Microbiology & Immunology - Theses
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ItemRole of IFN-y induced genes in cell autonomous defence against Legionellae( 2021)L. pneumophila and L. longbeachae are ubiquitous environmental bacteria that can cause a severe pneumonia, known as Legionnaires’ Disease, when contaminated aerosols are inhaled by susceptible humans. The co-evolution of the bacteria with their environmental hosts has equipped the bacteria with the ability to subvert the cell intrinsic host defence mechanisms in human cells, thereby allowing the pathogens to survive and replicate within the lung macrophages. During infection, Legionella establishes an intracellular niche, known as the Legionella containing vacuole (LCV). The biogenesis of the LCV is dependent on the defective organelle trafficking/intracellular multiplication (Dot/Icm) type IV secretion system, which translocates a large arsenal of bacterial effector proteins into the host cell. These effectors are known to modulate host metabolism and cell-autonomous defence, protect the integrity of the LCV and allowing the bacteria to acquire nutrients from the host to ensure intracellular survival and enable intracellular replication of the pathogen. It is known that during Legionella lung infection, the mammalian host mounts a robust inflammatory response, producing cytokines, such as TNF-alpha, IL-1-alpha, IL-6, IL-12 as well as type I interferons and IFN-gamma, which usually leads to the restriction of intracellular replication and culminates in the clearance of the infection. It was previously shown that IFN-gamma is crucial for host defence against Legionella in mice, since disruption of IFN-gamma signalling or IFN-gamma deficiency results in a high replication of Legionella in the lung as well as a failure to clear the infection from the host, despite the activity of other inflammatory cytokines. Exposure of cells to interferons (IFN), including IFN-gamma, results in the induction of a network of genes that combat infection, leading to so-called IFN-mediated cell-autonomous defence. This network is finely-tuned to balance efficient pathogen control while preventing collateral tissue damage. However, which interferon-induced genes and through what mechanism this strikingly potent restriction is mediated remains elusive for Legionella. In this study, we shed some light on the mode of action of the IFN-gamma induced host defence against Legionella. We identified a new mechanism of host defence mediated by interferon stimulated genes (ISGs), that results in the disruption of effector translocation into host cells by the Dot/Icm secretion system. We demonstrated that this mechanism is uniquely triggered by interferon signalling and is independent of well-known host defence mechanisms such as host cell death, direct bactericidal activities, inflammasome activation as well as proteasome and autophagy-mediated degradation. By utilising mRNA sequencing of IFN-gamma and type I interferon-stimulated macrophages, we identified possible factors that mediate this inhibition: ISG15 and PARPs. These proteins have not previously been implicated in Legionella host defence and represent a unique opportunity to increase our knowledge of interferon mediated cell-autonomous host defence. Currently, more than 65 Legionella species are known and roughly half of them have been clinically associated with infection, frequently in immune compromised patients. After L. pneumophila, L. longbeachae is the second most common causative agent of Legionnaire’s Disease worldwide and is the leading causative agent in Australia and south-east Asia. Despite this, knowledge about the pathogenesis of L. longbeachae is minimal. Therefore, during this study, we also aimed to provide new insights into the pathogenesis of L. longbeachae infection and characterise the impact of IFN-gamma on immune control. We observed unique features of L. longbeachae infection in comparison to L. pneumophila, such as the ability to survive within a wider range of lung phagocytes, dampening of the cytokine response of the host and translocation of effectors into all lung phagocytes tested. These unique features may enable L. longbeachae to subvert the host defence more efficiently than L. pneumophila and thus replicate to higher numbers. Furthermore, we were able to show that IFN-gamma is crucial for host defence against L. longbeachae in vivo, with neutrophils and monocyte derived cells dependent on IFN-gamma signalling to mediate their bactericidal properties. In addition, we were able to demonstrate that IFN-gamma stimulation restricts L. longbeachae Dot/Icm secretion system effector translocation into host cells. Overall, this study substantiates the importance of IFN-gamma in host defence against Legionella and supports the need to broaden research efforts to non-L. pneumophila species. Investigation and deeper understanding of critical host defence mechanisms can be used as a starting point to develop anti-infective agents against pathogens targeting the process of effector translocation or effector mediated manipulation of host function and cell-autonomous defence.
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ItemIdentification and characterization of proteins and mechanisms involved in the uptake and traffic of vitamin B related antigens( 2021)Major histocompatibility complex, class I-related (MR1) presents Vitamin B-related antigens (VitBAg) at the cell surface to activate mucosal associated invariant T (MAIT) cells, directing homeostasis and immune responses. Although previous work has suggested endocytosis as a participant in MR1 presentation, how these antigens are captured by the cell is currently unknown. It is likely that MR1 ligands are uptake as metabolites for they have several structural similarities with molecules known to be transported through solute carrier (SLC) transporters. Here, we shown that flavins are pathway-specific inhibitors of MR1-5-OP-RU, and do not inhibit MR1-Ac-6-FP upregulation. We revealed that 5-OP-RU, ribityl lumazine (RL) and bacterial VitBAg, but not folate derived ligands, enter the cell through SLC52A family of riboflavin transporters, as their expression increases MR1 presentation and MAIT cell activation in a riboflavin modulated manner. In contrast, knock-outs models SLC52A family drastically reduce the incorporation of RLs but do not abolish the capacity to present 5-OP-RU through MR1. In fact, pathway specific inhibitors of MR1-5-OP-RU and MR1-Ac-6-FP extend to nucleosides, nucleobases and other drugs, arguing for the contribution of more SLC transporters in their uptake. Likewise, MR1 presentation during infection is increased by ligand-producing bacteria located in the cytosol, stating a cytosolic step to reach empty MR1 molecules. Finally, we showed that 5-OP-RU alters the metabolome of cells like LPS, leading to changes in their transcriptome profile. Our results unveil a new route for 5-OP-RU, RL and bacterial VitBAg uptake through SLC52A transporters, contributing to their capture and modulating MR1 presentation, together with a new potential role of 5-OP-RU as a pathogen-associated molecular patterns (PAMPs) molecule.
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ItemT cell response to an MHC-II restricted epitope of rodent malaria( 2021)Malaria is caused by different Plasmodium species that can infect a variety of animals including humans and rodents. The life cycle of these parasites is complex and includes a liver stage followed by a blood-stage in their vertebrate hosts. While the host’s immune response against each of these stages is incompletely understood, CD4 T cells are known to play an important role in immunity to Plasmodium infection during both stages. This project aims to examine the specific CD4 T cell response to a novel MHC II-restricted epitope in Plasmodium infection in C57BL/6 mice, and to characterise the protective capacity of these T cells. To this end, we made use of a recently generated TCR transgenic mouse line, termed PbT-II, which responds to a so far unknown Plasmodium derived epitope. In this project, the PbT-II epitope was identified as derived from heat shock protein 90, residues 484 to 496 (Hsp90484-496 or abbreviated DIY). Different priming methods, such as injection of an anti-Clec9A antibody attached to the Hsp90 epitope (aClec9A-DIY), infection with P. berghei ANKA (PbA) infected red blood cells (iRBCs) or immunisation with radiation attenuated PbA sporozoites (RAS), were used to characterise PbT-II memory cell formation. Results revealed the formation of memory PbT-II cells expressing surface markers associated with central memory T cells (TCM), effector memory T cells (TEM) and tissue resident memory T cells (TRM). Given the importance of tissue-resident memory T cells in peripheral immunity, mainly studied in CD8 T cells, we focused our study on the formation and function of CD4 TRM cells in the liver. Parabiosis studies using RAS vaccinated mice confirmed the liver residency of a CD69+ PbT-II cell population. Gene expression profile analysis revealed that these CD4 T cells expressed a core gene signature similar to that of CD8 resident memory T cells. Furthermore, differences in the gene expression profile of PbTII TRM cells generated via different protocols, suggested lineage specific effector mechanisms, such as IL-4 production or perforin expression, for subsets of CD4 TRM cells in the liver. As CD4 T cells can potentially act against both the liver and blood-stage of Plasmodium infection, we sought to investigate the protective potential of PbTII effector and memory cells for both of these stages. While none of the PbT-II priming methods resulted in a reduction of liver parasite burden upon sporozoite infection, mice injected with large numbers of in vitro polarized PbT-II Th1 or Th2 cells showed reduced parasitemia after PbA blood-stage infection. Surprisingly, most of these mice were protected from experimental cerebral malaria (ECM), although they were not able to clear PbA blood-stage infection.
