Microbiology & Immunology - Theses
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ItemRole of the chemokines CCL17 and CCL22 in the immune defence against Salmonella infection( 2019)The chemokines CCL17 and CCL22 are both ligands of the chemokine receptor CCR4, which is expressed on dendritic cells (DC) and a variety of different effector T cells including regulatory T cells (Treg). Both chemokines are mainly produced by DC, but also by macrophages. CCL17 promotes numerous inflammatory and allergic diseases, whereas CCL22 is rather associated with an immunosuppressive milieu. These differential roles are reflected by preferential recruitment of distinct subsets of T cells to site of inflammation. While CCL17 facilitates chemotaxis of effector T cells and supports DC-T cell interactions as well as DC migration towards CCR7-ligands, CCL22 induces chemotaxis of Treg cells. In addition, CCL22 signalling induces a more rapid desensitisation and internalisation of CCR4 than CCL17, suggesting biased agonism of CCL17 and CCL22. The functionality of CCL17 and CCL22 should, therefore, be considered in combination as well as individually in the context of immune-related diseases. The role of CCL17 and CCL22 in infectious diseases has not been well understood. The central hypothesis was that CCL17 and CCL22 play important but potentially different roles during bacterial infection. This was modelled using a well-studied bacterial pathogen, Salmonella enterica serovar Typhimurium (STM). It was hypothesised that CCL17 expression may direct the migration of STM-infected DC from the gut to draining lymph nodes a key bottleneck in early infection that controls bacterial dissemination to systemic sites. It was further hypothesised that CCL22 may play a role in immune regulation through the induction of Treg cells. These regulatory cells may have downstream effects on Th1 responses, which are critical for the control of Salmonella infection. In the first part of the thesis, the role of CCL17+ DC in the transmission of STM was investigated. Histological analysis of CCL17 reporter mice revealed that CCL17-expressing cells co-localised with Salmonella in the dome area of Peyer’s patches (PP). Further, CCL17-expressing DC contributed to dissemination of STM from PP to the mesenteric lymph nodes (mLN). Within the mLN, STM were found within CCL17+ DC as well as in other DC, monocytes and macrophages. Analysis of the STM+ DC subpopulations revealed that all DC subsets carried STM, but the CD103+ CD11b- DC could be identified as the main STM-containing population. STM infection triggered upregulation of CCL17 expression in specific intestinal DC subsets in a tissue-specific manner. Interestingly, the CD103+ DC subsets upregulated CCL17 in the PP, whereas CD103- DC subsets upregulated CCL17 in the mLN. In the second part of this thesis, the role of CCL17 and CCL22 in the induction of antigen-specific CD4+ T cell responses was investigated. CCL17/CCLL22 double-deficient, CCL17- and CCL22 single-deficient, and wild type mice were analysed after live-attenuated STM TAS2010 vaccination, vaccination/challenge and in steady-state. Mice deficient in both chemokines, CCL22 and CCL17, demonstrated a reduction of effector Treg cells. This promoted an enhanced STM-specific Th1 immune response characterised by an expansion of Th1 T cells, resulting in a more favourable effector Treg/activated Tconv ratio and a significantly improved vaccine efficacy to challenge with virulent Salmonella. In conclusion, the work presented within this thesis showed the contribution of CCL17+ DC in the dissemination of STM and identified CCL22 as a potential target to improve vaccine approaches.
