RESUMEN

Toxoplasma gondii is an obligate intracellular protozoan parasite found worldwide that is able to chronically infect almost all vertebrate species, especially birds and mammalians. Chitinases are essential to various biological processes, and some pathogens rely on chitinases for successful parasitization. Here, we purified and characterized a chitinase from T. gondii. The enzyme, provisionally named Tg_chitinase, has a molecular mass of 13.7 kDa and exhibits a Km of 0.34 mM and a Vmax of 2.64. The optimal environmental conditions for enzymatic function were at pH 4.0 and 50 °C. Tg_chitinase was immunolocalized in the cytoplasm of highly virulent T. gondii RH strain tachyzoites, mainly at the apical extremity. Tg_chitinase induced macrophage activation as manifested by the production of high levels of pro-inflammatory cytokines, a pathogenic hallmark of T. gondii infection. In conclusion, to our knowledge, we describe for the first time a chitinase of T. gondii tachyzoites and provide evidence that this enzyme might influence the pathogenesis of T. gondii infection.

Asunto(s)

Quitinasas/inmunología , Activación de Macrófagos/inmunología , Macrófagos Peritoneales/inmunología , Proteínas Protozoarias/inmunología , Toxoplasma/inmunología , Secuencia de Aminoácidos , Animales , Quitinasas/genética , Quitinasas/metabolismo , Cromatografía Liquida , Citocinas/inmunología , Citocinas/metabolismo , Citoplasma/enzimología , Interacciones Huésped-Parásitos/inmunología , Concentración de Iones de Hidrógeno , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Cinética , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/parasitología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microscopía Confocal , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas Protozoarias/química , Espectrometría de Masas en Tándem , Temperatura , Toxoplasma/enzimología , Toxoplasma/fisiología

RESUMEN

Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), one of the most prevalent mycosis in Latin America. P. brasiliensis cell wall components interact with host cells and influence the pathogenesis of PCM. Cell wall components, such as glycosylphosphatidylinositol (GPI)-proteins play a critical role in cell adhesion and host tissue invasion. Although the importance of GPI-proteins in the pathogenesis of other medically important fungi is recognized, little is known about their function in P. brasiliensis cells and PCM pathogenesis. We cloned the PbPga1 gene that codifies for a predicted GPI-anchored glycoprotein from the dimorphic pathogenic fungus P. brasiliensis. PbPga1 is conserved in Eurotiomycetes fungi and encodes for a protein with potential glycosylation sites in a serine/threonine-rich region, a signal peptide and a putative glycosylphosphatidylinositol attachment signal sequence. Specific chicken anti-rPbPga1 antibody localized PbPga1 on the yeast cell surface at the septum between the mother cell and the bud with stronger staining of the bud. The exposure of murine peritoneal macrophages to rPbPga1 induces TNF-α release and nitric oxide (NO) production by macrophages. Furthermore, the presence of O-glycosylation sites was demonstrated by ß-elimination under ammonium hydroxide treatment of rPbPga1. Finally, sera from PCM patients recognized rPbPga1 by Western blotting indicating the presence of specific antibodies against rPbPga1. In conclusion, our findings suggest that the PbPga1gene codifies for a cell surface glycoprotein, probably attached to a GPI-anchor, which may play a role in P. brasiliensis cell wall morphogenesis and infection. The induction of inflammatory mediators released by rPbPga1 and the reactivity of PCM patient sera toward rPbPga1 imply that the protein favors the innate mechanisms of defense and induces humoral immunity during P. brasiliensis infection.

Asunto(s)

Proteínas Fúngicas/inmunología , Proteínas Ligadas a GPI/inmunología , Paracoccidioides/inmunología , Paracoccidioidomicosis/inmunología , Animales , Anticuerpos/inmunología , Pollos , Proteínas Fúngicas/genética , Proteínas Ligadas a GPI/genética , Regulación Fúngica de la Expresión Génica , Orden Génico , Glicosilación , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Óxido Nítrico/biosíntesis , Paracoccidioides/genética , Paracoccidioides/metabolismo , Pichia/genética , Pichia/metabolismo , Transporte de Proteínas , Factor de Necrosis Tumoral alfa/biosíntesis

RESUMEN

Agro-industrial wastes are mainly composed of complex polysaccharides that might serve as nutrients for microbial growth and production of enzymes. The aim of this work was to study polygalacturonase (PG) production by Aspergillus niveus cultured on liquid or solid media supplemented with agro-industrial wastes. Submerged fermentation (SbmF) was tested using Czapeck media supplemented with 28 different carbon sources. Among these, orange peel was the best PG inducer. On the other hand, for solid state fermentation (SSF), lemon peel was the best inducer. By comparing SbmF with SSF, both supplemented with lemon peel, it was observed that PG levels were 4.4-fold higher under SSF. Maximum PG activity was observed at 55°C and pH 4.0. The enzyme was stable at 60°C for 90 min and at pH 3.0-5.0. The properties of this enzyme, produced on inexpensive fermentation substrates, were interesting and suggested several biotechnological applications.