Aspergillus fumigatus Cal A Binds to 946;1 Integrins and Mediates Host Cell Invasion and Virulence

Ref ID: 19531

Author:

Liu1, QT Phan1, MJ Lee2, NV Solis1, DC Sheppard2, SG Filler1,3*

Author address:

1Division of Infectious Diseases, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical
Center, Torrance, USA
2Departments of Medicine, and Microbiology and Immunology, McGill University, Montreal, Canada
3David Geffen School of Medicin

Full conference title:

6th Advances Against Aspergillosis 2014

Abstract:

Purpose:
During the initiation of invasive disease, Aspergillus fumigatus adheres to, invades, and damages
pulmonary epithelial cells. However, the fungal factors and host cell receptors that mediate these
host cell interactions are currently poorly understood. The A. fumigatus CalA protein is predicted to
be an adhesin by bioinformatics analysis. Also, purified recombinant AfCalA binds to laminin. We
aim to identify the role of CalA in mediating adherence and host cell interactions of A. fumigatus.
Methods:
The cellular localization of CalA was determined by expression of a mCherry-tagged allele of calA.
A 916;calA deletion and 916;calA+calA complemented strains of A. fumigatus were constructed. The
capacity of these strains to adhere to, invade, and damage A549 pulmonary epithelial cells and human
umbilical vein endothelial cells (HUVECs) was analyzed. A549 cell membrane proteins that bound
to A. fumigatus were isolated and identified by NanoLC-MS/MS. Antibody blocking and siRNA
knock down were used to determine the role of β 1 integrin in A549 cell invasion by A. fumigatus. The
mouse fibroblast cell lines GD25 (β 1 integrin knock out) and β 1AGD25 (expressing functional β 1A
integrin) were used to investigate the role of CalA and β 1 integrin in mediating host cell invasion.
Finally, the virulence of the various strains was evaluated in the corticosteroid-treated mouse model
of invasive pulmonary aspergillosis.
Results:
CalA-mCherry localized mainly to the cell surface of swollen conidia and the proximal portion
of germlings. Scanning electron microscopy revealed that the 916;calA mutant formed abnormally
curved hyphal tips when grown on A549 cells. Both conidia and germlings of the 916;calA mutant
had wild-type (WT) adherence to A549 cells and laminin. However, the 916;calA mutant had 51%
lower A549 cell invasion (p<0.005) and caused 67% less A549 cell damage (p<0.001) than the WT or 916;calA+calA complemented strains. Furthermore, the 916;calA mutant displayed 70% less HUVEC invasion (p<0.001) compared to the control strains. Although expression of AfCalA alone in Saccharomyces cerevisiae had no effect on adherence to or invasion of A549 and HUVECs, coexpression of AfCalA and the Candia albicans adhesin Als1 increased adherence to HUVECs by 55% and enhanced invasion by 114%, as compare to in S. cerevisiae expressing CaAls1 alone. Wildtype A. fumigatus hyphae was found to bind to β 1 integrin in A549 membrane proteins extracts, while the 916;calA mutant bound poorly to this protein. Both siRNA knockdown and antibody inhibition of β 1 integrin inhibited A549 cell invasion by WT A. fumigatus by 40% (p<0.001). Also, both WT A. fumigatus and CalA-expressing S. cerevisiae had minimal invasion of GD25 cells, which lack β 1 integrins, but high invasion of β 1AGD25 cells in which β 1 integrin expression was restored. Finally, mice infected with the 916;calA mutant had significantly longer survival (p<0.05) than mice infected with the control strains. Conclusions: CalA is the first A. fumigatus invasin to be identified. It interacts with β 1 integrins on host cells to mediate host cell invasion and is necessary for maximal virulence during invasive pulmonary aspergillosis in mice. NOTE: THIS ABSTRACT HAS BEEN SELECTED FOR ORAL PRESENTATION.

Abstract Number: 58

Conference Year: 2014

Link to conference website: http://www.AAA2014.org

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