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Title: Modulation of neutrophil migration by the pneumococcal toxin, pneumolysin
Author: Mohamed, Z.
ISNI:       0000 0004 7970 3613
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2018
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Streptococcus pneumoniae is a common bacterial respiratory pathogen which often causes community-acquired pneumonia and is responsible for a huge burden of human disease and death. The initial innate immune response to promote clearance of this bacterium includes the heavy infiltration of neutrophils to the site of infection. However, excessive neutrophil recruitment, or uncontrolled neutrophil activity in response to the infection can result in massive damage to the host tissues. Pneumolysin (PLY), is a membrane-damaging toxin synthesized by S. pneumoniae, which plays a multifunctional role as a pneumococcal virulence factor during infection. It has various biological activities, with distinct cytolytic and sub-lytic effects that interfere with cell function. It has been suggested this toxin can directly modulate neutrophil migration. However, the precise effects on neutrophil motility, and the molecular mechanism underpinning these changes, remain unknown. The effects of PLY on neutrophil migration form the basis of this PhD project, with the aim to understand the molecular mechanism used by PLY to alter neutrophil migration and function in S. pneumoniae infection. We demonstrate that PLY inhibits murine neutrophil chemotaxis towards CXCL1/KC in 2- and 3-D, but does not competitively act as a chemoattractant for neutrophils. PdB, a toxoid derivative of PLY with greatly reduced haemolytic activity, did not inhibit neutrophil chemotaxis, suggesting full function of the toxin is required to inhibit chemotaxis. Consistent with this reduction in migration, we also show that PLY treated neutrophils have strong adhesion to tissue culture plastic, display enhanced actin polymerization, and express high levels of CD11b. Live bacteria were also able to inhibit neutrophil chemotaxis to varying degrees, but this did not strictly correlate with the haemolytic activity of the isolate. Quantitative label-free proteomics of PLY-treated neutrophils revealed four proteins related to cell migration that significantly changed in abundance in PLY-treated neutrophils: Tyrosine-protein kinase Fes/Fps (FES), Grancalcin (GCA), Tyrosineprotein kinase CSK (CSK) and FYVE, RhoGEF and PH domain-containing protein 3 (FGD3). We also found a group of proteins changed in abundance in PLY-treated but not LPS treated neutrophils. These were enriched for cellular responses to calcium, and the presence of EF-hand domains, and were; Grancalcin (GCA), Peflin (PEF1) and Programmed cell death6 (PDCD6) Calpain (CAPN) and Sorcin(SRI) These proteins may offer potential clues to understanding the inhibitory effects of PLY on neutrophil chemotaxis. Preliminary experiments using HerbimysinA to inhibit Fes, revealed that chemotaxis of PLY-treated neutrophils towards KC could be partially restored. The data presented here provide new insight into how PLY regulates neutrophil migration and function. In addition to advancing our understanding of the pathogenesis of S. pneumoniae, it is useful to understand how neutrophils migrate through inflamed tissues in order to develop therapies which can prevent neutrophilmediated damage to host tissues in sterile injury.
Supervisor: Coombes, Janine ; Kadioglu, Aras Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral