RESUMEN
Paracoccidioidomycosis (PCM), caused by the pathogenic fungus Paracoccidioides brasiliensis, is a systemic mycosis with severe acute and chronic forms. The pathology of PCM is not completely understood, and the role of proteases in the infection is not clearly defined. In this report, we describe a metallopeptidase activity in P. brasiliensis total and cytosolic protein extracts similar to that of mammalian thimet oligopeptidase (TOP). The analogous enzyme was suggested by analysis of P. brasiliensis genome databank and by hydrolytic activity of the FRET peptide Abz-GFSPFRQ-EDDnp which was completely inhibited by o-phenanthrolin and significantly inhibited by the TOP inhibitor, JA-2. This activity was also partially inhibited by IgG purified from patients with PCM, but not from normal individuals. As shown by high-performance liquid chromatography (HPLC), the hydrolysis of bradykinin had the same pattern as that of mammalian TOP, and anti-mammalian TOP antibodies significantly inhibited fungal cytosolic peptidase activity. Moreover, anti-mammalian TOP antibodies recognized a component of 80 kDa on fungal cytosol. A P. brasiliensis virulent isolate showed higher gene expression and TOP-like peptidase activity than a non-virulent strain. The release of enzyme following fungal lysis would be consistent with host antibody production and may have a role in the pathogenesis, inflammation and further development of the mycosis.
Asunto(s)
Perfilación de la Expresión Génica , Metaloproteasas/metabolismo , Paracoccidioides/enzimología , Paracoccidioides/patogenicidad , Animales , Bradiquinina/metabolismo , Cromatografía Líquida de Alta Presión , ADN de Hongos/genética , Inhibidores Enzimáticos/metabolismo , Humanos , Pulmón/microbiología , Masculino , Metaloproteasas/antagonistas & inhibidores , Metaloproteasas/genética , Metaloproteasas/aislamiento & purificación , Ratones , Ratones Endogámicos BALB C , Peso Molecular , Paracoccidioides/aislamiento & purificación , Paracoccidioidomicosis/microbiología , Filogenia , Homología de Secuencia de Aminoácido , VirulenciaRESUMEN
BACKGROUND: CD1d-restricted iNKT cells are protective against murine melanoma B16F10-Nex2 growing subcutaneously in syngeneic C57Bl/6 mice as inferred from the fast tumor development in CD1d-KO in comparison with wild type animals. CD1d glycoproteins are related to the class I MHC molecules, and are involved in the presentation, particularly by dentritic cells (DC), of lipid antigens to iNKT cells. In the present work we attempted to identify the endogenous lipid mediator expressed in melanoma cells inducing such immunesurveillance response and study the possibility of protecting animals challenged with tumor cells with lipid-primed DC. RESULTS: Crude cytosolic and membrane fractions from in vivo growing melanoma contained iNKT-stimulating substances. Lipids were then extracted from these cells and one of the fractions (i.e. F3A) was shown to prime bone marrow-derived dendritic cells (BMDC) to stimulate iNKT murine hybridoma (DN32D3) cells to produce IL-2. The active fraction was analyzed by electrospray ionization-mass spectrometry (ESI-LIT-MS) and both iGb3 and iGb4 were identified along with GM3. When iGb3 was incubated with BMDC and tested with DN32D3 cells, IL-2 was equally produced indicating iNKT cell activation. GM3 consistently inhibited this response. To assess the antitumor response-induced by iGb3, a cytotoxicity assay in vitro was used with [3H]-thymidine labeled B16F10-Nex2 cells. At target/effector (iGb3-activated iNKT) cell ratio of 100(-1)-100(-4) tumor cell lysis was shown. The antitumor activity in vivo was tested in mice challenged i.v. with B16F10-Nex2 cells and treated with iGb3- or alpha-galactosylceramide-primed DCs. A 4-fold lower tumor load in the lungs was observed with either treatment. CONCLUSION: Our results show the expression of globo and isoglobohexosylceramides in murine melanoma B16F10-Nex2. The expression of iGb3 and its precursor, iGb4, on tumor cells may prime an effective iNKT cell-dependent antitumor response, modulated negatively by GM3 which is also produced in these cells. iGb3-primed BMDC exerted a significant iNKT cell-mediated anti-tumor activity in mice challenged with melanoma cells.