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    An Analysis of the Role of Bacillus oleronius Proteins in the Pathogenesis of Ocular Rosacea

    McMahon, Frederick W. (2015) An Analysis of the Role of Bacillus oleronius Proteins in the Pathogenesis of Ocular Rosacea. PhD thesis, National University of Ireland Maynooth.

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    Rosacea is a chronic dermatosis of the central T-zone of the face and eyes. It can be characterised by the presence of inflammatory papules and pustules, persistent non-transient erythema, and flushing episodes. Factors such as alterations in the innate immune response, changes to the vascular network in the skin, the presence of reactive oxygen species within the skin, and neutrophil activation have been suggested as major players in the induction and persistence of the disease. The aetiology of the disease remains unknown but studies suggest a role for bacteria in the induction and persistence of the condition. The aim of the work presented here was to investigate the role of a bacterium, Bacillus oleronius, in the pathogenesis of this disfiguring disease. It was previously established that B. oleronius produces two immunogenic proteins that are capable of inducing an immune response in rosacea patients. As part of this project, the serologically reactive proteins were isolated and purified by anion exchange chromatography and ÄKTA FPLC™ for their use in further assays. The expression of the immuno-reactive 62 kDa GroEL protein was analysed in different culture conditions to determine a possible role in the pathogenesis of the disease, and it was observed that the B. oleronius increased expression of the 62 kDa GroEL protein in response to stress following alterations in temperature, pH, reactive oxygen species (ROS) presence, and oxygen availability of the local environment. To investigate the role of B. oleronius proteins in the induction of ocular rosacea, a human telomerase-immortalized corneal epithelial (hTCEpi) cell line was used. Exposure of corneal epithelial cells to B. oleronius proteins reduced growth in a dose-dependent manner and this corresponded to a decreased rate of cell proliferation and an increase in mean generation time of the epithelial cells. Through FACS analysis, it was shown that corneal epithelial cells stimulated by B. oleronius proteins inhibited the hTCEpi cell cycle at the G1/S transition. It was also observed using label-free mass spectrometry (LF/MS) that exposure of hTCEpi cells to B. oleronius proteins leads to alterations in the abundance of proteins that specifically function in poyadenylation (poly (A)) RNA binding. The effect of B. oleronius proteins on immune cells was investigated, and it was observed that exposure of neutrophils to B. oleronius protein resulted in neutrophil activation along the inositol 1, 4, 5-triphosphate (IP3) pathway with calcium (Ca2+) release, cytoskeletal rearrangement and conversion to filamentous (F)-actin, and increased chemotaxis. The effect of B. oleronius proteins on the secretion of monocyte-specific and T-cell-specific cytokines was determined by ELISpot/FluoroSpot assays, and it was observed that exposure of peripheral blood mononuclear cells (PBMC) to B. oleronius proteins significantly increased the secretion of a range of cytokines (p < 0.0001) including IL-1β, IL-10, IL-12p40, IL-23, tumor necrotic factor (TNF)-α, IL-5, IL-13, IL-17A, IL-22, and interferon (IFN)-γ. The serum from erythematotelangiectatic rosacea patients was analysed for sera reactivity to B. oleronius proteins. It was observed that 82% of erythematotelangiectatic rosacea patients were sera reactive to the B. oleronius proteins compared to 28% sera reactivity in controls. This population of rosacea patients demonstrated an increased density of Demodex folliculorum mites present in the skin but sera reactive rosacea patients also had reduced levels of sebum. Thus, demonstrating altered sebum levels in rosacea patients, and implicating a role for B. oleronius in the induction of the skin condition. The aetiology of rosacea remains unknown but the results presented in this thesis demonstrate that B. oleronius proteins may be implicated in the pathogenesis of rosacea, and that the action of these proteins can lead to the induction of an inflammatory response that is a hallmark of the disease.

    Item Type: Thesis (PhD)
    Keywords: Bacillus oleronius Proteins; Pathogenesis; Ocular Rosacea;
    Academic Unit: Faculty of Science and Engineering > Biology
    Item ID: 7085
    Depositing User: IR eTheses
    Date Deposited: 26 Apr 2016 13:59
      Use Licence: This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here

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