Atopic Dermatitis (AD) is a common chronic relapsing inflammatory skin disease affecting 15 - 20% of children and 2 - 10% of adults worldwide, with significant morbidity. A hallmark of AD is disruption of the critical barrier function of upper epidermal layers, causatively linked to environmental stimuli, genetics and infections. Another typical feature of AD is skin infections, especially from Staphylococcus aureus (S. aureus), which closely relates with the disease severity. Although not a normal flora, S. aureus is found on 75-100% of AD lesions (< 30% on healthy skin). S. aureus secrete a range of virulence factors, including extracellular toxins and proteases which contribute to disease pathogenesis. S. aureus serine protease A (SspA/V8) is a well-characterised extracellular protease widely expressed among different S. aureus strains. The pathogenic effect of V8 protease has been demonstrated in vivo, damaging murine skin integrity via effects on the stratum corneum (SC), but the targets for this V8-mediated damage remains unclear. The capacity of proteases to induce barrier dysfunction has been proposed as a key driving force in the initiation and exacerbation of AD. Thus, understanding the host factors that maintain barrier function is a priority in developing novel therapeutic approaches. This thesis therefore aimed at detecting host factors which can combat the barrier dysfunction caused by pathogenic proteases, assessing their relevance in vitro and ex vivo and elucidating the underlying mechanisms. Firstly, an in vitro skin barrier integrity model was developed, using both immortalized and primary keratinocytes, to evaluate the barrier damage mediated by pathogenic proteases. The results revealed that S. aureus protease SspA/V8 is the dominant secreted factor (in laboratory and AD clinical strains of S. aureus) inducing barrier integrity impairment. In addition, studies demonstrated that V8 protease itself was sufficient to induce barrier disruption, and this phenotype was not dependent on cell death, but rather on breaking down of cell-cell junctions. Key tight junction proteins including claudin-1 and occludin were found to be degraded by V8 protease. Next, a wide range of host and bacterial factors were investigated to determine whether they could promote protection of keratinocytes against V8 damage. Several factors, including IL-1β, TNF-α, heat-killed Staphylococcus epidermidis (which is the main skin normal flora), were found to induce protection against V8 protease, with IL-1β having the strongest effect. In addition, data indicated that this IL-1β-mediated protection was independent of effects on claudin-1 but occurred via secretion of a transferrable host factor. Induction of keratinocyte expression of the antimicrobial/host defence peptide human beta-defensin 2 (hBD2) was found to be the mechanism underpinning this IL-1β- induced protective effect. Endogenous hBD2 expression was required and sufficient for protection against V8 protease-mediated integrity damage, and exogenous application of hBD2 was also protective. An ex vivo model using human skin tissue was also established to address this novel function of hBD2, and preliminary data indicated that exogenous hBD2 protected against V8-mediated damage in this system. Overall, my data reveal a novel function for the antimicrobial/host defence peptide hBD2. This modulatory property of hBD2, independent of its antibacterial effects, gives new significance to the defective induction of hBD2 in the barrier-defective skin lesions of AD and indicates therapeutic potential to prevent S. aureus-mediated aggravation of skin barrier dysfunction in AD.

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.

This work was focused on the characterization of microparticles with particular emphasis on waterborne pathogens which pose a great health risk to human lives. The goal of this study was to develop microfluidic systems for enhanced characterization and isolation of bioparticles. Insulator-based dielectrophoresis (iDEP) is a promising technique for analyzing, characterizing and isolation of microparticles based on their electrical properties. By employing insulator-based constrictions within the microchannel in combination with microelectrodes within the vicinity of the electrodes, dielectrophoretic performance is enhanced. In this study, three dimensional insulator-based dielectrophoresis devices are fabricated using our in-house developed 3D micromachining technique. This technology combines the benefits of electrode-based DEP, insulator-based DEP, and three dimensional insulating features with the goal of improving trapping efficiency of biological species at low applied signals and fostering wide frequency range operation of the microfluidic device. The dielectric properties of bacteria as well as submicron polystyrene beads are discussed and the impact of these results on the future development of iDEP microfluidic systems is explored. / Master of Science

Bacteria are known to adhere to surfaces, which allows for the formation of biofilms, possibly causing a surge in hospital-offset infections, perilous diseases, and in some cases, death. Although certain bacteria are present in the natural flora of the human skin, some present extreme clinical significance due to the ability to transmit and adhere, and can be resistant to antibiotics. They also evolve over time to survive in harsh environmental conditions. Current research reveals that design of plastic surfaces containing submicron structures, is becoming a popular approach to tackle issues concerning infection transmission, with inspiration being derived from biomimetics and self-cleaning surfaces, such as the surface of a gecko skin, and the hydrophobic wax layer of forest leaves. Main barriers to adoption include that these surfaces alone are difficult to manufacture on 3D products, expensive to fabricate on a large scale and do not last long when subjected to environmental wear. Replication of nano-scale ridges was carried out using micro-injection, and the various samples were characterised using a range of tools to determine physical and biomechanical parameters. The sample surfaces were then cultured with the pathogenic bacterium Staphylococcus aureus under several environmental conditions, and the results were statistically analysed to reveal that anti-fouling LIPSS (laser induced periodic surface structures) ridges perform better to reduce bacteria cell-substrate adhesion, when compared to flat surfaces, or surfaces containing dual structures (anti-fouling ridges combined with anti-wear walls). It was therefore demonstrated that nanotextured polymeric surfaces with hydrophobic characteristics have exceptional non-fouling properties, preventing S. aureus, a very significant bacterial strain, from initial adhesion, a critical primary mechanism in its ability to proliferate. Collectively, the findings of this study strongly support the literature, suggesting that the bacteria struggle to adhere onto polymeric topography with increased water contact angles and simple nanostructures. However, the addition of certain anti-wear micro-features increased bacterial adhesion, reducing the efficacy of the non-fouling nanostructures from preventing biofilm formation.

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