Paracoccin is a constituent of Paracoccidioides brasiliensis, a human pathogen that causes paracoccidioidomycosis, the most prevalent systemic mycosis in Latin America. Paracoccin is a dual function protein exerting lectin and N-acetylglucosaminidase activities. Proteomic analysis of paracoccin preparation revealed its correspondence with a hypothetical protein from P. brasiliensis isolate Pb18 (Pb18), annotated as PADG-3347.1, which has a polypeptide sequence similar to the family 18

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... ases. These endochitinases have distinct lectin and enzymatic domains. The multi-exon assembly of the correspondent gene (PADG-3347) was cloned and expressed in E. coli, and the physical and biological features of the recombinant protein were compared to those of the native paracoccin. Moreover, recombinant PADG-3347.1 (rPCN) was evaluated for its immunomodulatory properties and its ability to confer protection against murine P. brasiliensis infection. Thus, we investigated the interference of prophylactic and therapeutic administration of rPCN on the course of P. brasiliensis infection in BALB/c mice. The pulmonary histopathology of the treated mice showed lower incidence of granulomas, which were also smaller than those observed in the control animals. Consistently with the observation of few yeasts in the center of the granulomas, the CFU count provided by lung homogenates of treated mice was lower than the provided by control mice. Furthermore, administration of rPCN was associated with higher levels of IL-12, IFN-γ, TNF-α, NO and IL-10, detected in the lung homogenates of animals. The high levels of cytokines produced in the rPCN treated mice prompted us to investigate the occurrence of interaction of the lectin with receptors present in innate immune cells, such as TLR2 and TLR4. We verified that rPCN activates TLR2,in homo or heterodimeric forms, and TLR4, in a manner that does not depend on CD14 and CD36 coreceptors. These data reveal a possible mechanism by which rPCN generates protection in mice against PCM. rPCN, administered therapeutic or prophylactically, induces TLRs activation and Th1 immunity, conferring protection against P. brasiliensis infection, LISTA DE ABREVIATURAS A/Sn: camundongos resistentes à infecção com P. brasiliensis APC: antigen-presenting cell B10.A: camundongos susceptíveis a infecção com P. brasiliensis BCA: bicinchoninic acid assay BHI: brain heart infusion BSA: bovine serum albumin CO 2 : gás carbônico Con A: concanavalina A CRD: carbohydrate recognition domain DEMEM: Dulbecco's modified Eagle's Medium DO: densidade óptica EDTA: Ethylene-diamine tetraacetic acid ELISA: Enzyme linked immuno sorbent assay FBS: fetal bovine serum FSL-1: Pam2CGDPKHPKSF GlcNAc: N-acetilglicosamina GMS: Grocott methenanime silver gp43: glicoproteína de peso molecular de 43 kDa. HE: hematoxilina-eosina HEK: human embryonic kidney HSP: heat shock protein IFN-γ: Interferon-gamma IgY: Immunoglobulin from egg-yolk IL: interleucina iNOS: inducible nitric oxide synthase IPTG: isopropil-β-tiogalactopíranosídeo kDa: quilodaltons kV: quilovolts LB: Lúria-Bertani LPS: lipopolissacarídeo bacteriano Min: minuto MR: mannose receptor I.v.: