Year

2021

Degree Name

Doctor of Philosophy

Department

Illawarra Health and Medical Research Institute

Abstract

Invasive infections due to group A Streptococcus (GAS) advance rapidly causing tissue degradation and unregulated inflammation. Neutrophils are the primary immune cells that respond to GAS infection, where it is known that the degree of the neutrophil inflammatory response can influence the outcome of infection. The response of human neutrophils to GAS has not been described in depth. This thesis aimed to characterise the neutrophil response to GAS using in vitro and in vivo models of invasive infection, to further understand the contribution of neutrophils to invasive GAS disease. The current project optimised methodology to investigate the presence of intracellular neutrophil proteins via immunoblot, including caspases, where increased proteases and phosphatase inhibitors decreased protein degradation in neutrophil lysates. Methods of immunoblot normalisation were assessed and revealed total protein is the most appropriate method when undertaking densitometric quantification of neutrophil proteins.

Previous research identified a lytic form of neutrophil death in response to emm98.1 GAS isolate NS88.2 (isolated from invasive infection, covS mutant), however the neutrophil phenotype was largely uncharacterised. Taking into account recent developments in the understanding of cell death, the neutrophil response to GAS was further characterised in vitro using emm98.1 GAS strain NS88.2 and avirulent variant NS88.2rep. Co-incubation of neutrophils with NS88.2 resulted in GAS proliferation and reduced the production of reactive oxygen species when compared with NS88.2rep. In the presence of NS88.2, neutrophil death was delayed compared with NS88.2rep. Infection with either GAS strain induced expression of inflammatory caspases-1 and -4 in neutrophils, with increased detection of activated inflammatory caspases in response to NS88.2rep compared with NS88.2. NS88.2 infection caused differential expression of cell-surface CD66b, CD16 and CD31, when compared to NS88.2rep.

FoR codes (2008)

060501 Bacteriology, 060103 Cell Development, Proliferation and Death, 110707 Innate Immunity, 110801 Medical Bacteriology

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Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.