Influenza A virus (IAV) causes annual epidemics and sporadic pandemics of respiratory disease in humans. One of the main complications of primary IAV infection is increased susceptibility to secondary bacterial co-infection, with Staphylococcus aureus being the most common co-infecting species. Previous work identified secreted proteases from S. aureus as a pro-viral factor, leading to specific cleavage of the IAV surface hemagglutinin and increase in infectious viral titre. The aim of this study was to investigate the effect of bacterial proteases, and other secreted bacterial proteins, on IAV replication. Supernatants from the S. aureus community-associated epidemic clone USA300 were separated by size exclusion chromatography and each fraction was tested for an impact on IAV replication in primary chicken embryo fibroblast (CEF) cells. A fraction that increased viral titre by at least 10-fold was identified, but this effect was independent of known secreted proteases. Through the use of mass spectrometry fingerprinting and bacterial mutagenesis, a single protein, S. aureus lipase 1, was identified to be responsible for the pro-viral effect. Lipase 1 is expressed by an array of diverse S. aureus strains of distinct clonal origins. Both the native and recombinant form of lipase 1 were pro-viral only during the infection of primary cells, including primary human lung fibroblasts. Further validation of this interaction indicated lipase 1 was pro-viral in a concentration dependant manner and for a range of IAV strains. Investigation into the mechanism of action of lipase 1 revealed the protein acts during a single infectious cycle in a manner dependent on its active site. Time of addition studies and western blot analysis showed lipase 1 affects the later stages of virus replication, but there is no direct interaction with the virus particle; rather, the protein manipulates the cell, resulting in an increased number of infectious particles being produced. This work has identified and validated a single S. aureus protein, which affects IAV replication. Thus, it has elucidated some of the complex interactions that occur between the virus and bacteria during co-infection. It has also demonstrated a novel role for a bacterial enzyme in IAV replication, the study of which can further our understanding of both IAV and cell biology.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:739092 |
| Date | January 2017 |
| Creators | Goncheva, Mariya Ilieva |
| Contributors | Fitzgerald, Jonathan ; Dutia, Bernadette |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/29535 |
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