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ItemThe SseK effector proteins of Salmonella Typhimurium target host cell signaling proteins( 2019)Pathogenic serovars of Salmonella are the causative agents of a variety of disease states, including typhoid fever, self-limiting gastroenteritis, and invasive bacteremia. To achieve infection, Salmonella relies on two type-three secretion systems (T3SS) to deliver distinct cohorts of effector proteins into host cells. These effector proteins interact with specific human proteins to subvert normal cellular processes, thus impairing the ability of host cells to respond to the invading bacteria. To date, more than 40 different effector proteins have been identified, though many remain poorly characterised. This thesis focused on the SseK family of effector proteins, which had a largely unknown molecular mechanism and role in Salmonella infection. The aim of this thesis was to identify the host proteins that are targeted by the SseK effectors in order to determine how these effectors contribute to Salmonella virulence. The SseK effectors show strong sequence similarity to NleB1, a unique T3SS effector of enteropathogenic E. coli, which functions as an arginine glycosyltransferase and catalyses the addition of N-acetylglucosamine (GlcNAc) to arginine residues of the mammalian signaling adaptors FADD and TRADD. Based on strong sequence homology to NleB1, we predicted that the SseK effectors would similarly catalyse arginine glycosylation. Here, we determined that SseK1 and SseK3, but not SseK2, also function as arginine glycosyltransferases. We showed that these effectors catalyse arginine glycosylation of different host proteins and appear to play different roles during infection. We developed a mass spectrometry-based strategy to enrich for arginine glycosylated peptides from host cells infected with Salmonella Typhimurium (S. Typhimurium). Using this approach, we identified the preferred substrate of SseK1 as the signaling adaptor TRADD, which participates in a range of innate immune signaling pathways. We also showed that overexpression of SseK1 broadens the range of glycosylated substrates, and that SseK1 was capable of glycosylating both mammalian and bacterial proteins under these conditions. Further, we identified the site of glycosylation within TRADD, and using a mutagenesis approach we showed that SseK1 is also capable of glycosylating secondary sites within TRADD. Collectively, these data show that the preferred substrate of SseK1 is TRADD, and highlight the importance of studying effectors in the natural context of infection. Next, we applied our strategy for enriching arginine glycosylated peptide to identify the substrates of SseK3. We identified the host signaling receptors TNFR1 and TRAILR as the preferred substrates of SseK3 during S. Typhimurium infection, and conducted a range of experiments to validate the glycosylation of these receptors and identify the specific residues that are modified. We also conducted preliminary analyses to explore the contribution of these glycosylation events to virulence in vivo. Together, the data presented in this thesis demonstrate that the S. Typhimurium effectors SseK1 and SseK3 function as arginine glycosyltransferases that target different innate immune signaling proteins during infection. We showed that SseK1 prefers the adaptor protein TRADD while SseK3 targets the signaling receptors TNFR1 and TRAILR. These observations provide new mechanisms by which Salmonella may manipulate innate immune signaling during infection.
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ItemImmunological checkpoints in the control of murine Salmonella enterica infection: IFN-γ pathways and early dendritic cell death( 2017)Salmonella enterica is a Gram-negative intracellular pathogen, which can cause typhoid fever and non-typhoidal salmonellosis. Every year ~22 million cases and ~200,000 deaths are reported for typhoid fever and ~93 million cases and ~155,000 deaths for non-typhoidal salmonellosis. Innate immunity provides the very early protection against Salmonella, a better understanding of which may lead to a progress in treatment and prevention to Salmonella infection. Dendritic cells (DC) are one of the first cells to sense Salmonella in vivo, and play an important role in initiating a cascade of innate immune control, including phagocytising bacteria and the activation of inflammasomes, which further induces cell death and the production of pro- inflammatory cytokines, such as IFN-γ. In addition, dendritic cells are potent antigen presenting cells (APC) that induce the development of protective adaptive immunity against Salmonella. However, it has been reported that Salmonella possesses various mechanisms, including regulating phagolysosomal fusion and delaying vacuole acidification, and down-regulating flagellin expression to prevent antigen presentation, highlighting the dynamic and complex nature of DC-Salmonella interactions. In recent years, the critical role of DCs in immunity against Salmonella has gained increased attention, however the cellular and molecular mechanisms of the DC-Salmonella interactions are not fully understood. The first aim of this study was to study the survival and death in infected DCs during Salmonella infection, utilising murine bone marrow-derived DCs (BMDCs), which are sensitive to Salmonella-induced cell death within hours of infection. It is found that several virulence factors such as lipopolysaccharide (LPS), Type III secretion system 1 (SPI-1) and flagellin