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Generation and evaluation of stable Lifeact-expressing A549 cell lines for investigation of A.fumigatus interaction with pulmonary epithelial cells


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A human cancer-derived A549 lung epithelial cell line is widely used in studies analyzing the interaction of A. fumigatus with pulmonary epithelial cells. Previous studies showed that conidia can adhere to A549 cells and enforce formation of actin-rich pseudopods followed by conidia invagination and endocytosis (1-4). Moreover, secreted fungal proteases were suggested to cause loss of actin fibers and focal adhesions in A549 cells, followed by cell detachment and necrosis (5). The aim of our study is to investigate the interaction of A.fumigatus conidia with alveolar epithelial cells and changes of the actin cytoskeleton. Using lenti-viral transduction A549 –derived cell lines that stably express LifeAct (GFP) (6) or LifeActRed (mCherry) were generated. These cell lines were then infected with either a GFP-expressing strain of A.fumigatus (3) or a wild type strain (ATCC 46645) for fluorescence live cell video microscopy. Our preliminary data suggest that the formation of actin-rich pseudopods around cell membrane attached conidia is a rare and a highly conidia concentration-dependent event. In line with previous studies, in A549 cells infected with high conidia numbers (108 conidia/ml), pseudopod formation could be observed along with a strong depolymerisation of F-actin. However, when cells were infected with lower numbers of conidia (103-104 conidia/ml), the actin cytoskeleton remained intact. Moreover at the sites of conidia-membrane interactions we could observe an increase in stress fiber formation, suggesting activation of possible “protective” mechanisms against conidia internalization. Using the WST-1 assay, we also observed that treatment of A549 with either A.fumigatus culture filtrates or with A.fumigatus cellular extracts caused strong inhibition of metabolic activity in a concentration- and time- dependent manner. Interestingly, the effect of culture filtrates and cellular extracts was strongly decreased when A549 were seeded on CELLBind (Corning®) plates, suggesting the importance of cellular adhesion for the A.fumigatus interaction. Our preliminary data suggest that cell systems where A549 are confronted with a high amount of A.fumigatus conidia may not be optimal to investigate cell-pathogen interactions. Further use of live microscopy and stable A549 cell lines may help reinvestigation of initial interactions of conidia /epithelial cells with high optical resolution. References: 1: Bromley IM and Donaldson K., Thorax. 1996. PMID: 8994516 2. DeHart DJ et al., J Infect Dis. 1997. PMID: 8985209 3. Wasylnka JA and Moore MM., Infect Immun. 2002. PMID: 12011010 4. Wasylnka JA and Moore MM., J Cell Sci. 2003. PMID: 12640041 5. Kogan TV et al., J Infect Dis. 2004. PMID: 15143461 6. Riedl J et al., Nat Methoda. 2008 PMID: 18536722


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© Copyright 2020 Morressier GmbH.
All rights reserved.