title: Recombinant paracoccin reproduces the biological properties of the native protein and induces protective Th1 immunity against Paracoccidioides brasiliensis infection.
abstract: 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.
  
  
  RESULTS
  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
  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-α 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-γ. 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.
authors.affiliation: Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.
authors.forename: Ana Claudia Paiva
authors.initials: AC
authors.lastname: Alegre
authors.affiliation: Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.
authors.forename: Aline Ferreira
authors.initials: AF
authors.lastname: Oliveira
authors.affiliation: Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.
authors.forename: Fausto Bruno
authors.initials: FB
authors.lastname: Dos Reis Almeida
authors.affiliation: Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.
authors.forename: Maria Cristina
authors.initials: MC
authors.lastname: Roque-Barreira
authors.affiliation: Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.
authors.forename: Ebert Seixas
authors.initials: ES
authors.lastname: Hanna
mesh_terms: D000118:Acetylglucosaminidase / Q000378:metabolism; D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D005657:Fungal Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*; D015870:Gene Expression; D037102:Lectins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D008168:Lung / Q000276:immunology / Q000382:microbiology / Q000473:pathology; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000276:immunology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009569:Nitric Oxide / Q000378:metabolism; D010228:Paracoccidioides / Q000235:genetics / Q000276:immunology*; D010229:Paracoccidioidomycosis / Q000473:pathology / Q000517:prevention & control*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D018417:Th1 Cells / Q000276:immunology*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D005657:Fungal Vaccines; D037102:Lectins; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; C548066:paracoccin, Paracoccidioides brasiliensis; D009569:Nitric Oxide; D000118:Acetylglucosaminidase
references.citation: PLoS One. 2011;6(12):e29216
references.pmid: 22216217
references.citation: Nat Rev Immunol. 2003 Feb;3(2):133-46
references.pmid: 12563297
references.citation: Am J Pathol. 2009 Dec;175(6):2489-500
references.pmid: 19893050
references.citation: Anal Biochem. 1976 May 7;72:248-54
references.pmid: 942051
references.citation: Am J Pathol. 2000 May;156(5):1811-20
references.pmid: 10793093
references.citation: Glycobiology. 2001 Dec;11(12):1035-42
references.pmid: 11805076
references.citation: Clin Exp Immunol. 1993 Aug;93(2):189-94
references.pmid: 8348743
references.citation: Am J Pathol. 2008 Aug;173(2):423-32
references.pmid: 18599609
references.citation: Infect Immun. 2010 May;78(5):2302-11
references.pmid: 20231412
references.citation: Mycopathologia. 1982 Sep 17;79(3):153-8
references.pmid: 6983033
references.citation: Hum Vaccin Immunother. 2012 Oct;8(10):1450-3
references.pmid: 22894948
references.citation: Braz J Med Biol Res. 1998 May;31(5):615-23
references.pmid: 9698765
references.citation: Microbes Infect. 2006 Mar;8(3):704-13
references.pmid: 16476564
references.citation: Crit Rev Microbiol. 1984;11(2):101-27
references.pmid: 6329600
references.citation: Yeast. 2010 Feb;27(2):67-76
references.pmid: 19908201
references.citation: PLoS One. 2009;4(2):e4519
references.pmid: 19229338
references.citation: J Immunol. 1999 Mar 15;162(6):3504-11
references.pmid: 10092807
references.citation: J Mol Biol. 1996 Sep 20;262(2):243-57
references.pmid: 8831791
references.citation: Dermatol Clin. 2008 Apr;26(2):257-69, vii
references.pmid: 18346557
references.citation: Infect Immun. 2011 Aug;79(8):2964-73
references.pmid: 21576331
references.citation: Med Mycol. 2002 Jun;40(3):225-42
references.pmid: 12146752
references.citation: Res Immunol. 1998 May-Jun;149(4-5):407-17; discussion 499-500
references.pmid: 9720958
references.citation: J Infect Dis. 2007 Oct 1;196(7):1108-15
references.pmid: 17763336
references.citation: Med Mycol. 2010 Sep;48(6):792-9
references.pmid: 20392144
references.citation: J Agric Food Chem. 2007 Apr 18;55(8):2911-7
references.pmid: 17362028
references.citation: Poult Sci. 2007 Feb;86(2):400-7
references.pmid: 17234857
references.citation: Microbes Infect. 2007 May;9(6):695-703
references.pmid: 17400504
references.citation: Am J Trop Med Hyg. 1987 May;36(3):603-8
references.pmid: 3555139
references.citation: Med Mycol. 2003 Oct;41(5):391-9
references.pmid: 14653515
references.citation: J Med Vet Mycol. 1992;30(3):261-4
references.pmid: 1517962
references.citation: J Immunol. 2007 Oct 15;179(8):5367-77
references.pmid: 17911623
references.citation: Glycoconj J. 2013 Oct;30(7):641-57
references.pmid: 23299509
references.citation: Mycopathologia. 1979 Feb 28;66(3):175-7
references.pmid: 375098
references.citation: Immunol Invest. 2011;40(7-8):825-38
references.pmid: 21815776
references.citation: PLoS One. 2013 May 31;8(5):e65616
references.pmid: 23741502
references.citation: FEMS Yeast Res. 2009 Feb;9(1):103-14
references.pmid: 19076240
references.citation: Arch Med Res. 1995 Autumn;26(3):305-6
references.pmid: 8580685
references.citation: Mycopathologia. 2008 Apr-May;165(4-5):223-36
references.pmid: 18777631
references.citation: An Bras Dermatol. 2011 May-Jun;86(3):516-24
references.pmid: 21738969
references.citation: J Immunol. 2004 Mar 15;172(6):3712-8
references.pmid: 15004175
references.citation: Arthritis Rheum. 2005 Aug 15;53(4):618-20
references.pmid: 16082636
references.citation: Infect Immun. 1998 Feb;66(2):800-6
references.pmid: 9453644
references.citation: Clin Microbiol Rev. 1993 Apr;6(2):89-117
references.pmid: 8472249