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Title: Survival and replication of Group A Streptococcus in human macrophages
Author: O'Neill, Alan
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2016
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As key components of innate immune defence, macrophages are essential in controlling bacterial pathogens, including Group A Streptococcus (GAS). Despite this, only a limited number of studies have analysed the recovery of GAS from within human neutrophils and macrophages. The purpose of this study was to determine the intracellular fate of GAS in human macrophages using several quantitative approaches. The first part of this work involved the set-up of a reliable and reproducible infection system for GAS. In both U937 and primary human macrophages, the appearance over time of long GAS chains revealed replication that despite GAS-mediated cytotoxicity, occurred in viable macrophages. Whereas the major virulence factor M1 did not contribute to bacterial growth, a GAS mutant strain deficient in streptolysin O (Δslo) was impaired for intracellular replication. SLO was required for vacuolar rupture and bacterial escape into the host cytosol. Despite evidence of efficient cytosolic growth, up to half of cytosolic GAS were targeted by autophagy and decorated with ubiquitin and p62. Our studies reveal that GAS can replicate within the cytosol of viable human macrophages and that components of antibacterial autophagy machinery, despite being recruited to the bacteria, fail to restrict overall growth. To my knowledge, this study provides the first direct visualisation of GAS replication inside human cells. GAS is also persistent coloniser of the oropharynx. Persisters are a subpopulation of non- or slow-growing bacteria that are multidrug tolerant, and thought to be involved in the recalcitrance of chronic infections. In the final section of this study, I investigated the in vitro persister formation of clinical isolates of GAS in response to antibiotics. I found that GAS produce a high level of persisters, but no significant difference was detected between different isolates. GAS isolates did not produce more persisters upon uptake in epithelial cells or by macrophages. I also characterized several putative toxin/antitoxin (TA) modules as candidate persister genes of GAS and a subset were shown to encode functional toxins, which when overexpressed in E. coli resulted in bacterial growth arrest.
Supervisor: Holden, David Sponsor: European Commission
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral