Identification and characterization of a laminin-binding protein of Aspergillus fumigatus: extracellular thaumatin domain protein (AfCalAp)

1 Institute of Genomics and Integrative Biology, Mall Road, Delhi 110007, India 2 Department of Biosciences, Jamia Millia Islamia, New Delhi 110025, India 3 National Institute for Research in Reproductive Health (NIRRH), Parel, Mumbai 400012, India Correspondence Taruna Madan taruna_m{at}hotmail.com Received August 20, 2008 Accepted February 10, 2009 Aspergillus fumigatus , an opportunistic fungal pathogen, infects the human host via inhalation of airborne conidia. Adhesion of fungal conidia, to host cells and extracellular matrix (ECM) components associated with host tissue surfaces, is thought to be the primary step in the pathogenesis and dissemination of infection. To identify novel adhesion proteins (adhesins) of A. fumigatus , we screened its proteome in silico using SPAAN (software program for prediction of adhesins and adhesin-like proteins using neural networks). One of the predicted adhesin-encoding genes with a P ad (probability of being adhesin) value >0.9, the gene encoding extracellular thaumatin domain protein (AfCalA), was cloned and expressed in Escherichia coli . Recombinant AfCalAp showed significant binding with laminin and murine lung cells. Anti-AfCalAp antibodies inhibited the binding of AfCalAp to laminin in a dose-dependent manner. Significant binding of anti-AfCalAp antibodies to 2 h swollen conidia suggests the presence of AfCalAp on the conidial surface. AfCalA transcript was not detectable in resting conidia but was detected in conidia incubated with RPMI 1640 medium in the presence and absence of lung epithelial cell line (A539)-derived ECM. Elevated levels of IgE antibodies specific to AfCalAp were observed in the sera of two out of seven patients with allergic bronchopulmonary aspergillosis. The study confirms the relevance of the bioinformatic approach for predicting fungal adhesins and establishes AfCalAp as a novel laminin-binding protein of A. fumigatus . Abbreviations: ABPA, allergic bronchopulmonary aspergillosis; DAPI, 4',6-diamidino-2'-phenylindole dihydrochloride; ECM, extracellular matrix; FITC, fluorescein isothiocyanate; GPI, glycosylphosphatidylinositol; GST, glutathione S -transferase; P ad , probability of being adhesin. Figures showing protein sequence alignments for AfCalAp and expression analysis of the RNA are available as supplementary data with the online version of this paper.