Microbiology & Immunology - Theses

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    Dissecting the role of gd T cells in T cell priming for liver stage immunity
    Le, Shirley ( 2023-11)
    Liver resident memory T cells (TRM) are poised for protection against repeat infection and rapidly form a robust defence against tissue-specific insults such as liver stage malaria. A direct correlation between liver stage immunity and gd T cells has been observed both in mice (Zaidi et al. 2017) and in humans (Seder et al. 2013; Ishizuka et al. 2016), but the precise molecular mechanisms by which these gd T cells exert their protective effect are yet to be defined. In mice, intravenous injection with radiation-attenuated sporozoites (RAS) confers sterile protection against challenge with live sporozoites. This protection is mediated by responding antigen-specific CD8+ and CD4+ T cells that migrate to the liver and form resident-memory T cells (TRM). In the absence of gd T cells, protective CD8+ liver TRM are not generated, leaving mice susceptible to reinfection. Using Plasmodium-specific T cells as a readout for effective immunity, we determined that IL-4 is important for the accumulation of CD8+ and CD4+ T cells. By utilising complex in vivo systems including mixed-bone marrow chimeras and adoptive transfer of gd T cells, we revealed that gd T cell-derived IL-4 is crucial for the expansion of antigen-specific CD8+ T cells. In addition, in vivo neutralisation of IL-12 or IFN-g confirmed a partial dependency for these cytokines, despite their traditionally opposing function to IL-4. Given IL-4, IFN-g and IL-12 all have a clear role in CD8+ T cell priming following RAS vaccination, we hypothesised that IL-4 and IFN-g synergise to enhance cDC1 activity. These findings led to our development of a novel model to reconstitute cDC1-deficient mice using CRISPR-edited primary dendritic cells. This model enabled the investigation of the mechanism by which gd T cell derived IL-4 leads to DC activation and therefore effective CD8+ T cell expansion for memory development. Collectively, this project has shown a significant role for IL-4 in the priming of malaria-specific CD8+ T cells and demonstrates a novel pathway for collaboration between gd T cells, cDC1s, and CD8+ T cells, revealing the potential for harnessing gd T cells in vaccination strategies against malaria.
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    Antibody-mediated passive immunity against Helicobacter pylori
    Gorrell, Rebecca Jane. (University of Melbourne, 2008)
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    Induction of immune responses by lipopeptide vaccines
    Lau, Yuk Fai. (University of Melbourne, 2006)
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    Control of pulmonary immunity by physical exercise
    Michla, Marcel ( 2023-03)
    Sedentary lifestyles combined with high caloric nutrition are widely known to severely contribute to the rise of metabolic diseases like diabetes, atherosclerosis, or obesity in recent years. While endurance training has been shown to induce the secretion of adipokines, and so-called myokines (muscle-derived cytokines), the impact of physical exercise on the host immune response in the context of bacterial or viral infections remains largely unknown. Here, we aimed to investigate the impact of voluntary wheel running (VWR), mimicking an active lifestyle, on the pulmonary immune system and to which extent a lack of exercise might affect the severity of pneumonia induced by bacterial L. longbeachae or viral influenza A virus (IAV) infection. We observed that VWR enhanced stamina to exercise and reduced visceral adipose tissue. Moreover, VWR induced the expression of myokines and lipolysis-associated genes and decreased the number of circulating monocytes. Notably, neither acute nor long-term (2 and 8 weeks, respectively) physical exercise significantly affected the abundance or metabolism of pulmonary immune cells in healthy mice. However, upon infection with L. longbeachae, acute physical training reduced pathogen burden, dampened anorexia-induced weight loss, and decreased the recruitment of neutrophils and monocytes to the airways. Additionally, pro-inflammatory cytokines associated with bacterial clearance, including IFN-g & TNFa increased in the lungs of exercised mice. Notably, VWR enhanced the potential to produce TNFa in both alveolar macrophages and infiltrating monocytes early and late in infection with L. longbeachae. Furthermore, in running mice we found increased mitochondrial and glucose dependency in myeloid cells, crucial for the inhibition of pathogen replication. Our results also show that CD4+ T cells from trained animals display reduced IFN-g production, suggesting that exercise may predominantly boost the innate immune response. In contrast, during IAV infection, we observed significantly decreased numbers of activated type 1 helper T (Th1) cells in running mice, critical for viral clearance. However, the frequency of tetramer+ CD4+ and CD8+ T cells was increased, indicating higher antigen specificity of the immune response. Additionally, we found higher viral RNA content in lung tissue from exercising mice, and elevated production of pro-inflammatory cytokines including type I interferons. Moreover, we found higher expression of interferon-signalling genes in the pulmonary tissue of exercising mice. VWR increased the gene expression of Ifng in CD44+ CD4+ T cells in running mice, suggesting an enhanced capacity of CD4+ T cells to produce IFN-g. Notably, VWR increased the expression of tissue-residency markers on CD8+ T cells. Taken together, our results suggest that VWR might have opposing effects on pulmonary immunity during infection. Hence, we conclude that acute physical exercise might enhance protection against bacterial invasion (L. longbeachae) by specifically boosting the innate immune response. In contrast, VWR reduces Th1-mediated anti-viral responses and increases the pulmonary viral RNA content, suggesting that physical exercise might enhance the susceptibility to IAV infection. However, by boosting the adaptive IFN-g-mediated anti-viral response, antigen-specificity, and increasing tissue residency, exercise may enhance the defence against secondary viral infections.
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    The Development, Homeostasis, and Function of Unconventional T Cells
    Xu, Calvin ( 2023-09)
    Unconventional T cells detect non-peptide antigens presented by MHC class-I-like molecules, such as MR1 and CD1. MR1-reactive T cells, CD1-reactive T cells, and gamma-delta T cells represent three broad unconventional T-cell lineages which collectively are abundant and play key roles in the immune response. The frequencies of these cells vary widely between individuals, and the factors that govern their numbers and diverse effector functions are not well-understood. This thesis investigates the mechanisms that controls the development, homeostasis, and function of unconventional T cells and their subsets in the thymus and peripheral tissues. In chapter 3, MR1 and group 1 CD1 (CD1a, CD1b, and CD1c) tetramers were used to isolate and characterise rare unconventional T-cell populations in the human thymus. Using tetramer-mediated enrichment, thymic MR1-reactive T cells were found to comprise Valpha7.2+ and diverse Valpha7.2- subsets that differed in phenotype, binding to antigen-loaded MR1 tetramers, TCR repertoire diversity, frequency, and post-natal expansion. Whilst tetramer-mediated enrichment allowed for isolation of thymic CD1a- and CD1b-reactive T cells, blockade of CD36 expressed on thymocytes was additionally needed in order to detect CD1c-reactive T cells. Group 1 CD1-reactive T cells were highly rare after enrichment and generally resembled other CD4+ thymocytes. These findings highlight diverse MR1 and CD1-reactive T cell subsets in the thymus and forms the basis for understanding their intrathymic generation and developmental pathways. The expansion of MAIT cells in NKT- and gamma-delta T cell-deficient mice was investigated in chapter 4. Although MAIT cells were highly elevated in mice deficient in both NKT and gamma-delta cells, they phenotypically and functionally resembled their counterparts in wildtype mice. Mechanistically, this MAIT cell expansion was due to: 1) increased rearrangement of the MAIT cell TCR alpha chain within developing Tcrd-/- thymocytes, and 2) a higher capacity of peripheral MAIT cells to proliferate in the absence of NKT and gamma-delta cells. Overall, this chapter provides evidence for a shared niche in which MAIT, NKT, and gamma-delta T cells reside and compete for common homeostatic factors, revealing a novel interplay between their steady-state frequencies. In chapter 5, the regulation of peripheral unconventional T cells by the purinergic P2RX7 receptor was examined. Human unconventional T cells expressed P2RX7, whereas their mouse T-bet+, but not RORgt+, counterparts highly co-expressed the ADP-ribosyltransferase, ARTC2, and P2RX7. P2RX7 activation in response to ATP induced death of both mouse and human unconventional T cells ex vivo. Mouse T-bet+ unconventional T cells were highly susceptible to the effects of ARTC2-dependent P2RX7 activation in response to nicotinamide adenine dinucleotide, which resulted in their cell death ex vivo and depletion in vivo. By blocking ARTC2 or P2RX7, this chapter demonstrated the existence of IFN-gamma/IL-4 co-producing unconventional T cells, including MAIT and other non-MAIT/NKT alpha-beta T cell populations, which were selectively regulated by ARTC2-dependent P2RX7 activation. In contrast, this axis did not affect IL-17-producing unconventional T-cell subsets. These findings reveal a unique mechanism that controls the number and functional diversity of unconventional T cells via the selective regulation of their T-bet+ and IFN-gamma/IL-4 co-producing subsets. Overall, this thesis has examined the presence of diverse unconventional T cells in the human thymus, their regulation within a homeostatic niche, and their modulation by P2RX7 activation. Whilst unconventional T cells are collectively abundant, understanding their development and homeostasis will provide insight into why their frequencies vary widely in humans and how their numbers can be finely-controlled. The homeostatic relationships between unconventional T cells and how their functional subsets are regulated will be important considerations in the targeting of one or more unconventional T-cell populations within the immune response. Given their production of functionally-opposing cytokines, findings in this thesis will guide the development of future immunotherapies that leverage the abundance and potent effector functions of unconventional T cells in treating disease.
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    A multi-omic approach to understanding the mechanisms of daptomycin resistance in Enterococcus faecium
    Turner, Adrianna Marie ( 2023-11)
    Healthcare-associated infections caused by multi-drug resistant organisms such as vancomycin-resistant Enterococcus faecium (VREfm) are a public health threat. Daptomycin is a ‘last-resort’ antibiotic for VREfm infections with a novel membrane targeting mode-of-action, but for which resistance has surprisingly been reported in clinical strains. Despite the importance of daptomycin for treating VREfm infections, the genetic changes and molecular mechanisms leading to daptomycin resistance are poorly characterised in VREfm. This thesis aims to better understand these mechanisms using a ‘multi-omic’ approach – combining genomics, transcriptomics, proteomics, and lipidomics – on defined isogenic mutants and clinical VREfm strains. To understand which genetic mutations were associated with daptomycin resistance in clinical VREfm, we completed a genomic epidemiological and phenotypic study in Chapter 2. We demonstrate that daptomycin resistance is linked with the presence of novel mutations (G482D, H486Y, and S491F) in the B subunit of the bacterial RNA polymerase (RpoB) that confer resistance to rifaximin and cross-resistance to daptomycin. Surprisingly, these RpoB mutations emerge in VREfm following exposure to rifaximin, an unrelated antibiotic that is commonly prescribed to liver disease patients, a cohort at high-risk for VREfm colonisation and infection. Clinical VREfm isolates with these RpoB mutations were spread globally, across 20 countries and 5 continents, making this a major mechanism of resistance. Our study shows that rifaximin use may be compromising the clinical use of daptomycin through the selection RpoB mutations in VREfm. To understand how mutations in the B subunit of the bacterial RNA polymerase cause resistance to daptomycin, a cell membrane active antibiotic, we utilised a multi-omics approach in Chapter 2. Our analyses show that the G482D, H486Y, and S491F RpoB mutations mediate daptomycin resistance via a conserved mechanism, with each of these RpoB mutations causing similar transcriptional reprograming in VREfm. The dysregulation of a single, previously uncharacterised genetic locus was found to be solely responsible for daptomycin resistance. The locus, that we have named the Phenotypic Resistance to Daptomycin (prd) operon consists of a transcriptional regulator (PrdR) and two putative membrane proteins (PrdA and PrdB). Upregulation of the prdRAB operon leads to cell membrane remodelling, with decreased levels of anionic phospholipids (PG and CL) and increased levels of cationic phospholipids (Lys-PG). This ultimately decreases the negative cell surface charge and reduces daptomycin binding, which renders VREfm resistant to daptomycin. Mutations in the cell membrane stress response system LiaFSR are a major mediator of daptomycin resistance in VREfm, however, the molecular mechanism is unknown. We therefore created a panel of isogenic VREfm mutants and applied these same multi-omic approaches to understand how the clinically common mutations (namely LiaR W73C and LiaS T120A) in LiaFSR lead to changes in daptomycin susceptibility in Chapter 3. Our analyses show that mutations in LiaRS result in the dysregulation of several effector proteins, such as PrdRAB, LiaX, and a HD domain protein, all associated with daptomycin resistance. The LiaRS mutants displayed changes in cell membrane remodelling (decreased PG and CL and increased Lys-PG) and decreases in negative cell surface charge, leading to daptomycin resistance through reduced binding of the antibiotic. Through this work, we have identified a previously uncharacterised, but globally disseminated, daptomycin resistance mechanism mediated through mutations in the rpoB gene. Further, by understanding the molecular mechanism of daptomycin resistance in two major systems, RpoB and LiaRS, we demonstrate that although these mutations (RpoB G482D, H486Y, and S491F as well as LiaR W73C and LiaS T120A) occur in unrelated genes, they cause upregulation of the same operon (PrdRAB), leading to similar phenotypic changes to the cell membrane. These data indicate that electrostatic repulsion is an important mechanism of daptomycin resistance in VREfm.
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    Characterisation of Inflammasome-Induced Extracellular Vesicles, Their Uptake by and Effect on Bystander Cells
    Standke, Lena ( 2023-03)
    Inflammasomes are multimeric complexes whose activation triggers caspase-1 cleavage resulting in processing of the cytokines interleukin (IL)-1b and IL-18, and the pore-forming protein gasdermin D. This, in turn, initiates programmed inflammatory cell death (pyroptosis). However, even in the absence of IL-1b and IL-18 signalling, inflammation develops, pointing towards the importance of other factors released upon inflammasome activation, such as activated inflammasome itself or extracellular vesicles (EVs). We therefore hypothesised that EVs enhance the paracrine inflammatory effects of inflammasome-activated cells. To investigate this, we isolated EVs from inflammasome-activated cells using differential centrifugation and size-exclusion chromatography. We next analysed the ribonucleic acid (RNA) and protein content of EVs and investigated their uptake by and their effect on different bystander cells. Our results show that EV secretion is increased in macrophages stimulated with inflammasome activators relative to controls. Inflammasome-elicited EVs can be identified by the presence of N-terminal gasdermin D, as well as by their distinct RNA signatures. Thus, they have the potential to be used as biomarkers in clinical settings. EVs can exert their effects over long distances and may therefore contribute to the propagation of inflammatory signals from one part of the body to cells at distant sites. We could show that inflammasome-elicited EVs are taken up by diverse recipient cells, including macrophages, endothelial cells, epithelial cells, fibroblasts, and T cells (primarily activated T cells). We then used transcriptomics to determine the effect of inflammasome-elicited EVs on these recipient cells. They for example induced inflammatory gene sets in endothelial cells and led to the upregulation of marker genes of activated endothelium, including adhesion molecules, which are known to facilitate the attachment and tissue invasion of immune cells. Upon interaction with fibroblasts EVs have the potential to induce inflammatory signalling, further propagating inflammation. Gene sets such as interferon (IFN)-a/-g, and tumour necrosis factor (TNF)-a signalling were enriched in these cells. Taken together, these findings indicate that EVs may not only serve as diagnostic markers for inflammatory disease, but also play an important role in the systemic response towards inflammasome activation.
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    Programmed cell death regulation during Salmonella Typhimurium infections
    Engel, Sven Julian ( 2023-04)
    Programmed cell death (PCD) is a highly regulated process that is vital for the development and homeostasis of multicellular organisms. It functions as an essential mechanism to remove dispensable or unfavourable cells in a coordinated manner under physiological as well as pathophysiological conditions. Importantly, this type of cellular suicide also serves as an effective defence strategy to control intracellular pathogens that aim to repurpose host cells as replicative niche and to evade extracellular immune responses. Recent findings indicate that host cells utilise multiple PCD pathways to fight invading pathogens. However, the organisation and regulation of this complex cell death network consisting of pyroptosis, apoptosis and necroptosis and their relative importance for the control and clearance of intracellular infection is not completely understood. In this thesis, we systematically investigated the cellular and molecular requirements for PCD induction and the contribution of different PCD pathways to immunity against intracellular bacteria. We infected novel genetically modified mouse strains deficient for various combinations of PCD mediators, such as different cysteine-dependent aspartate-directed proteases (caspases), with the bacterial model organism Salmonella enterica serovar Typhimurium (S. Typhimurium) to analyse their ability to control intracellular infections. Additionally, we established in vitro assays to disentangle the complex interactions of the PCD network by analysing the cell death kinetics of bone-marrow derived macrophages obtained from various mouse strains in real-time and determining the intracellular bacterial burden following infection with S. Typhimurium. Our findings unveiled that the PCD pathways pyroptosis and apoptosis are highly interconnected and regulated with a remarkable level of redundancy. We identified that pyroptosis and apoptosis are essential for the control of S. Typhimurium and that molecular components of these pathways, such as caspase-1 and -8, can be used interchangeably to counteract bacterial evasion strategies. Furthermore, we investigated the underlying mechanisms that coordinate the flexible induction of pyroptosis and apoptosis during S. Typhimurium infection. We demonstrated that caspase-2 neither plays a significant primary nor compensatory role in the regulation of cell death and control of intracellular infections. Upon excluding caspase-2 as link between PCD pathways, we evaluated means of extracellular cell death induction. The results presented in this thesis imply critical functions for cytotoxic CD4+ T cells and interferon gamma (IFN-g) in extrinsic apoptosis induction of S. Typhimurium infected cells. In conclusion, our results highlight the relative importance, interconnectivity and redundancy of different PCD pathways. This study provides detailed insights into the highly complex network of PCD by unravelling new functions for several of its components and thereby defining novel mechanisms of cell death induction during infections with S. Typhimurium. These findings aid to discover new drug targets and develop novel treatment strategies to fight intracellular infections by enhancing essential host immune responses.