Staphylococcus aureus is a clinically significant human pathogen which poses an increasing healthcare threat due to the spread of antibiotic resistance. To better understand the process of S. aureus pathogenesis, a vertebrate model for infection, using zebrafish embryos, was previously pioneered at The University of Sheffield.
In this study I have utilised this systemic embryonic model of S. aureus infection in combination with a recently developed fluorescence microscopy technique – light sheet fluorescence microscopy, in order to investigate the real-time dynamics of S. aureus infection within a living host.
The first aim of this project was to develop methodology that enables the imaging of infected, living transgenic embryos, over extended time scales. Having established mounting and imaging parameters, infection progression was followed using fluorescent S. aureus reporter strains and fluorescently labelled host phagocytes.
The 4D imaging of these interactions identified macrophages as the host-niche in which bacterial expansion, followed by phagocyte escape, occurs. Furthermore, by using bacterial population studies it was confirmed that depletion of macrophages abolishes the immune bottleneck which proceeds clonal, population expansion of S. aureus.
When imaging embryos in the terminal stages of infection it was apparent that the large bacterial aggregates which form within the host, have biofilm-like characteristics. As such, the role of staphylococcal proteins involved in biofilm formation, during infection progression was investigated using fluorescent reporters for gene expression. It was determined that S. aureus nuclease is produced both inside of host phagocytes and later by bacteria associated with large aggregates. Nuclease was also identified as a novel virulence factor in the zebrafish embryo model of S. aureus infection.
Light sheet fluorescence microscopy has proven a useful tool to gain further insight into the temporal and spatial dynamics of S. aureus pathogenesis and to dissect real-time host-pathogen interactions on a cellular level.
