Identificação de atividade metalo-oligopeptidásica Thimet-like em Paracoccidioides brasiliensis: um novo fator de patogenicidade fúngica?

• Autor(a) principal: Gravi, Ellen Tihe [UNIFESP]
• Data de Publicação: 2010
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: Repositório Institucional da UNIFESP
• Texto Completo: http://repositorio.unifesp.br/handle/11600/9696
• topic: Patogenicidade; Peptidase intermediária de mitocôndria; Saccharolisina; Thimet oligopeptidase; Paracoccidioides brasiliensis

Paracoccidioidomycosis (PCM), caused by the pathogenic fungus Paracoccidioides brasiliensis (Pb) is a systemic mycosis with severe acute and chronic forms. Proteases or peptidases are proteolytic enzymes that occur in all organisms and constitute 1-5% of their genetic contents. These enzymes are involved in biological processes such as blood clotting, cell death and tissue differentiation. Several important proteolytic steps occur during the invasion of metastatic tumors, as well as in the infection of a large number of viruses and pathogens. To date, a small number of Pb proteases were isolated and characterized, also, their activities during the development of the disease was not determined, and an oligopeptidase activity was not detected in this fungus. In the present work, we demonstrated a metallopeptidase thimet oligopeptidase (TOP)-like activity in the cytosolic extract of Pb18 yeasts. Our results shown a major hydrolysis of the fluorescence resonance energy transfer (FRET) peptide Abz-GFSPFRQ-EDDnp, preferentially cleaved by TOP from mammals, and the inhibition of the hydrolysis of this peptide by orthophenantrolin and JA-2, selective inhibitors of metalloproteases and TOP, respectively. The presence of neurolysin- like and neprilysin-like, serinepeptidases, cysteine-peptidases and angiotensin converting enzyme I was discarded by analyzing selective FRET peptides and inhibitors. The higher peptidase activity of cytosolic extracts over the membrane/cell wall and total yeast lysate preparations may indicate that this enzyme is localized in the yeast cytosol. The metallo-oligopeptidase activity was not detected on in vitro culture supernatants, even after addition of fetal calf serum. However, the peptidase with TOP-like activity of P. brasiliensis seems to be secreted in vivo, or released after fungal lysis by immune factors, since antibodies that can inhibit this enzymatic activity were found in sera from paracoccidioidomycosis patients, and serum with highest titer in immunodiffusion contains higher concentrations of enzymespecific antibodies. Bradykinin, an important inflammatory mediator in vivo, is cleaved by several enzymes from the M3 family. The same fragments observed after hydrolysis by TOP were observed after cytosolic extract hydrolysis of bradykinin and the substrate Abz-GFSPFRQ-EDDnp. MIP and bacterial OpdA hydrolysis of these peptides generate different fragments, and this is an additional indicator of a major TOP-like activity in P. brasiliensis yeast cells Bradykinin hydrolysis by the TOP-like metallopeptidase of P. brasiliensis may occur in inflammatory processes and this suggests that the enzyme may be involved in the inhibition of a protective anti-fungal response induction, limiting fungal elimination. We also observed that the expression of the TOP homologous gene in P. brasiliensis has almost a two-fold increased in the virulent isolate 18 compared to the non-virulent isolate. Increased hydrolysis of the substrate Abz-GFSPFRQ-EDDnp was also observed in the most virulent isolate compared to the non-virulent. The possible correlation between TOP-like peptidase expression and fungal virulence suggests that this peptidase could be classified as a fungal virulence factor, however, additional experiments are needed to confirm this hypothesis. Gp43 expression was also analyzed in both isolates, and it was observed a thirteen-fold increase in the expression on the virulent isolate. In order to better characterize the P. brasiliensis TOP-like activity, we attempted to obtain the purified recombinant or the native protein, isolated from fungal lysate. However, we were not successful in the expression of recombinant proteins and neither on the isolation of the native protein using chromatographic methods. Our results suggest the presence of a TOP-like activity in the cytosolic fraction of P. brasiliensis yeasts. In vivo release of this enzyme after fungal lysis, or host factors-stimulated secretion, may have a role in inflammation and development of the disease.