title: Paracoccidoides brasiliensis 30 kDa adhesin: identification as a 14-3-3 protein, cloning and subcellular localization in infection models.
abstract: Paracoccidoides brasiliensis adhesion to lung epithelial cells is considered an essential event for the establishment of infection and different proteins participate in this process. One of these proteins is a 30 kDa adhesin, pI 4.9 that was described as a laminin ligand in previous studies, and it was more highly expressed in more virulent P. brasiliensis isolates. This protein may contribute to the virulence of this important fungal pathogen. Using Edman degradation and mass spectrometry analysis, this 30 kDa adhesin was identified as a 14-3-3 protein. These proteins are a conserved group of small acidic proteins involved in a variety of processes in eukaryotic organisms. However, the exact function of these proteins in some processes remains unknown. Thus, the goal of the present study was to characterize the role of this protein during the interaction between the fungus and its host. To achieve this goal, we cloned, expressed the 14-3-3 protein in a heterologous system and determined its subcellular localization in in vitro and in vivo infection models. Immunocytochemical analysis revealed the ubiquitous distribution of this protein in the yeast form of P. brasiliensis, with some concentration in the cytoplasm. Additionally, this 14-3-3 protein was also present in P. brasiliensis cells at the sites of infection in C57BL/6 mice intratracheally infected with P. brasiliensis yeast cells for 72 h (acute infections) and 30 days (chronic infection). An apparent increase in the levels of the 14-3-3 protein in the cell wall of the fungus was also noted during the interaction between P. brasiliensis and A549 cells, suggesting that this protein may be involved in host-parasite interactions, since inhibition assays with the protein and this antibody decreased P. brasiliensis adhesion to A549 epithelial cells. Our data may lead to a better understanding of P. brasiliensis interactions with host tissues and paracoccidioidomycosis pathogenesis.
authors: Department of Clinical Analyses, Faculty of Pharmaceutical Sciences, São Paulo State University - University Estadual Paulista Araraquara, São Paulo, Brazil.
authors: Julhiany de Fatima
authors: Jde F
authors: da Silva
authors: Haroldo César
authors: HC
authors: de Oliveira
authors: Caroline Maria
authors: CM
authors: Marcos
authors: Rosângela Aparecida Moraes
authors: RA
authors: da Silva
authors: Tania Alves
authors: TA
authors: da Costa
authors: Vera Lucia García
authors: VL
authors: Calich
authors: Ana Marisa Fusco
authors: AM
authors: Almeida
authors: Maria José Soares
authors: MJ
authors: Mendes-Giannini
mesh_terms: D048948:14-3-3 Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002473:Cell Wall / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D004847:Epithelial Cells / Q000382:microbiology / Q000473:pathology; D005656:Fungal Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010228:Paracoccidioides / Q000166:cytology / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology*; D010229:Paracoccidioidomycosis / Q000382:microbiology* / Q000473:pathology; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism
chemical_list: D048948:14-3-3 Proteins; D005656:Fungal Proteins; D011994:Recombinant Proteins
references: Front Microbiol. 2011 Oct 10;2:207
references: 22013430
references: Med Mycol. 2005 Nov;43(7):637-45
references: 16396249
references: Mycopathologia. 2008 Apr-May;165(4-5):331-9
references: 18777637
references: J Proteome Res. 2012 Mar 2;11(3):1676-85
references: 22288420
references: BMC Microbiol. 2009 Dec 24;9:272
references: 20034376
references: Cell Microbiol. 2006 Jan;8(1):55-71
references: 16367866
references: Mycopathologia. 1990 Jan;109(1):13-7
references: 2139178
references: Curr Top Med Mycol. 1993;5:115-49
references: 8242798
references: Microbes Infect. 2006 Oct;8(12-13):2686-97
references: 16962356
references: Cell Microbiol. 2008 Aug;10(8):1695-710
references: 18419773
references: J Med Vet Mycol. 1987 Feb;25(1):5-18
references: 3553526
references: Infect Immun. 1994 Apr;62(4):1465-9
references: 8132354
references: PLoS One. 2011;6(7):e22810
references: 21829521
references: Curr Opin Cell Biol. 1999 Oct;11(5):561-6
references: 10508653
references: J Med Vet Mycol. 1987 Dec;25(6):377-87
references: 3437374
references: Genome Biol. 2001;2(3):REVIEWS1009
references: 11276429
references: Microbiol Mol Biol Rev. 2000 Dec;64(4):746-85
references: 11104818
references: Infect Immun. 1995 May;63(5):1777-83
references: 7729885
references: Med Mycol. 2012 Jul;50(5):533-7
references: 21988701
references: Nature. 1970 Aug 15;227(5259):680-5
references: 5432063
references: FEMS Yeast Res. 2007 Dec;7(8):1381-8
references: 17714474
references: Annu Rev Microbiol. 2006;60:281-303
references: 16753032
references: Infect Immun. 2005 Apr;73(4):2486-95
references: 15784595
references: FEMS Yeast Res. 2004 Jan;4(4-5):369-76
references: 14734017
references: Mycopathologia. 2008 Apr-May;165(4-5):183-95
references: 18777628
references: Annu Rev Pharmacol Toxicol. 2000;40:617-47
references: 10836149
references: FEMS Yeast Res. 2012 Aug;12(5):557-70
references: 22443156
references: Annu Rev Genet. 2009;43:1-24
references: 19640229
references: Methods Mol Biol. 2000;132:185-219
references: 10547837
references: Mem Inst Oswaldo Cruz. 2009 Aug;104(5):749-54
references: 19820837
references: Trends Microbiol. 2006 Jan;14(1):15-21
references: 16356720
references: Yeast. 2008 Feb;25(2):141-54
references: 18098122
references: Bioessays. 2001 Oct;23(10):936-46
references: 11598960
references: Electrophoresis. 1988 Jun;9(6):255-62
references: 2466658
references: Mol Microbiol. 2006 Nov;62(4):970-83
references: 17038119
references: Microbes Infect. 2007 Apr;9(5):583-90
references: 17387029
references: Mycopathologia. 2011 Jan;171(1):1-9
references: 20669049
references: Fungal Genet Biol. 2011 Dec;48(12):1087-95
references: 21945996
references: FEBS J. 2011 Jul;278(13):2318-32
references: 21535474
references: Rev Inst Med Trop Sao Paulo. 1998 May-Jun;40(3):125-35
references: 9830725
references: Curr Opin Microbiol. 1998 Aug;1(4):381-9
references: 10066511
references: FEMS Microbiol Lett. 2002 Apr 23;210(1):61-6
references: 12023078
references: Microbes Infect. 2004 May;6(6):549-58
references: 15158188
references: Yeast. 2006 Feb;23(3):159-71
references: 16498703
references: Eukaryot Cell. 2008 Jan;7(1):58-67
references: 18039940
references: Microbes Infect. 2001 Jun;3(7):535-42
references: 11418327
references: Mol Biol Cell. 2003 Jun;14(6):2314-26
references: 12808032
references: FEMS Yeast Res. 2008 Mar;8(2):300-10
references: 17927766
references: Microbiology (Reading). 1997 May;143 ( Pt 5):1673-1680
references: 9168616
references: Microbes Infect. 2005 May;7(5-6):875-81
references: 15862780
references: Biochim Biophys Acta. 2008 Mar;1778(3):670-91
references: 18201548
references: Infect Immun. 2003 Nov;71(11):6534-42
references: 14573675
references: Microbes Infect. 2000 Jul;2(8):877-84
references: 10962270
references: Curr Opin Cell Biol. 1994 Oct;6(5):752-8
references: 7833055
references: Infect Immun. 2010 Dec;78(12):5280-6
references: 20876288
references: Infect Immun. 2006 Jan;74(1):382-9
references: 16368993
references: Mol Plant Pathol. 2011 Jun;12(5):507-14
references: 21535355
references: Immunobiology. 2011 Jul;216(7):821-31
references: 21257226
references: Eukaryot Cell. 2004 Apr;3(2):420-9
references: 15075272
references: Infect Immun. 2005 Nov;73(11):7190-7
references: 16239513
references: Nucleic Acids Res. 1997 Sep 1;25(17):3389-402
references: 9254694
references: Mol Biol Cell. 2005 Oct;16(10):4792-813
references: 16030248
references: Microbes Infect. 2005 Apr;7(4):666-73
references: 15857804
references: Med Mycol. 2000;38 Suppl 1:113-23
references: 11204137
references: Cell Immunol. 2002 Jul-Aug;218(1-2):87-94
references: 12470616
references: Microbes Infect. 2006 May;8(6):1550-9
references: 16698299
references: Med Mycol. 2009 Nov;47(7):734-44
references: 19888806
references: Eukaryot Cell. 2003 Feb;2(1):34-48
references: 12582121
references: J Med Microbiol. 2009 Jun;58(Pt 6):706-713
references: 19429745
references: FEMS Yeast Res. 2006 Nov;6(7):1085-93
references: 17042758
references: Front Biosci (Schol Ed). 2009 Jun 01;1(1):61-7
references: 19482683
references: Eukaryot Cell. 2011 Mar;10(3):343-51
references: 21216942
references: Cell Microbiol. 2010 Jul;12(7):863-72
references: 20482553
references: Future Microbiol. 2010 Feb;5(2):253-65
references: 20143948
references: Yeast. 2003 Feb;20(3):263-71
references: 12557278