Upadhyay Santosh Kumar, Mahajan Lakshna, Ramjee Sandhya, Singh Yogendra, Basir Seemi Farhat, Madan Taruna
Department of Biosciences, Jamia Millia Islamia, New Delhi 110025, India.
Institute of Genomics and Integrative Biology, Mall Road, Delhi 110007, India.
J Med Microbiol. 2009 Jun;58(Pt 6):714-722. doi: 10.1099/jmm.0.005991-0.
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.
烟曲霉是一种机会性真菌病原体，通过吸入空气中的分生孢子感染人类宿主。真菌分生孢子与宿主细胞以及与宿主组织表面相关的细胞外基质（ECM）成分的黏附，被认为是感染发病机制和传播的首要步骤。为了鉴定烟曲霉的新型黏附蛋白（黏附素），我们使用SPAAN（利用神经网络预测黏附素和黏附素样蛋白的软件程序）对其蛋白质组进行了计算机模拟筛选。预测的黏附素编码基因之一，其P(ad)（作为黏附素的概率）值>0.9，即编码细胞外奇异甜蛋白结构域蛋白（AfCalA）的基因，被克隆并在大肠杆菌中表达。重组AfCalAp与层粘连蛋白和小鼠肺细胞表现出显著结合。抗AfCalAp抗体以剂量依赖性方式抑制AfCalAp与层粘连蛋白的结合。抗AfCalAp抗体与2小时肿胀分生孢子的显著结合表明AfCalAp存在于分生孢子表面。在静止分生孢子中未检测到AfCalA转录本，但在存在和不存在肺上皮细胞系（A539）衍生的ECM的情况下，在与RPMI 1640培养基孵育的分生孢子中检测到。在七名过敏性支气管肺曲霉病患者中的两名患者血清中观察到针对AfCalAp的IgE抗体水平升高。该研究证实了生物信息学方法在预测真菌黏附素方面的相关性，并将AfCalAp确立为烟曲霉一种新型的层粘连蛋白结合蛋白。
