The Role of Glycans in Influenza A Virus Mucosal Trapping

PROJECT SUMMARY The mucosal barrier lining the respiratory epithelium serves as the first line of defense against inhaled particulates and pathogens such as influenza A virus (IAV). The mucus barrier is composed of large O-linked glycan decorated gel-forming mucins. These glycans are terminated with different glycans such as sialic acid (Sia), fucose, and sulfates. Terminal glycans on mucins have an important role in the barrier function of airway mucus. For example, IAV penetrates the mucus layer through the coordination of its envelope proteins hemagglutinin (HA), and neuraminidase (NA) which bind and cleave Sia respectively. Fucose and sulfation are not involved in IAV binding, but contribute hydrophobic and anionic properties of the mucus gel layer, respectively. Previous research within our lab has demonstrated the importance of mucus pore sizes on IAV trapping, but the glycan dependence of mucus architecture has yet to be explored. Building upon this, we propose development of glycotransferase knockout human airway epithelium cultures that lack terminal glycans (sialic acid, fucose, sulfation) present in the mucus gel to investigate the following aims: In Aim 1, we will determine how removal of terminal glycans impacts the biophysical properties and transport of mucus as well as influenza A diffusion and infection. In Aim 2, will determine how removal of these glycans impacts antibody (Ab) mediated trapping of IAV. To accomplish this, we will employ CRISPR/Cas9 editing targeting the exons of Fut2, SLC35A1, and PAPSS2, which contribute to mucin fucosylation, sialylation, and sulfation respectively. We will use fluorescence microscopy based analyses to study the diffusion of IAV and Ab within mucus depleted of terminal glycans. Additionally, we will use live cell imaging and particle velocimetry to determine how the removal of these terminal glycans impact mucus clearance efficiency. For the second objective, we will add neutralizing IAV antibodies to normal and glycan knockout mucus to determine how glycans impact Ab-mediated neutralization of IAV. Insights into the role of terminal glycans in their maintenance of physical architecture and viral trapping capabilities of mucus will greatly enhance our understanding of host defense mechanisms critical to lung health.