment failure and biofilm formation on implanted materials (catheters), which increase the resistance against antifungals. The biofilm formation depends on proteins expressed in the cell surface, called adhesins, which mediate cell-environment interactions, knowingly as important virulence factors, which are totally uncharacterized in Trichosporon spp.. Thus, the present work aims to characterize putative genes encoding proteins with potential adhesion function. For this, we searched (tBLASTn-NCBI) for hypothetical adhesin genes in the reference genome of T. asahii CBS2479, deposited in a genomic in public databases, from 23 well-known adhesins of Candida albicans and Cryptococcus neoformans. In addition, its complete proteome was analyzed through adhesin prediction programs (FungalRV and Faapred). From the CBS prediction server, signal peptide prediction, secretion pathway, presence of transmembrane helices, glycosylations, mannosylations and acetylations were performed. After this selection, total DNAs was extracted from four isolates of T. asahii (CBS2479, CBS7631, L2585 and L773) and conventional PCR, to verify the existence of the complete ORFs. Then, total RNA extraction from the four planktonically cultivated isolates was performed for expression analysis by RT-PCR. As results, by tBLASTn only 1 protein was found with 30% identity and 85% CFL1p similarity to C. neoformans. Through FungalRV and Faapred, 16 sequences were predicted in common as adhesins, being selected 17 by the 3 programs of analysis. After applying other inclusion criteria such as: protein size (≥300 amino acids), presence of conserved regions indicating adhesion function, presence of signal peptide and absence of transmembrane helices, 4 genes were selected for our study. All selected putative genes possess protein chain predicted with more than 300 amino acids, signal peptide, absence of transmembrane helices and are secreted by the classical secretory pathway. The first one was named CFL1p-like protein and contains 325 amino acids, exhibits O-glycosylations on 4 serine/threonine residues (ser/thr), with no prediction for N-glycosylations, with presence of 14 residues of trioptophan (Trp) predicted with mannosylation and no prediction for N-acetylation. Beta-like protein contains 1383 amino acids with 463 possible residues of O-glycosylations and 21 sites predicted for N-glycosylations, showing no C-mannosylation and N-acetylation sites. The third putative protein was called Restin-like and contains 507 amino acids with 101 residues likely to receive O-glycosylation, without N-glycosylation sites and N-acetylation, but presented only one residue with C-mannosylation. The last selected protein contains 406 amino acids, with 65 residues with predicted O-glycosylation and 12 with N-glycosylation, without sites of C-mannosylation and N-acetylation. The four genes were amplified in the four isolates by conventional PCR, and it was verified by RT-PCR that all selected genes were expressed in the four T. asahii strains. Three of the four genes, MAR-like, RES- like and BETA-like were more expressed in the CBS2479 strain from skin mycosis, demonstrating that the isolation site may influence their expression in planktonic growth. It was not possible to relate expression levels to the biofilm formation capacity of blood culture isolates during planktonic growth. However, expression analyzes demonstrate that the gene CFL1-like gene was more expressed in blood cultures CBS7631 and L773 which had predominant morphology of artroconids, rather than those with filamentous morphology. This work also contributed to the genomic annotation of the four ORFs, changing their status from hypothetical to verified sequences, by identifying genes that may encode novel adhesins of the Basidiomycota phylum using as model, the emerging pathogen T. asahii.