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Title: Phenotypic, transcriptomic, and proteomic comparison of clinically-derived Group A streptococcus isolates in the United Kingdom population
Author: Alsahag, M. A.
ISNI:       0000 0004 7970 4624
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2019
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Group A Streptococcus (GAS), also known as Streptococcus pyogenes, is an important cause of human diseases ranging from minor conditions such as pharyngitis to severe, life-threatening infections such as necrotising fasciitis. Between 2010 and 2012, a GAS outbreak (n=14 cases) caused by the emm32.2 GAS subtype occurred in Merseyside, which led to severe invasive disease with 29% case fatality. The outbreak was confined to adults and IV drug users; homelessness and alcohol abuse were identified as risk factors. We sought to investigate the genomic features of this subtype to explain its clinical phenotype. A first objective in my work was to carry out in vitro examination of the emm32.2 outbreak strain to identify whether phenotypic differences may be associated with specific genomic features hence explaining the epidemiological features of this emm subtype. A series of in vitro experiments were exploited and developed to determine whether and how emm32.2 might behave differently from other non-outbreak strains. We carried out a comparative examination of invasive emm32.2 isolates (n=14), against invasive and non-invasive isolates, i.e., emm6.0, emm89.0, and emm1.0, with respect to their capsule thickness, resistance to complement deposition, proteinase, NAD+-glycohydrolase (NADase) and Streptolysin O (SLO) activity, and biofilm formation. Our results demonstrated that phenotypic heterogeneity exists within the emm32.2 isolates as evidenced by variability in capsule thicknesses, proteolytic activity, SLO Activity, and biofilm formation. Interestingly, NADase activity was found to be below detection in all emm32.2 strains. More importantly, the emm32.2 isolates were less susceptible to complement deposition which may be translated to reduced phagocytosis in the host. In addition, RNA sequencing (RNA-Seq) and shotgun mass spectrometry approaches were used in order to identify differences in gene expression and expressed proteins levels of emm32.2 strain (112327) compare to emm1.0 (101910 invasive) and emm89.0 (127746 non-invasive) strains collected during the early, mid, and late exponential growth phases. My results displayed that a total of 76 GAS genes showing significantly different gene expression (p-value =< 0.01; log2-fold change > 4 or<- 4) in emm32.2 vs. emm1.0 and emm89.0. Notably, our data highlighted 16 virulence associated genes which showed different patterns of expression. This list included known virulence factors such as slo and nga which showed higher expression in emm32.2 isolates when compared to emm1.0 and emm89 isolates. In addition, proteomic analysis exhibited several recognised GAS virulence factors which were more abundant (q < 0.05 and fold change values = 2) in emm32.2 compared to emm1.0 and emm89 isolates, including SLO and streptococcal NAD+ glycohydrolase (SPN). My project provides evidence that virulence factors such as SLO and SPN are essential for the virulence of emm32.2 GAS. Overall, characterisation of group A streptococcus outbreak isolates using high-throughput phenotypic, transcriptomic and proteomic analysis reveal genes associated with highly invasive strains and gives new insights into the development of potential GAS vaccine strategies.
Supervisor: Kadioglu, Aras Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral