Comparative transcriptome analysis of Paracoccidioides brasiliensis during in vitro adhesion to type I collagen and fibronectin: identification of potential adhesins.
Paracoccidioidomycosis is caused by the dimorphic fungus Paracoccidioides brasiliensis. The extracellular matrix (ECM) plays an important role in regulation of cell adhesion, differentiation, migration and proliferation of cells. An in vitro binding assay of P. brasiliensis yeast cells adhering to type I collagen and fibronectin was performed in order to identify novel adhesins. Representational difference analysis (RDA) was employed to identify genes upregulated under adhesion-inducing conditions. Expressed sequence tags (ESTs) from cDNA libraries generated by the RDA technique were analyzed. Genes related to functional categories, such as metabolism, transcription, energy, protein synthesis and fate, cellular transport and biogenesis of cellular components were upregulated. Transcripts encoding the P. brasiliensis protein enolase (PbEno) and the high-affinity cooper transporter (PbCtr3) were identified and further characterized. The recombinant enolase (rPbEno) and a synthetic peptide designed for PbCtr3 were obtained and demonstrated to be able to bind ECM components. Immunofluorescence assays demonstrated that rPbEno specifically binds to the macrophage surface, reinforcing the role of this molecule in the P. brasiliensis interaction with host cells. In addition, upregulation of selected genes was demonstrated by qRT-PCR. In synthesis, the strategy can be useful in characterization of potential P. brasiliensis adhesins.
Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, 74001-970 Goiânia, GO, Brazil.
Alexandre Melo
AM
Bailão
Sarah Veloso
SV
Nogueira
Sheyla Maria
SM
Rondon Caixeta Bonfim
Kelly Pacheco
KP
de Castro
Julhiany
J
de Fátima da Silva
Maria José Soares
MJ
Mendes Giannini
Maristela
M
Pereira
Célia Maria
CM
de Almeida Soares
D002448:Cell Adhesion; D024042:Collagen Type I / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D020224:Expressed Sequence Tags; D005353:Fibronectins / Q000378:metabolism*; D020869:Gene Expression Profiling; D006801:Humans; D010228:Paracoccidioides / Q000235:genetics / Q000502:physiology*; D060888:Real-Time Polymerase Chain Reaction
D024042:Collagen Type I; D018076:DNA, Complementary; D005353:Fibronectins