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Title: Streptococcus pyogenes protease SpyCEP impairs neutrophil signalling
Author: Goldblatt, Jennifer
ISNI:       0000 0004 9350 1452
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2018
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The enzyme Streptococcus pyogenes Cell Envelope Protease (SpyCEP) is critical to the pathogenesis of necrotizing fasciitis. SpyCEP is known to cleave the C-terminus of ELR+ CXC chemokines, notably the neutrophil attractant CXCL8 which binds to the cell-surface receptors CXCR1 and CXCR2. Cleavage of CXCL8 leaves the N-terminus and disulphide bonds of the chemokine intact, but removes a small portion of an α-helix known to be important in chemokine binding to glycosoaminoglycans (GAGs) on the surface of cells. The work presented in this thesis examined the interaction between SpyCEP-cleaved CXCL8 and its receptors CXCR1 and CXCR2, considered the role of glycosaminoglycan binding in the receptor:chemokine interaction and probed the impact of SpyCEP upon neutrophil death. In vitro studies in this thesis demonstrated that the chemotaxis of CXCR1+ and CXCR2+ cells towards a source of CXCL8 was impaired by SpyCEP cleavage of the chemokine. Similarly, SpyCEP-cleaved CXCL8 was found to have markedly reduced affinity for both CXCR1 and CXCR2 in ligand binding assays. Thus, SpyCEP-cleavage of CXCL8 prevents an effective interaction with its receptors and subsequent downstream signalling. SpyCEP-cleavage of CXCL8 was also found to impair the interaction of CXCL8 with GAGs, with cleaved CXCL8 unable to form multimers on heparin-coated beads. This leads us to hypothesize that SpyCEP functions in vivo by impeding the ability of CXCL8 to bind to GAGs present on the neutrophil surface, leading to a loss of receptor binding and a lack of neutrophil recruitment to the site of infection. Neutrophil survival in vitro and the impact of SpyCEP was found to be highly context-dependent, with the magnitude of difference due to SpyCEP varying markedly between culture conditions. Exposure of neutrophils to high concentrations of affinity-purified SpyCEP appeared to be associated with increased neutrophil necrosis, whilst exposure to supernatant from SpyCEP+ S.pyogenes cultures increased neutrophil death compared to SpyCEP- control supernatant. Exposure of neutrophils to live SpyCEP+ S.pyogenes may afford marginal protection to neutrophils from necrotic death induced by live S.pyogenes. Antibiotic killed bacteria did not appear to influence neutrophil survival regardless of the presence of SpyCEP. Thus, in the presence of intact bacteria, SpyCEP appears not to be a major determinant of neutrophil survival or death. Evaluation of clinical samples taken from different sites and at different timepoints from a patient with S.pyogenes necrotising fasciitis demonstrated the presence of SpyCEP in infected tissue. A range of SpyCEP concentrations within the tissue were detected, which were comparable to the SpyCEP concentrations used in the in vitro assays of neutrophil survival. From the work performed here, a greater understanding of the mechanisms by which CXCL8 interacts with its receptors has been gained, notably highlighting the importance of the chemokine C-terminal α-helix in GAG-mediated presentation of CXCL8 to its receptors. The impact of SpyCEP activity upon CXCR1 and CXCR2 signalling is more clearly understood, whilst the complexity and context-dependent nature of the relationship between CXCL8, SpyCEP, S.pyogenes and neutrophil death is better appreciated.
Supervisor: Sriskandan, Shiranee ; Pease, James Sponsor: Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral