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Interactions with extracellular matrix and epithelial cellular cytoskeletal involvement during Paracoccidioides brasiliensis adhesion and invasion

THESIS

Interactions with extracellular matrix and epithelial cellular cytoskeletal involvement during Paracoccidioides brasiliensis adhesion and invasion

S. A. Hanna

Correspondence Correspondence to S. A. Hanna Departamento de Doenças Tropicais e Diagnóstico por Imagem, Faculdade de Medicina de Botucatu, UNESP, Distrito de Rubião Júnior s/n, 18.618-000, Botucatu, SP, Brasil Email: crisnog@fmb.unesp.br

THESIS. S. A. Hanna submitted this dissertation for her Masters in Tropical Diseases at Botucatu School of Medicine, São Paulo State University, UNESP, Botucatu, São Paulo, Brazil, 2001.

Advisor: Professor Maria José Soares Mendes Giannini

ABSTRACT. Microbial virulence is a set of mechanisms that enable infectious agents to penetrate the host protection barriers and survive against defense mechanisms, multiply, and cause disease. Paracoccidioidomycosis presents a variety of clinical manifestations; the fungus Paracoccidioides brasiliensis can reach many tissues, most importantly the lungs. The understanding of dissemination mechanisms is based on indirect evidence, and the polymorphic aspects of disease suggest that several virulence mechanisms are involved. Pathogen ability to interact with the host superficial structures is essential to its virulence, but little is known about interactions between cells and P. brasiliensis. For this reason, interactions between P. brasiliensis and epithelial Vero and A549 cells were evaluated, with emphasis on interactions with extracellular matrix and induction of cytoskeletal alterations during fungus-cell interaction. Interaction kinetics showed that adhesion occurred after 30 minutes of contact between the two cell lines and yeast, and the number of adhesion points increased on the cells after that. Fungi were inside the Vero cells after two hours and inside A549 after one hour. Interaction between P. brasiliensis and epithelial cells and the components of the extracellular matrix (MEC) were evaluated through immunoperoxidase in situ and indirect immunofluorescence. Recognition patterns of infected cells against anti-fibronectin, anti-laminin, and anti-collagen type I sera were different from those of uninfected cells. Characteristic distributions of bound anti-fibronectin and anti-laminin on yeast cell surface were detected, indicating the presence of specific binding sites for each one. Cytochalasin D, an inhibitor of actin polymerization and colchicine, which disrupts microtubules, substantially reduced invasion, indicating the participation of microfilaments and microtubules in this mechanism. Cytokeratin filaments were not involved in fungus adhesion. However, filament disarrangement and disruption occurred after longer times of fungus-cell contact. The gp 43 activity at concentrations ranging from 25 to 3.125µg/ml changed the actin cytoskeletal arrangement; at concentrations ranging from 32.5 to 12.5µg/ml the integrity of cytokeratin filaments was destroyed. Reactivity of the infected culture supernatant against anti-actin serum showed 43 and 94 kDa bands and anti-cytokeratin serum 40, 43, 67, and 94 kDa bands, suggesting that some components are produced during fungus-cell interaction which bind to cytoskeleton components. Through an immunoblot assay, gp43 was also recognized by anti-actin and anti-cytokeratin sera, but not by anti-tubulin serum. Thus, it seems that this antigen could be a ligand for some cytoskeleton components and may be a candidate invasin. Using the TUNEL with fluorescent probe technique to label cells undergoing DNA fragmentation, P. brasiliensis was shown to induce apoptosis in infected cells. Nuclear fragmentation appeared after one hour and characteristic apoptotic cells were observed after hours of contact between fungus and cells. After five hours, apoptotic bodies and rare yeasts were found. Adhesion and invasion of epithelial cells by P. brasiliensis may represent strategies used to thwart the host immune response, and it may help in pathogen dissemination.

KEY WORDS:Paracoccidioides brasiliensis, virulence, extracellular matrix, adhesion, cytoskeletal.

  • Correspondence to
    S. A. Hanna
    Departamento de Doenças Tropicais e Diagnóstico por Imagem, Faculdade de Medicina de Botucatu, UNESP, Distrito de Rubião Júnior
    s/n, 18.618-000, Botucatu, SP, Brasil
    Email:
  • Publication Dates

    • Publication in this collection
      09 Dec 2003
    • Date of issue
      2003
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