Effect of N-glycosylation inhibition on the transcriptional and proteomic profiles of Paracoccidioides brasiliensis

Abstract

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, the most prevalent systemic mycosis in Latin America. Fungus can able to increase their pathogenicity by factors as lipids, polysaccharides and glycoproteins. Glycosylation is important for protein secretion, function, stability and immunogenicity. N-glycans linked to proteins have been characterized in many species of yeast to mammals. In P. brasiliensis yeast, N-glycans play important role in the activity of N-acetyl-beta-D-glucosaminidase (NAGase) produced by yeast, and are directly involved in fungal growth and morphogenesis. N-glycans also exert considerable influence on the regulation of expression of NAGase, ²-1,3-glucanase, ±-1,4-amylase and other enzymes related to biosynthesis/degradation of the P. brasiliensis cell wall. Due the fact of N-glycans influence on the biological activities of P. brasiliensis proteins, have motivated us to investigate the effects of inhibition of N-glycosylation on the transcriptome and proteome of yeast. Therefore, we propose to do the following aims: (1) evaluate the effect of inhibition of N-glycosylation (provided by using tunicamycin) on the transcriptional profile of P. brasiliensis by HSS (Subtractive Hybridization deleted), (2) validate the differentially expressed genes by real-time PCR (RT-qPCR), (3) evaluate the inhibition effect of N-glycosylation on the proteomic profile of P. brasiliensis by 2D-DIGE (two-dimensional - Differential Gel Electrophoresis). Our expectation is that the achievement of these aims will allow the elucidation of the existence of a direct association between the N-glycosylation and regulation of genes that coding to proteins involved on growth, morphogenesis and pathogenicity of P. brasiliensis. (AU)