Microbiology & Immunology - Theses
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Functional and cellular heterogeneity of the myeloid cell system
Cells of the myeloid lineage form the innate part of the immune system and are characterized by a high level of functional plasticity, which is required to address the diverse set of functions of these mononuclear cells. Monocytes, Macrophages and dendritic cells (DC) are collectively categorized as the mononuclear phagocyte system (MPS), to highlight their functional equipment that specializes them to the phagocytosis of pathogens as a starting point to elicit an immune response. Besides this role, cells of the MPS are also involved in a wide variety of homeostatic functions including early development and regulation of physiological processes. However, the multitude of mechanisms required to acquire this functional plasticity remains poorly understood. The work that has been performed in the scope of this dissertation aimed to advance current knowledge of the causes and consequences of functional and cellular plasticity of the myeloid immune system. High-dimensional characterization of the effects of a Western diet on myeloid immune cell progenitor cells revealed a long-term transcriptional and epigenetic reprogramming of the myeloid cell compartment. The formation of an innate immune memory in myeloid progenitor cells leads to lasting inflammatory priming of monocytes, which may directly contribute to the progression of myeloid cell-associated diseases. In addition, single-cell RNA-seq elucidated unreported cellular heterogeneity of the monocyte and dendritic cell compartment in human peripheral blood. A combination of phenotypic and transcriptional analyzes resulted in a precise categorization of the human DC compartment consisting of pDCs, cDC1, two cDC2 subsets, and a deeply characterized preDC subset. Furthermore, a universal strategy for the integration of cellular atlases was conceptualized and applied to establish a consensus map of the human DC and monocyte cell space. This thesis provides mechanistic insights into the cellular composition of myeloid cells and their functional plasticity, which will form the foundation for further investigations into the dynamic changes of the immune cell compartment during diseases and will be critically relevant for designing effective treatments for a wide variety of pathologies linked to myeloid cells.
Investigating the ability of mammalian Membrane Associated RING-CH proteins to modulate respiratory virus infections
Respiratory viruses generally infect epithelial cells lining the upper and lower airways, however subsets of airway immune cells are also susceptible to infection. Of interest, airway epithelial cells (AEC) and airway macrophages (AM) are both susceptible to influenza virus infection, but only AEC support productive virus replication. Constitutive expression and/or induction of intracellular host proteins to limit or block virus replication is a well-known antiviral mechanism, however few such “restriction factors” have been well characterized and the antiviral activity of many putative restriction factors against respiratory viruses has not been reported. A recent RNA-seq study performed in our laboratory compared gene expression between AEC and AM in the presence or absence of influenza infection, allowing us to identify protein families with differential expression between these two cell types in steady-state and/or following virus infection. We hypothesized that amongst genes differentially expressed between AEC and AM would be those that represent novel restriction factors and these might contribute to the abortive versus productive phenotype observed in AM and AEC, respectively, in regard to influenza replication. One of the protein families identified were the membrane-associated RING-CH (MARCH) family proteins. MARCH proteins are E3 ubiquitin ligases involved in the final step of the ubiquitination process. Amongst their functions, MARCH proteins modulate host immune responses by virtue of their ability to control the turnover of multiple immune molecules, including major histocompatibility complex (MHC) proteins. More recent evidence indicates that they can also modulate virus infection, acting as restriction factors against viruses such as human immunodeficiency virus (HIV)-I, or as proviral factors for viruses such as hepatitis C virus (HCV). Based on these findings, this thesis aimed to investigate the ability of MARCH family proteins to modulate infections caused by influenza and other respiratory viruses. First, we generated stable cell lines with doxycycline (DOX)-inducible expression of several human MARCH proteins (MARCH1/2/3/5/6/7/8/9) and evaluated their ability to modulate either the early or late stages of infections caused by influenza A viruses (IAV), respiratory syncytial virus (RSV) or human metapneumovirus (hMPV). Although none of the MARCH proteins tested affected the early stages of IAV infection (as measured by flow cytometry for newly-synthesized viral proteins), inducible expression of MARCH8 was associated with a significant reduction in titres of infectious IAV released from infected cells. Moreover, MARCH8 expression was also associated with a reduced percentage of RSV-infected cells, consistent with its ability to restrict at an early stage of the RSV replication cycle, although this was not explored further in this thesis. We did not observe any significant differences in the ability of any MARCH protein tested to modulate early or late stages of hMPV infection. Subsequent studies have focussed on understanding MARCH8-mediated restriction of influenza virus infections. Next, we characterized the antiviral activity of MARCH8 against influenza viruses. MARCH8 was shown to mediate antiviral activity against a range of influenza viruses relevant to human health, including A/H1N1, A/H1N1pdm09 and A/H3N2 viruses, as well as against an influenza B virus. We demonstrated that expression of MARCH8, but not the closely related MARCH1, was associated with a reduction in the specific infectivity of virus particles released from IAV-infected cells. Moreover, virus particles released from IAV-infected cells in the presence of MARCH8 exhibited altered protein composition and virion morphology, and MARCH8 was incorporated into the nascent virions. Of interest, MARCH8 expression did not alter cell-surface expression of the viral proteins HA, NA and M2, indicating no major defect in their synthesis and transport during viral replication. Overall, these findings are consistent with MARCH8 acting to block a step late in the virus replication cycle, possibly during virus assembly and/or budding from the cell surface. Studies described in this thesis also attempted to determine features of MARCH8 and IAV relevant to MARCH8-mediated restriction of IAV. We used site-directed mutagenesis and reverse genetics to modify lysine residues in the cytoplasmic tails of the viral hemagglutinin (HA), neuraminidase (NA) and matrix protein (M)2 proteins, either alone or in combination. However, none of these substitutions were sufficient to overcome MARCH8-mediated restriction of IAV. We also modified several lysine residues in the viral M1 protein but again did not identify specific residues to abrogate MARCH8-mediated restriction. Currently, the identity of the particular viral and/or host proteins targeted by MARCH8 which result in inhibition of late stage IAV replication are not known. To study MARCH8 itself, we generated mutants lacking E3 ligase activity and confirmed that this was essential for its anti-IAV activity. Furthermore, given that MARCH8, but not the closely related MARCH1, mediated potent anti-IAV activity against some strains of IAV, we generated cell lines with DOX-inducible expression of MARCH1-MARCH8 chimeric proteins in an effort to determine particular domains of MARCH8 critical to its anti-IAV activity. Chimeric proteins contained one or more domains/regions of MARCH1 substituted into the MARCH8 backbone. While all chimeric proteins retained activity against a common target protein (CD86), all of them also retained ability to restrict IAV replication. Based on these studies, we were unable to identify critical domains of MARCH8 that were essential for its antiviral activity against IAV. Finally, we used a MARCH8 knockout (KO) mouse to evaluate the role of endogenous MARCH8 in modulating IAV infection in vivo. Following intranasal infection, MARCH8 KO mice exhibited enhanced weight loss and viral replication in the lungs at day 5 post- infection, although no major differences in virus titres or virus clearance were observed at later time-points. Moreover, we did not observe major differences in soluble inflammatory mediators or inflammatory cells at day 7 or 10 post-infection. These findings are consistent with a role for endogenous MARCH8 in controlling the early stages of IAV infection in vivo but suggest that it does not have a major impact on virus clearance or the development of adaptive immunity in this model. Of interest, siRNA knockdown of endogenous MARCH8 in a human epithelial cell line also resulted in a modest, but significant, reduction in titres of IAV released from infected cells in vitro. Together, the studies presented in this thesis describe and characterize the antiviral activity of MARCH8 against influenza viruses. In addition, they provide preliminary data to indicate that MARCH8 can also mediate antiviral activity against the pneumovirus RSV, albeit by a distinct mechanism. Overall, these findings contribute to a growing body of evidence that MARCH8 plays an important role in modulating infections caused by a range of different viruses with relevance to human health.
Coxiella burnetii control of the host transcription factors TFEB and TFE3
Coxiella burnetii, the etiological agent of the zoonotic disease Q fever, is an obligate Gram-negative intracellular bacterial pathogen that replicates inside the lysosome-derived CCV (Coxiella-containing vacuole) within mammalian hosts. The CCV maintains the degradative and acidic nature of the host lysosome despite C. burnetii directing the massive expansion of this compartment to accommodate the replicating pathogen. To establish this unique replicative niche, C. burnetii requires the Dot/Icm type IV secretion system (T4SS). This T4SS translocates approximately 150 effectors into the host cell to modulate various cellular processes. To date, the functional role of very few of these effectors have been defined. Given the CCV’s origins it is not surprising that C. burnetii infection increases host autophagy and lysosome biogenesis. To investigate this at the protein level, we employed an elegant SILAC based proteome analysis of human cells infected with C. burnetii. This validated that many proteins involved in these processes are increased in abundance during infection. This prompted us to examine the role of the human transcription factor EB (TFEB) and its close homologue TFE3 during C. burnetii infection. TFEB is a master transcription regulator directly controlling the expression of a network of genes responsible for autophagy and lysosome biogenesis. 3 day’s post-infection with C. burnetii, TFEB/TFE3 is activated as demonstrated by TFEB/TFE3 trafficking from the cytoplasm into the nucleus. The nuclear translocation of TFEB/TFE3 appears to be controlled by C. burnetii as blocking bacterial translation with chloramphenicol leads to TFEB/TFE3 movement back into the cytoplasm. siRNA silencing of tfeb and tfe3 additionally demonstrated their contribution towards the intracellular success of C. burnetii. Interestingly, these host factors did not contribute to the replication of C. burnetii but in the absence of TFEB and TFE3 the CCV did not undergo its typical massive expansion. This research was able to demonstrate that C. burnetii induced activation of TFEB/TFE3 was dependent on the Dot/Icm T4SS thus we hypothesized that an effector(s) of this system may manipulate TFEB/TFE3. An unbiased visual screen was conducted to identify effectors that influence this process and two putative C. burnetii effector proteins, namely CBU1701 and CBU2016, were identified. We demonstrated that ectopic expression of these proteins leads to nuclear localisation of TFEB. Subsequent characterisation of the impact of CBU1701 and CBU2016, demonstrated that they influence the host proteome in similar ways but with surprisingly little impact on TFEB- regulated proteins. These results indicated that nuclear localisation of TFEB in response to CBU1701 and CBU2016 may be uncoupled from activation of this host transcription factor. In addition to characterising the impact of these effector proteins on host cellular function, we set out to understand the role they play in intracellular replication and virulence of C. burnetii. Genetic manipulation of this pathogen is in its infancy and remains technically challenging however this study reports the successful production of multiple mutant strains. Initial characterisation experiments demonstrate that CBU1701 and CBU2016 likely make important contributions to the establishment of the C. burnetii intracellular niche. Overall, the research carried out in this thesis has worked towards elucidating the contribution of TFEB and TFE3 to C. burnetii infection and developing an understanding of the molecular players in this process. Significant tool development and foundational findings reported here will pave the way to a deeper understanding of the interplay between intracellular bacterial pathogens and the host response to infection. Additionally, using C. burnetii effector proteins as a novel biological toolbox may uncover important insights that will impact our understanding of a range of human molecular pathways that impact human health.
Advancing the ferret as an immunological model to study B-cell responses
Introduction Influenza is a clinically significant disease, causing 24000-62000 deaths alone in the United States during the 2019-2020 season. While annual vaccines are available, variable efficacies have been reported and annual updates are required due to antigenic drift. Ferrets are a useful model for studying human respiratory viruses and have been widely used to evaluate vaccines and transmission of influenza. Sera from ferrets infected with different influenza strains are used in HI assays as part of strain determination of seasonal influenza vaccines. A key limitation of the ferret model is the paucity of immunological reagents to characterise immune responses and a lack of knowledge regarding the ferret immune system. This PhD thesis aims to advance the ferret as an immunological model to study human respiratory viruses by developing methods and reagents which will enable in-depth interrogation of ferret B-cell responses. Methods While a draft copy of the ferret genome is available, immunoglobulin sequence information is not well-annotated. Hence, we first annotated the ferret genome with immunoglobulin variable, diversity, joining and constant chain genes by inferring homology using human and canine orthologs (Chapter 3). Novel PCR primers targeting 5’- leader, 3’- joining and 3’- constant chain immunoglobulin genes were derived, enabling the recovery of functional, paired heavy and light chain transcript sequences from single sorted ferret B-cells. Ferret immunoglobulin constant sequences were validated by RNA-seq, which enabled the development of ferret IgG expression plasmids. Using this technique, HA-specific B-cell responses were characterised for the first time in ferrets at the transcript level (Chapter 4). Candidate ferret mAbs were derived from the recovered sequences, expressed and screened for HA binding specificity and in-vitro influenza virus neutralisation activity. We noted poor recovery of ferret HA specific mAbs and subsequently sought to improve flow cytometric panels available for ferrets. We established a methodology using previously developed murine single-cell BCR sequencing methods to recover murine anti-ferret mAbs (Chapter 5). First, coding sequences of ferret B and NK-cell reagents were identified on the ferret genome and validated by sequence and structural comparisons with other mammalian homologs. C57BL/6 mice were subsequently immunised with these antigens and candidate mAbs were recovered for examination by ELISA and flow cytometry. Results Ferret variable, diversity, joining and constant chain coding genes were identified on the draft copy of the ferret genome and show good sequence similarity to human and canine variants. Our novel ferret immunoglobulin specific PCR primers enabled the recovery and characterisation of germline ferret immunoglobulin genes from single sorted ferret B-cells. RNA-seq validation of ferret immunoglobulin constant chain genes subsequently enabled the construction of ferret IgG/IgL expression plasmids. This facilitated the expression of chimeric human-ferret CR9114 IgG antibody retaining HA binding specificity. Subsequently, using previously developed trimeric HA probes, clonally expanded sequences were recovered from single sorted HA-specific B-cells derived from infected ferrets. Screening of candidate ferret monoclonal antibodies enabled the identification of two novel antibodies, belonging to the same clonal family showing HA binding specificities. Further examination by HAI and microneutralization assays revealed the ability of the mAbs to neutralise influenza virus in vitro. Viral escape mapping revealed binding epitope to previously reported Sa site of the HA head domain, showing proof of concept for mapping HA epitopes using these recombinant ferret mAbs. We next attempted to improve flow cytometric panels for ferrets which will enhance recovery of ferret immunoglobulin transcripts. As there are currently no mAbs targeting B and NK-cell markers in ferrets, we identified key markers for murine mAb development including CD19, IgD, CD138, NKp46 and LAMP-1. We identified candidate anti-ferret CD19 and IgD mAbs which bound to cognate recombinant antigens by ELISA, validating this method for generating anti-ferret mAbs to improve panels for flow cytometry and confocal microscopy. As the mAbs in this thesis lacked the capacity to resolve ferret cell populations by flow cytometry, we identified and discussed key steps in the process which will inform future use of this approach to develop anti-ferret mAb reagents. Conclusion The body of work presented in this thesis forms the proof of concept of studying antigen-specific B-cell responses at the mAb level in ferrets. Future improvements in tools developed in this thesis and future development of reagents will enable detailed interrogation of the ferret immune system.
Investigation of rare actinomycetes for novel antimicrobials
Nocardia are a genus of ubiquitous environmental bacteria belonging to the phylum Actinobacteria. Genomics has revealed that Nocardia species are endowed with extensive and varied arrays of secondary metabolite biosynthetic gene clusters with the potential to produce natural products that have antibiotic properties. Furthermore, the abundance of such gene clusters within the Nocardia rivals that of Streptomyces, the signature genus among the Actinobacteria, owed to the fact that Streptomyces species have yielded many clinically used antibiotics. This project aimed to address the current antibiotic resistance crisis and the shortfall in new compounds within the drug discovery pipeline. A range of natural product discovery techniques were utilised amongst different Actinobacteria with a particular focus on a collection of species within the generally overlooked genus Nocardia. This study had three primary objectives, the first was to use a traditional, high-throughput, empirical screen of 169 pathogenic actinomycetes predominantly from the genus Nocardia. These isolates were screened for antibiotic activity on 19 distinct growth media against a panel of five highly prevalent, multidrug resistant pathogens (Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecium and Acinetobacter baumannii). Secondly, whole genome sequencing and bioinformatic interrogation of 100 Nocardia species was conducted to assess their genetic potential to biosynthesise natural products. This facilitated the selection of a single Nocardia isolate which possessed a non-ribosomal peptide synthetase locus that appeared to be unique amongst other Nocardia species. The locus was also transcriptionally silent. Bioengineering using promoter refactoring was employed to activate expression of this gene cluster, the product of which might have potential as a novel antimicrobial. Thirdly, by utilisation of liquid chromatography-mass spectrometry (LC-MS), bioinformatics and molecular networking, a metabolomic approach was employed to gain a global secondary metabolic footprint of ten predicted “biosynthetically talented" Nocardia species grown on five distinct media types. This project identified: (i) A Nocardia sp. with activity against multidrug resistant Acinetobacter baumannii. (ii) Two Streptomyces isolates (Streptomyces cacaoi and Streptomyces sp.) which exhibited antimicrobial activity against multidrug resistant Escherichia coli and Acinetobacter baumannii respectively. Secondary metabolite extracts from each of these producing isolates were investigated by LC-MS/MS and the resulting spectra was assessed for uniqueness through a dereplication data platform developed specifically for bacterial natural product identification. No hits for previously discovered metabolites were obtained suggesting that the antimicrobials discovered within this project appear to be unique and have potential as new drug leads for today’s ever-decreasing antibiotic discovery pipeline. (iii) Four distinct families of bioactive secondary metabolites that were produced by multiple Nocardia species following LC-MS/MS and molecular network analysis. The identified secondary metabolites were correlated with genome sequence data to identify their probable biosynthetic origin in Nocardia species.
Neuroimmune responses in viral infection
The regulation of the immune responses is important in maintaining good health. Interactions between the nervous and immune systems are increasingly studied and widely appreciated to be influential in orchestrating immune responses. T cells express adrenergic receptors (AR) that enable them to respond to neurotransmitters produced by the sympathetic nervous system (SNS), noradrenaline (NA) and adrenaline, inducing downstream signalling and modulating cell functions, although whether this is stimulatory or inhibitory in T cell antiviral responses is unclear. In this thesis, I examine the effects of SNS in various models of viral infection through chemical sympathectomy and treatment with AR agonists. Modulation of sympathetic signals in systemic infections with LCMV had minor influences on T cell responses but resulted in increased viral loads. Notably, the infection results in a loss of tyrosine hydroxylase (TH) positive sympathetic fibres in the spleen as early as day 3 post infection and is reflected by decreased NA splenic NA. The immune response may play a role with interferon g partially contributing to the depletion. Additionally, this thesis also investigates the capability of long-lasting resident memory T cell (TRM) responses in the highly innervated, immune-privileged cornea. Using a model of herpes infection of the cornea, I showed that T cells are effectively recruited to the cornea with a small heterogenous population able to persist following cessation of immune responses. These cells express CD69 and CD103, canonical markers of tissue residency, to varying degrees. Persistence, but not recruitment, of these cells is dependent on antigen availability at the cornea. These memory cells are capable of responding to secondary encounters with antigen. Moreover, circulating memory cells are also able to infiltrate the immune-privileged cornea more efficiently following infection. Together, these results highlight the important nuances in the regulation of immune responses by the nervous system.
The dynamics of the B cell response during influenza A virus infection
Although vaccination remains the most effective method of managing influenza epidemics, there is still much that remains to be characterized about humoral immunity against the varying contexts in which influenza infection can occur. With the continuous subversion of humoral immunity by seasonal influenza through antigenic drift and the potential of zoonotic influenza viruses adapting and spreading through human populations through antigenic shift, improving our understanding of B cell immunity against different types of influenza infection could provide important insights into improving management of epidemics and vaccine formulations. In order to understand B cell responses during influenza infection, the well-characterized C57BL/6 mouse model was used to investigate and compare humoral responses in the context of different influenza infection histories. Markers that identified specific B cell subsets such as germinal centre (GC) B cells and plasmablasts were analysed by flow cytometry paired with influenza virus-specific B cell ELISPOT assays to investigate strain-specific antibody secreting responses within the same experiment. As the surface glycoprotein HA is thought to be the immunodominant response for B cell responses against influenza virus, the prediction is that the greater the antigenic differences between the HA of the first and second infecting strains, the more primary-like the response to the second strain would be. Primary and homologous secondary B cell responses in the mediastinal lymph node (MLN) and spleen were first characterized using this model to establish baseline responses against influenza virus before heterosubtypic infection was studied through infection of mice with H1N1A/Puerto Rico/8/34 (PR8) virus followed by H3N2 A/Udorn/305/72 (Udorn) virus 7 weeks later. Unexpectedly, a secondary-like plasmablast, GC B cell and Udorn-specific antibody secreting cells was observed during heterosubtypic infection, with earlier and higher magnitude B cell responses. These findings suggested a possible role for cross-subtype T cell memory in modulating B cell responses. The effect of antigenic drift on the B cell responses during influenza infection was then analysed with the same model. Mice were infected with H3N2 strains isolated between 1972 and 1979, representing different antigenic distance from a virus isolated in 1982 (Ph82). Seven weeks post infection mice were reinfected with Ph82 and the B cell response over the course of infection examined. It was found that infection of strains up to 10 years apart appeared to induce a secondary-like B cell response in the secondary lymphoid organs when compared to baseline primary and secondary responses against Ph82 virus. Prior infection with any H3N2 strain also resulted in minimal viral replication during the secondary challenge when compared to primary infection groups. However, data from both primary antibody inhibition and HA-specific B cell responses appears to suggest a narrower threshold of recognition, around a maximum of 3 years drift before serum and HA-specific responses cease to bind with other strains. Taken together, secondary-like B cell responses in both heterosubtypic and drift models of infection and in the case of drift responses, irrespective of reactivity of HA-specific B cells, appear to refute the hypothesis that virus-specific B cell responses would reflect antigenic relatedness between the HA of the infecting strains. Overall, data from this study identifies the diversity of the overall B cell response against influenza infection in the context of prior exposure to strains of different antigenic properties. Further study into the reactivity of these B cells against different influenza virus components and the role of memory T cells in the observed responses may provide important insights into the nature of host immunity against the ever-shifting target of influenza virus.
Understanding the mechanisms utilised by Coxiella burnetii for intracellular replication within the host phagolysosome
The human pathogen Coxiella burnetii is the causative agent of Q fever, a febrile illness which may lead to chronic disease in a small percentage of infected individuals. C. burnetii is a unique Gram negative intracellular bacterium which replicates within a host cell lysosome derived vacuole, termed the Coxiella containing vacuole (CCV). Currently, the exact mechanisms which allow C. burnetii to replicate within this normally hostile compartment are unknown. In order to understand how C. burnetii survives within this intracellular niche, this research investigated carbon metabolism of both intracellular and axenically cultivated bacteria, using steady state metabolic profiling and 13C stable isotope labelling. Both C. burnetii populations were shown to assimilate exogenous [13C]glutamate and [13C]glucose, with concomitant labelling of intermediates in glycolysis and gluconeogenesis, and in the TCA cycle. Significantly, the two populations displayed metabolic pathway profiles reflective of the nutrient availabilities within their propagated environments. Disruption of the C. burnetii glucose transporter, CBU0265, by transposon mutagenesis led to a significant decrease in [13C]glucose utilisation but did not abolish glucose usage, suggesting that C. burnetii express additional hexose transporters that may be able to compensate for the loss of CBU0265. This was supported by intracellular infection of human cells and in vivo studies in the Galleria mellonella insect model showing loss of CBU0265 had no impact on intracellular replication or virulence. Using this mutagenesis and [13C]glucose labelling approach, this study identified a second glucose transporter, CBU0347, the disruption of which also showed significant decreases in 13C label incorporation. Despite maintaining a relatively small genome, C. burnetii have retained seemingly redundant strategies to obtain glucose. This suggests that glucose may be an important metabolite for C. burnetii. Together, these analyses indicate that C. burnetii may use multiple carbon sources in vivo and exhibits greater metabolic flexibility than expected. In addition, this thesis also investigated the novel and unique C. burnetii protein CBU2072, a small, 18.3 kDa protein, which has subsequently been named essential for intracellular replication A (EirA), as loss of this protein prevents replication of C. burnetii within the CCV. Intracellular replication of the EirA mutant can be restored during co infection of the same vacuole with C. burnetii wild type, which is analogous to the phenotype observed for mutants of the Dot/Icm type 4B secretion system in C. burnetii. EirA localises to the C. burnetii inner membrane, and absence of this protein leads to the loss of Dot/Icm effector translocation. These data together contribute important understanding of the unique mechanisms involved in C. burnetii pathogenesis within the host phagolysosome.
The molecular and cellular basis of antigen recognition by unconventional T cells
Unconventional T cells are evolutionarily conserved populations of T cells, many of which recognise non-peptide antigens in the context of monomorphic antigen presentation molecules related to the major histocompatibility complex (MHC). These MHC related molecules, known as CD1 and MR1 present lipid and small metabolite antigens respectively. Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T cells that are highly abundant in humans and recognise the potent riboflavin biosynthesis derived metabolite 5-OP-RU presented by MR1. The majority of MAIT cells in the peripheral blood of humans express the CD8 co-receptor, a molecule also expressed by other T cells that contributes to antigen recognition and T cell activation. In contrast to other T cells, a role for CD8 on MAIT cells has not been formally tested. The first chapter of results in this thesis examines whether CD8 is able to bind MR1, and if CD8 is important for the activation of MAIT cells and other MR1-reactive T cells. The data revealed that CD8 binds directly to MR1, in a manner concordant with MHC class I, and that this interaction is important for the functional response of MAIT cells. Specifically, both isoforms of CD8 (CD8aa and CD8ab) bound to MR1 tetramers and the CD8-MR1 interaction could be abrogated by mutating MR1 in the putative CD8 binding site. The effects of the CD8-MR1 interaction were examined on primary MAIT cells, where MR1 tetramers bound more strongly to CD8+ compared CD8- MAIT cell subsets. Importantly, mutating MR1 tetramers reduced CD8+ MAIT cell engagement to CD8- MAIT cell levels. To determine the effect of the CD8-MR1 interaction on function, primary MAIT cells were activated in the presence of wild type or mutant MR1. In line with the importance of CD8 on MR1 recognition, the cytokine secretion by CD8+ MAIT cells was decreased in the absence of CD8 binding to MR1. Furthermore, the data here establishes that CD8 is vital for the recognition of MR1 presenting less potent, lower affinity antigens such as folate derivatives. Similarly, low affinity MR1-antigen recognition and cytokine secretion by other MR1-reactive T cells was completely abrogated in the absence of CD8 binding. Thus, CD8 is an important co-receptor for the function of MAIT cells and may expand the diversity of ligands recognised by MAIT and other MR1-reactive T cells. CD1b-restricted T cells are a subset of unconventional T cells in humans that recognise lipid antigens derived from endogenous and microbial sources that are presented by CD1b. Comparatively little is known about the biology of CD1b-restricted T cells, particularly autoreactive CD1b-restricted T cells that recognise endogenous lipid antigens such as phospholipids. The second chapter of results examines autoreactive TCR recognition of CD1b, including the breadth of permissive endogenous lipid antigens that are bound by mammalian CD1b. Autoreactive CD1b-restricted T cells were identified from healthy blood donors using CD1b tetramers presenting heterogeneous mammalian lipids and the autoreactive T cells expressed distinct T cell receptors (TCRs) which were cloned to generate autoreactive T cell lines. Different CD1b restricted T cell lines exhibited altered staining reactivity with CD1b tetramers loaded with different mammalian phospholipid antigens and strikingly, some autoreactive T cell lines recognised CD1b in an antigen independent manner. An activation assay using the autoreactive T cell lines cocultured with a series of mutant CD1b expressing cell lines revealed several CD1b binding ‘hotspots’ along the a1 and a2 helices that were critical for TCR-mediated activation. Using soluble proteins, autoreactive TCR permissive ligands were isolated from mammalian CD1b by separating ternary TCR-CD1b-lipid complexes from binary CD1b-lipid complexes that did not bind to the TCR using size-exclusion chromatography. Mass spectra analysis of the fractionated proteins revealed an abundance of phospholipids, particularly phosphatidylcholine as permissive CD1b lipid antigens. These data suggest autoreactive TCR antigen-specificity is more diverse than previously described and that these TCRs may adopt novel docking modes onto CD1b to recognise distinct endogenous antigens. In the final chapter of results, the phenotype and functional characteristics of M. tuberculosis-reactive CD1b-restricted T cells was investigated. Glucose monomycolate (GMM) is a cell wall lipid expressed by pathogenic Mycobacteria, including M. tuberculosis, that is presented by CD1b to T cells. GMM-reactive T cells were first isolated directly ex vivo from the peripheral blood of latent M. tuberculosis infected donors using CD1b tetramers. Herein, an optimised CD1b tetramer enrichment was developed to isolate very small frequencies of GMM-reactive T cells from healthy donor blood directly ex vivo. In contrast to M. tuberculosis infected patients, GMM-reactive T cells from healthy donors expressed a diverse TCR repertoire. GMM-reactive T cells were exclusively CD4+CD8- and most cells expressed the memory marker CD45RO. Similar to other unconventional T cells, GMM-reactive T cells from healthy donors expressed the transcription factor promyelocytic leukaemia zinc finger (PLZF). In vitro stimulation revealed GMM-reactive T cells secrete both TNF and IFNg, similar to MAIT cells. To characterise these cells further, single-cell transcriptomic analysis was performed on GMM-reactive T cells isolated using CD1b-GMM tetramers. GMM-reactive T cell expressed unique transcriptomic signatures that were accurately distinguishable from the transcriptomes of natural killer T (NKT) cells and CD4+ T cells, indicating that these cells may be functionally distinct from other unconventional and conventional T cells populations. In summary, these data describe a role for the CD8 co-receptor on MAIT and other MR1-reactive T cells and expand on the limited knowledge of CD1b-restricted T cells in healthy blood in regard to TCR repertoire, antigen-specificity and phenotype.
Amoebae as a host for Legionella replication
Legionella pneumophila (L. pneumophila) is a gram-negative bacterium found ubiquitously in natural water sources where it replicates within amoebae, such as Acanthamoeba castellanii (A. castellanii). The evolution in amoebal hosts allows L. pneumophila to ‘accidentally’ infect human lungs after inhalation of aerosols containing L. pneumophila. In both human macrophages and amoebae, L. pneumophila replicates intracellularly within a vacuole known as the Legionella containing vacuole (LCV) that avoids fusion with the endocytic pathway. Fundamental to this process is the translocation of over 330 proteins by L. pneumophila into host cells by the Dot/Icm type 4 secretion system. To date, only a subset of Dot/Icm effector proteins has been characterised. These proteins are involved in manipulating host cellular processes such as ER vesicle recruitment, post-translational modifications and host cell survival. The majority of L. pneumophila effector proteins remain uncharacterised due to functional redundancy of effector proteins and complex effector protein regulation. In addition, despite the ecological and evolutionary significance of the intra-A. castellanii stage of the L. pneumophila life cycle, very little is known about this host-pathogen interaction. In this study, we constructed in-frame markerless mutants in L. pneumophila 130b according to genomic regions enriched for putative Dot/Icm effector genes and revealed host-specific genomic regions that are required for optimal L. pneumophila replication in A. castellanii. Specifically, by identifying a putative glutamate transporter necessary for replication in A. castellanii, but not in macrophages, this study suggests an aspect of L. pneumophila metabolism adaptation in different hosts. A. castellanii presents two life stages: active trophozoites and dormant cysts. Trophozoites act as reservoirs for L. pneumophila and are hijacked by L. pneumophila for bacterial replication. Cysts are resistant to anti-microbial treatments and were previously thought to facilitate L. pneumophila persistence and dissemination. However, in this study, we showed that the infection of A. castellanii with L. pneumophila led to inhibition of the transition from trophozoites to cysts in a Dot/Icm dependent manner. This inhibition partially required the gene letA, which encodes the regulator of two-component system LetAS. Pathogenic amoeba cysts prose a human health problem. This study provides a biological model for anti-cyst research and provides clues into critical encystment pathways. L. pneumophila can interfere with a broad range of host cellular pathways to establish the replication niche. Here, we investigate A. castellanii transcriptional response with L. pneumophila infection by RNA sequencing analysis. As a result, A. castellanii genes associated with mitosis, DNA replication and cell wall synthesis were significantly downregulated at late infection phase, which might contribute to the inhibition of A. castellanii proliferation and encystment. Compared with transcriptional profile in macrophage, L. pneumophila displayed different regulation on host eEF1a expression and genes associated with ATP production. Furthermore, a gene encoding sirtuin family protein, Sir6f, was upregulated during L. pneumophila infection, and silencing of sir6f led to decreased bacterial replication, suggesting that Sir6f is a host factor that facilitates L. pneumophila replication in A. castellanii. Evolution of L. pneumophila in amoebal hosts is hypothesized to allow its adaptations to survive in macrophages. Overall, this study highlights a number of strategies utilised by L. pneumophila during A. castellanii infection, including host-specific virulence factors, inhibition of A. castellanii encystment, affect host transcriptome and hijack of host factors. These microbial strategies will add valuable insights and provide potential targets regarding the development of new mechanisms for L. pneumophila control and prevention.
Molecular studies of Staphylococcus epidermidis
Staphylococcus epidermidis is a conspicuous member of the human microbiome, widely present on human skin. The shift in modern medicine towards invasive procedures has favoured its emergence as a significant nosocomial pathogen, especially in the setting of prosthetic devices. Despite being the most genetically diverse of the staphylococcal species, with 989 S. epidermidis multilocus sequence types characterised thus far, a single lineage, ST2, accounts for most clinical disease. Unfortunately, existing typing schemes lack the sensitivity to offer any epidemiological insights beyond this. Furthermore, the absence of dedicated molecular tools coupled with the genetically intractable nature of S. epidermidis has hindered the study of its molecular genetics, pathogenesis and treatment. This thesis utilised new genome sequencing technologies to provide a modern perspective on the global molecular epidemiology of S. epidermidis with a focus on antimicrobial resistance. The project began with the functional analysis of the first complete genome of a multidrug resistant ST2 S. epidermidis. Then extended globally, to uncover the previously unrecognised international spread of three multidrug-resistant, hospital-adapted lineages of S. epidermidis (two ST2 and one ST23) that emerged in recent decades. Acquisition of a dual D471E plus I527M RpoB substitution was common to all three lineages and demonstrated to have become fixed in the populations. Analysis of isolates from 96 institutions in 24 countries identified the RpoB D471E plus I527M combination as the most common cause of rifampicin resistance in S. epidermidis, accounting for 86.6% of mutations. Furthermore, these substitutions were found to occur almost exclusively in isolates from the ST2 and ST23 lineages. By breaching lineage-specific DNA methylation restriction modification (RM) barriers, then performing site-specific mutagenesis, these rpoB mutations were shown to confer both rifampicin resistance and reduced susceptibility to the glycopeptide antibiotics vancomycin and teicoplanin. Importantly, these findings suggest that current clinical practice has contributed to the generation of rifampicin and vancomycin resistance and spread of the three multidrug-resistant S. epidermidis lineages, warranting the review of staphylococcal treatment guidelines. The final work in this thesis is the first comprehensive analyses of the barriers to the uptake of foreign DNA in S. epidermidis. Contrary to current assumptions, analyses revealed that the type I RM systems in S. epidermidis are not lineage specific like Staphylococcus aureus, instead demonstrating considerable diversity even within a single genetic lineage. This diversity can be attributed to marked differences in the gene arrangement, chromosomal location and movement of type I RM systems in S. epidermidis compared to S. aureus. Findings indicated that genetic manipulation of S. epidermidis must be tailored to each strain of interest. Using Escherichia coli plasmid artificial modification to express S. epidermidis hsdMS, the restriction barriers in S. epidermidis were readily overcome, with electroporation efficiencies equivalent to modification-deficient mutants. Through functional experiments, the use of genomic data to predict both the activity of type I RM systems and the potential for a strain to be electroporation proficient were demonstrated, and an efficient approach to the genetic manipulation of any S. epidermidis strain of interest, including those that have hitherto been intractable is presented. Collectively, the works in this thesis offer new insights into the molecular epidemiology, resistance mechanisms, treatment recommendations and genetic manipulation of S. epidermidis. The method outlined for the genetic manipulation of any given S. epidermidis isolate should prove valuable in assisting future molecular studies and advancing knowledge about this significant nosocomial pathogen.
Understanding influenza B humoral immunity to improve vaccine design
Annual influenza epidemics cause significant morbidity and mortality globally. Although influenza B virus (IBV) is responsible for approximately 25% of the global influenza burden, it remains understudied compared to influenza A virus (IAV). Current influenza vaccines elicit mostly strain- specific antibody responses, so vaccine efficacy is dramatically reduced when mutated variants dominate in circulation. Improved IBV vaccines require a better understanding of humoral immunity against IBV in order to inform improved vaccine design. In this thesis, we examined the human IBV-specific humoral response following influenza vaccination and investigated the potential utility of ferritin nanoparticles and IBV HA stem antigens to induce broader protective immune responses. IBV-specific antibody responses have been described in previous studies, but the knowledge we have regarding the specificities, protection and epitopes of cross-reactive antibodies remains limited. Using a flow cytometry-based approach, we delineated different B cell populations with either single-lineage or cross-lineage specificities from samples collected following seasonal influenza immunization clinical trials. Both neutralizing and non-neutralizing antibodies protected mice from lethal challenges with IBV, but protection by non-neutralizing antibodies was non-sterile and dependent on Fc-effector functions. We also localized neutralizing epitopes of both lineage-specific and cross-lineage antibodies on IBV HA by sequencing viral escape mutants. The comprehensive information we gathered from this study may guide future efforts to design broadly protective IBV vaccines. Nanoparticles as a novel vaccine carrier system has drawn increasing attention over the past decade. The self-assembling ferritin nanoparticles loaded with IAV HA have been proven to induce robust and broad humoral response in mice and ferrets. We investigated the feasibility and protective potential of displaying IBV HA on ferritin nanoparticles. The results demonstrated that ferritin nanoparticles significantly boosted the immunogenicity of IBV HA. Furthermore, we found that co-loading HAs of both IBV lineages led to antibody responses against both lineages and conferred similar level of protection in comparison of mixing two monovalent nanoparticles. Overall, ferritin nanoparticles presenting IBV HA, either monovalent or multivalent, may provide some immunological advantages over the conventional influenza vaccines, hence are promising foundations to build improved IBV vaccines upon. The highly conserved stem domain of HA has long been considered as a major target for generating cross-reactive antibody response. Due partially to the difficulty in preparing stable IBV stem proteins, the protective potential of the IBV stem is poorly understood. Despite problematic expression and/or misfolding, IBV stem proteins we generated were immunogenic in mice and elicited robust cross-lineage antibody responses. Mice vaccinated with IBV stem proteins were partially protected from simultaneous challenge with IBV of both antigenic lineages. The broad protection of IBV stem demonstrated by these experiments suggests that it is a promising candidate for universal IBV vaccines. In summary, this thesis improves the understanding of humoral immunity against IBV both by elucidating important aspects of HA-specific antibodies and exploring different approaches to improve current influenza vaccines. Our findings add to the field of IBV research and inform development of better IBV vaccines.