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ItemNanoparticle interactions with the immune system( 2021)Vaccination has been an incredibly successful public health intervention, saving the lives of 2-3 million people each year. Despite this success, we still lack effective vaccines for many infectious diseases including HIV, tuberculosis and malaria. Nanoparticles (ordered structures within the range of 10-1000nm) have great potential to supplement traditional vaccines based upon pathogen subunits, or killed or attenuated microorganisms, as demonstrated by the successful licensure of virus-like particle vaccines for human papillomavirus and liposomal mRNA vaccines for SARS-CoV2. However, the immunological mechanisms that explain the potent immunity of nanoparticle vaccines and the factors dictating their interaction with the immune system are poorly defined. This thesis studies how nanoparticle characteristics affect their interaction with the immune system with a view to improving vaccine strategies. First, the contribution of the protein corona on the association of engineered nanoparticles with primary human blood cells was assessed. The association of high protein binding (high-fouling) mesoporous silica (MS) particles and low-fouling zwitterionic poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) particles with human white blood cells was assessed by flow cytometry in the presence or absence of plasma proteins. The effect of precoating nanoparticles with serum albumin, IgG and complement protein C1q was also assessed. The differential association of low and high-fouling nanoparticles was found to be largely a consequence of the de novo formed, not pre-adsorbed, biomolecular corona. Specifically, an enrichment of complement proteins within the corona resulted in an increased association with B cells. Second, the immune mechanisms that give rise to the improved immunogenicity of a prototypic nanoparticle vaccine were investigated. Humoral immune responses to a self-assembling protein nanoparticle vaccine for influenza (HA-ferritin) were contrasted to a subunit influenza vaccine (soluble HA) in mouse and non-human primate models. Antibody titres and protective efficacy of the vaccines were compared followed by a detailed study of lymph node germinal centre B cell and T follicular helper cell responses. Vaccination of C57BL/6 mice with HA-ferritin nanoparticles elicited higher serum IgG titres and greater protection against experimental influenza challenge compared with soluble HA vaccination. Within the antigen-draining lymph nodes, germinal centre reactions were expanded and persistent following HA-ferritin vaccination. This augmented humoral immunity was not driven by ferritin-specific T follicular helper cells but rather driven by expanded antigen colocalization with follicular dendritic cells. However, this immune enhancement did not translate from mice to pigtail macaques where antibody titres and lymph node immunity following HA-ferritin nanoparticle vaccination were comparable to soluble HA protein vaccination. And thirdly, we explored innate immune activation by HA-ferritin and soluble HA in mice. This was achieved through in vitro assessment of antigen glycosylation and complement activation and in vivo through serum IgM titres and cell trafficking to the lymph nodes following vaccination. HA-ferritin vaccination of mice was found to elicit an early enhancement of antigen-specific serum IgM however in vitro complement activation was not detected. Trafficking of immune cells to the lymph nodes was found to be influenced by antigen glycan composition in conjunction with purification methods. The findings of this thesis suggest that nanoparticle interaction with the immune system is driven by the complex interplay of nanoparticle physiochemical properties, antigen glycosylation, corona formation and pattern-recognition receptors of innate immune cells. Further improvements in understanding the relationship between these features and how they may differ between animal species will speed the rational design of next-generation nanoparticle vaccines against diverse pathogens.