contribute to, and in combination maximise, death in BMDCs. Intriguingly, BMDCs that were not directly infected with Salmonella were killed upon infection of neighbouring cells in culture. An apparently similar ‘bystander’ cell death was induced by co-culturing with filtered supernatant from infected BMDCs, suggesting a role for contact-independent mechanisms. Infected BMDCs released several cytokines, including IL-6, MCP-1 and TNF-α. However, blockade of intracellular protein transport and secretion of cytokines by monesin did not alter Salmonella-induced cell death in uninfected bystanders, suggesting that the bystander effect is not dependent on mediators released from infected BMDCs. BMDCs from mice with gene knockouts in key pathways that are involved in DC immune responses against Salmonella were also tested, and decreased death was observed in ICE-/- BMDCs, suggesting that caspase-1/caspase-11-mediated pyroptosis could be responsible for direct as well as bystander BMDC death. The second aim was to determine the contribution of IFN-γ and the IFN-γ induction pathways in Salmonella infection. Previous studies in our lab have shown that flagellin-induced NLRC4 inflammasome activation in splenic DCs triggers non- cognate memory CD8+ T cells to produce IFN-γ, a critical mediator of innate immunity against Salmonella. It was shown in the present study that deletion of individual components of the NLRC4 inflammasome pathway, e.g. caspase-1 or IL- 18, can lead to a moderate reduction of IFN-γ production, but the impact on the control of Salmonella in infected mice is minimal, suggesting that NLRC4 pathway is not the only source of IFN-γ and that low level of IFN-γ may be sufficient for full protection against Salmonella. In the studies presented here, it was shown that LPS- induced activation of the TLR4 pathway is also an important source of IFN-γ and that mice deficient in components of TLR4 pathway has poor early control of bacterial load during S. Typhimurium BRD509 infection. Interestingly, deficiency in the TLR4 pathway led to an increase rather than reduction of IFN-γ, suggesting that IFN-γ is regulated by different pathways and that TLR4 pathway may be involved in other immune responses that are important for early control of Salmonella.
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ItemCharacterisation of a family of novel glycosyltransferases from enteropathogenic Escherichia coli and Salmonella( 2016)Enteropathogenic Escherichia coli (EPEC) is a diarrhoeal pathogen of children that utilises a type III secretion system (T3SS) to inject virulence effector proteins into enterocytes during infection. NleB1 is a novel glycosyltransferase effector from EPEC that catalyses the addition of a single GlcNAc moiety in an N-glycosidic linkage to arginine. NleB1 modifies arginine-117 (Arg117) of the Fas associated death domain (DD) protein, FADD, which prevents assembly of the canonical death inducing signalling complex (DISC) and inhibits FasL-induced cell death. NleB1 also modifies the equivalent arginine residues in the DD proteins, tumour necrosis factor receptor type 1 (TNFR1)-associated death domain (TRADD) and receptor-interacting protein kinase 1 (RIPK1). Apart from the DxD catalytic motif of NleB1, little is known about other functional sites in the protein and the regions required for substrate binding and specificity. Here a library of 22 random transposon-based, in-frame, linker insertion mutants of NleB1 were tested for their ability to block caspase-8 activation in response to FasL during EPEC infection. Immunoblot analysis of caspase-8 cleavage products showed that 14 mutant derivatives of NleB1 no longer inhibited caspase-8 activation, including the catalytic DxD mutant. Regions of interest around the linker insertion sites were examined further with multiple or single amino acid substitutions. Coimmunoprecipitation studies of 34 site-directed mutants showed that the NleB1 derivatives with the E253A, Y219A, and PILN(63– 66)AAAA (in which the PILN motif from residues 63 to 66 was changed to AAAA) mutations bound to but did not GlcNAcylate FADD. A further mutant derivative, the PDG(236 –238)AAA mutant, did not bind to or GlcNAcylate FADD. Further testing of these mutants with TRADD and RIPK1, showed that NleB1 bearing the mutations E253A and Y219A could still bind to FADD and RIPK1 but not to TRADD. Infection of mice with the EPEC-like mouse pathogen Citrobacter rodentium expressing NleBE253A and NleBY219A showed that these 2 strains were attenuated, indicating the importance of the residues E253 and Y219 in NleB1 virulence in vivo. In summary, we identified new amino acid residues critical for NleB1 activity and confirmed that FADD GlcNAcylation was critical for NleB1 function. Close homologues of NleB1 are found in Salmonella enterica serovar Typhimurium and these are termed SseK1, SseK2 and SseK3. We hypothesized that the SseK effectors would also bind to DD proteins and inhibit apoptotic or inflammatory signalling. The SseK effectors did not appear to play a strong role in the inhibition of death receptor signaling given that we could not detect binding of the SseK effectors to the death domain proteins FADD, TRADD and RIPK1, which are targets of NleB1. A further survey of DD proteins revealed that SseK3 bound to TNFR1. However S. Typhimurium did not appear to inhibit TNF-induced IL-8 production and the biological significance of this interaction is still unknown. We conclude that the SseKs have an alternative function during S. Typhimurium infection to NleB1 in EPEC.
