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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

Recombinant Paracoccin Reproduces the Biological Properties of the Native Protein and Induces Protective Th1 Immunity against Paracoccidioides brasiliensis Infection

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Author(s):
Paiva Alegre, Ana Claudia [1] ; Oliveira, Aline Ferreira [1] ; Dos Reis Almeida, Fausto Bruno [1] ; Roque-Barreira, Maria Cristina [1] ; Hanna, Ebert Seixas [1]
Total Authors: 5
Affiliation:
[1] Univ Sao Paulo, Fac Med Ribeirao Preto, Dept Biol Celular & Mol & Bioagentes Patogen, Sao Paulo - Brazil
Total Affiliations: 1
Document type: Journal article
Source: PLoS Neglected Tropical Diseases; v. 8, n. 4 APR 2014.
Web of Science Citations: 17
Abstract

Author Summary 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 with domains for lectin and N-acetylglucosaminidase activities. Proteomic analysis of paracoccin preparation revealed its correspondence to a hypothetical protein from the P. brasiliensis isolate Pb-18, annotated as PADG-3347, which has a polypeptide sequence similar to family 18 endochitinases. These endochitinases are multi-functional proteins with distinct lectin and enzymatic activity 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 was compared to those of the native paracoccin. Moreover, recombinant PADG-3347 was evaluated for its immunomodulatory properties and its ability to confer protection against murine P. brasiliensis infection. The results presented herein showed that mice treated with recombinant paracoccin displayed lower pulmonary fungal burdens and reduced pulmonary granulomas. These protective effects were associated with augmented pulmonary levels of IL-12 and IFN-gamma. Background Paracoccin is a dual-function protein of the yeast Paracoccidioides brasiliensis that has lectin properties and N-acetylglucosaminidase activities. Proteomic analysis of a paracoccin preparation from P. brasiliensis revealed that the sequence matched that of the hypothetical protein encoded by PADG-3347 of isolate Pb-18, with a polypeptide sequence similar to the family 18 endochitinases. These endochitinases are multi-functional proteins, with distinct lectin and enzymatic domains. Methodology/principal findings The multi-exon assembly and the largest exon of the predicted ORF (PADG-3347), was cloned and expressed in Escherichia coli cells, and the features of the recombinant proteins were compared to those of the native paracoccin. The multi-exon protein was also used for protection assays in a mouse model of paracoccidioidomycosis. Conclusions/Significance Our results showed that the recombinant protein reproduced the biological properties described for the native protein-including binding to laminin in a manner that is dependent on carbohydrate recognition-showed N-acetylglucosaminidase activity, and stimulated murine peritoneal macrophages to produce high levels of TNF-alpha and nitric oxide. Considering the immunomodulatory potential of glycan-binding proteins, we also investigated whether prophylactic administration of recombinant paracoccin affected the course of experimental paracoccidioidomycosis in mice. In comparison to animals injected with vehicle (controls), mice treated with recombinant paracoccin displayed lower pulmonary fungal burdens and reduced pulmonary granulomas. These protective effects were associated with augmented pulmonary levels of IL-12 and IFN-gamma. We also observed that injection of paracoccin three days before challenge was the most efficient administration protocol, as the induced Th1 immunity was balanced by high levels of pulmonary IL-10, which may prevent the tissue damage caused by exacerbated inflammation. The results indicated that paracoccin is the protein encoded by PADG-3347, and we propose that this gene and homologous proteins in other P. brasiliensis strains be called paracoccin. We also concluded that recombinant paracoccin confers resistance to murine P. brasiliensis infection by exerting immunomodulatory effects. (AU)

FAPESP's process: 10/01112-9 - Evaluation of paracoccin interaction with cell receptors of innate immunity, in order to determine its role in the acute phase of paracoccidioidomycosis
Grantee:Ana Claudia Paiva Alegre
Support Opportunities: Scholarships in Brazil - Doctorate