Barbosa Mônica Santiago, Báo Sônia Nair, Andreotti Patrícia Ferrari, de Faria Fabrícia P, Felipe Maria Sueli S, dos Santos Feitosa Luciano, Mendes-Giannini Maria José Soares, Soares Célia Maria de Almeida
Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiānia, Goiás, Brazil 74001-970.
Infect Immun. 2006 Jan;74(1):382-9. doi: 10.1128/IAI.74.1.382-389.2006.
The pathogenic fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a pulmonary mycosis acquired by inhalation of fungal airborne propagules, which may disseminate to several organs and tissues, leading to a severe form of the disease. Adhesion to and invasion of host cells are essential steps involved in the infection and dissemination of pathogens. Furthermore, pathogens use their surface molecules to bind to host extracellular matrix components to establish infection. Here, we report the characterization of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of P. brasiliensis as an adhesin, which can be related to fungus adhesion and invasion. The P. brasiliensis GAPDH was overexpressed in Escherichia coli, and polyclonal antibody against this protein was obtained. By immunoelectron microscopy and Western blot analysis, GAPDH was detected in the cytoplasm and the cell wall of the yeast phase of P. brasiliensis. The recombinant GAPDH was found to bind to fibronectin, laminin, and type I collagen in ligand far-Western blot assays. Of special note, the treatment of P. brasiliensis yeast cells with anti-GAPDH polyclonal antibody and the incubation of pneumocytes with the recombinant protein promoted inhibition of adherence and internalization of P. brasiliensis to those in vitro-cultured cells. These observations indicate that the cell wall-associated form of the GAPDH in P. brasiliensis could be involved in mediating binding of fungal cells to fibronectin, type I collagen, and laminin, thus contributing to the adhesion of the microorganism to host tissues and to the dissemination of infection.
巴西副球孢子菌这种致病真菌可引发副球孢子菌病,这是一种通过吸入空气中的真菌繁殖体而获得的肺部真菌病,这些繁殖体可能扩散至多个器官和组织,导致病情严重。病原体对宿主细胞的黏附和侵袭是感染和传播过程中的关键步骤。此外,病原体利用其表面分子与宿主细胞外基质成分结合以建立感染。在此,我们报告了巴西副球孢子菌的甘油醛-3-磷酸脱氢酶(GAPDH)作为一种黏附素的特性,它可能与真菌的黏附和侵袭有关。巴西副球孢子菌的GAPDH在大肠杆菌中过表达,并获得了针对该蛋白的多克隆抗体。通过免疫电子显微镜和蛋白质印迹分析,在巴西副球孢子菌酵母相的细胞质和细胞壁中检测到了GAPDH。在配体远缘蛋白质印迹分析中发现重组GAPDH可与纤连蛋白、层粘连蛋白和I型胶原结合。特别值得注意的是,用抗GAPDH多克隆抗体处理巴西副球孢子菌酵母细胞以及用重组蛋白孵育肺细胞,可抑制巴西副球孢子菌对这些体外培养细胞的黏附和内化。这些观察结果表明,巴西副球孢子菌中与细胞壁相关的GAPDH形式可能参与介导真菌细胞与纤连蛋白、I型胶原和层粘连蛋白的结合,从而有助于微生物黏附于宿主组织并促进感染传播。