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Title: Building humanised models of Staphylococcus aureus infection
Author: Buchan, Kyle David
ISNI:       0000 0004 7428 1992
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2018
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Staphylococcal infection is shaped by a large repertoire of virulence factors, complicating both pathology and treatment. A large number of these factors attack the innate immune system directly, allowing S. aureus to resist phagosomal killing and target phagocytes using cytolytic toxins. Many of these factors display great species-specificity for humans, with as few as two amino acid changes reducing binding affinity by 100- fold. A lack of targetable components in existing in vivo models makes accurate representation of staphylococcal infection impossible, necessitating the creation of humanised models in order to fully understand the roles of these factors during infection. One component targeted by at least three virulence factors is the human C5a receptor (hC5aR), a critical chemotactic receptor in neutrophils. Another component targeted by S. aureus is the peroxidase enzyme myeloperoxidase (MPO), which potentiates the neutrophil respiratory burst to facilitate phagosomal killing. To investigate these adapted virulence factors, I generated two humanised zebrafish models that permit investigation of the interactions between human-adapted virulence factors and components of the innate immune system. I overexpressed the hC5aR and MPO as fluorescently-tagged fusion proteins in zebrafish neutrophils, and assessed the impact on neutrophil function and staphylococcal infection. Both constructs were successfully expressed in zebrafish neutrophils, with the hC5aR expressed at the cell membrane and MPO colocalising with the primary granules. Expression of MPO had no impact on neutrophil migration; however, expression of the hC5aR at the cell surface produced a broad defect in chemotaxis, likely due to disruption of endogenous chemotactic signals. Neutrophils expressing the hC5aR gained the ability to migrate to human C5a as a chemotactic agent and became susceptible to targeting by staphylococcal leukocidins, recapitulating human neutrophils. MPO was found to be enzymatically inactive in this model, potentially producing an in vivo marker of neutrophil granules that does not interfere with endogenous myeloperoxidase activity.
Supervisor: Renshaw, Stephen A. ; van Strijp, Jos A. G. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available