Identifying Aspergillus adhesins that bind ECM proteins

St. John'S University, Queens NY

Investigators

Abstract

[unreadable] DESCRIPTION (provided by applicant): The goal of this research is to identify Aspergillus proteins that are responsible for the binding of conidia to the extracellular matrix (ECM). A screen has been developed to identify proteins whose expression will cause the yeast S. cerevisiae to bind to fibronectin (FN) or laminin. it takes advantage of the fact that yeast lacking the cell surface flocculin, FLO11p are unable to bind to FN-coated or laminin-coated polystyrene. The specific aims are: 1. To construct cDNA libraries of Aspergillus fumigatus and Aspergillus nidulans in a yeast vector containing a strong promoter 5' to a multi-cloning site, and a URA3 selectable marker. Poly A+ RNA will be isolated from cultures of both A. fumigatus and A. nidulans and cDNAs will be prepared. The cDNA will be methylated, ligated to adaptors, restriction digested, and de-methylated. The resulting cDNAs will be ligated into the restriction digested plasmid. Plasmid libraries will be propagated in E. coli. 2. An adhesion-deficient Sigma 1278b flo11 strain of S. cerevisiae will be transformed with A. fumigatus and A. nidulans cDNA libraries. Transformants will be screened for their ability of bind to fibronectin (FN)- coated polystyrene. Adherent cells will be harvested and subjected to two additional rounds of selection in ECM protein-coated micro-titer plates to test for their ability to bind FN as well as other ECM and cell surface proteins. Cells will be cured of plasmid. Loss of adhesion will confirm that the ability to bind FN is dependent upon expression of an Aspergillus gene. 3. Plasmid DNA will be isolated from adherent transformants and the sequences of inserts determined. They wilt be compared with the A. fumigatus and A. nidulans genome databases to identify complete open reading frames (ORFs). Proteins encoded by the selected ORFs will be expressed in yeast in a secreted form with (His)6 sequences, and purified from the culture medium by Ni affinity chromatography. 4. Putative Aspergillus adhesins containing a (Histidine)6 sequence will be tested for their ability to adhere to A549 human lung epithelial cells in culture. Binding will be assessed by indirect immunofluorescence using antibodies directed against the His 6 tag. Patients receiving chemotherapy often develop fungal infections because their immune systems are not functioning. These infections can be fatal. There are very few effective anti-fungal drugs. Proteins that cause spores to bind to tissue could be important targets in the development of anti-fungal therapies. [unreadable] [unreadable] [unreadable]