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ItemSalmonella Typhimurium metabolism in the murine host and importance to virulence( 2014)The bacterial pathogen Salmonella enterica is responsible for considerable global morbidity and mortality, being the cause of several enteric and systemic diseases, including typhoid fever. Non- typhoidal Salmonella (NTS) serovars such as Typhimurium cause gastroenteritis in immunocompetent individuals, but in the context of HIV-co-infection can cause invasive disease with high fatality rates. Successive generations of antimicrobials have become ineffective against S. enterica pathogens due to the widespread development of resistance, and new drugs are critically needed. In addition, vaccines against typhoid fever have sub-optimal efficacy and no human NTS vaccines are currently available. Although the in vitro metabolism of S. enterica is relatively well defined, little is known about the specific nutrients that the pathogen consumes in its hosts, and the metabolic mechanisms by which S. enterica utilise these nutrients. Therefore, the central aim of this study was to investigate and characterise S. enterica serovar Typhimurium metabolism in the murine host. Attention was focused on two areas of S. Typhimurium metabolism which have been speculated to contribute to bacterial virulence: methylglyoxal detoxification and central carbon (sugar) catabolism. It was anticipated that this study may contribute to better defining the metabolic requirements of S. Typhimurium during infection and disease, and present opportunities for the identification of potential drug targets and metabolically-attenuated strains amenable to use as live vaccines. Several studies have ventured that methylglyoxal detoxification mechanisms are required for survival of bacterial pathogens in the mammalian host, but this hypothesis has not been thoroughly tested. In the current study, although S. Typhimurium mutants defective in the glutathione-dependent glyoxalase system (GDGS) or Kef-mediated potassium efflux were highly sensitive to methylglyoxal, they were not attenuated for intracellular replication and growth in mice, suggesting that these methylglyoxal detoxification mechanisms are not required for S.Typhimurium pathogenesis in the mammalian host and are not suitable targets for antimicrobial therapy against S. Typhimurium disease. While others have reported that the Embden-Meyerhof pathway (EMP) and the ability to utilise glucose are required for S. Typhimurium virulence in mice, the importance of other sugars and carbon catabolic pathways to S. enterica virulence is unclear. In this study, S. Typhimurium mutants blocked in several sugar catabolic pathways including the Entner-Doudoroff pathway (EDP) were found not to be attenuated for intracellular growth or fulminant infection of mice, demonstrating that gluconate, glucuronate, galacturonate are not essential carbon sources for S. Typhimurium in vivo. However, evidence was presented which suggested that the ability to utilise gluconate and glucose is required for optimal shedding of the pathogen in the murine faeces, revealing potential strategies for reducing the faecal-oral transmission of S. Typhimurium. EMP/EDP double mutants showed greater attenuation in mice than a EMP mutant, suggesting that the EMP mutant utilises the EDP in order to facilitate it’s modest growth in vivo. These findings demonstrated the functional redundancy of S. Typhimurium metabolism and suggested that combinational drug therapies targeting several bacterial pathways concurrently might be a viable option for treating S. Typhimurium disease. The S. Typhimurium EMP/EDP mutant TAS2010 was found to provide increased, long-term protection from virulent infection in a murine typhoid vaccination model than the prototypical aro-negative vaccine strain BRD509. Given that the lack of effective vaccines against S. enterica pathogens can be largely attributed to an insufficient understanding of the host immune response to the pathogen, the immunological response to the TAS2010 vaccine strain was characterised in mice to understand the mechanisms responsible for the increased protective capabilities of this strain. In comparison to BRD509, TAS2010 was found to replicate to higher numbers in murine organs and induce an increased immune response in the form of increased interferon-gamma secretion by splenic CD4+ T cells. Evidence was presented which suggested that the protection provided by TAS2010 is less reliant on T cells and more dependent on a CD4-CD8-Thy1+ lymphocyte subset, probably Thy1+ NK cells. In conclusion, this study has enhanced the understanding of S. Typhimurium metabolism in the murine host and introduced a live vaccine strain with improved protective and immunogenic properties.
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