Microbiology & Immunology - Theses
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ItemEstablishing two systems to study MAIT cell responses during bacterial infection( 2019)Mucosal-associated invariant T (MAIT) cells are a population of innate-like T cells, which are abundant in humans and also present in many mammals, including mice [1]. MAIT cells express semi-invariant T cell receptors (TCR) are activated by recognizing microbial vitamin B2 (riboflavin) metabolites presented on non-classical major histocompatibility complex (MHC) class I related protein 1 (MR1) [1-6]. Upon activation, MAIT cells can either directly kill infected cells or secrete IFN, IL-17, TNF and other functional molecules that enhance the anti-microbial activity of other immune cells [7, 8]. These characteristics suggest an important role played by MAIT cells in anti-bacterial immunity in mammals. Bacterial infections are major problems in clinical settings. While MAIT cells have shown antibacterial properties, clinically relevant in vivo animal models have not been extensively developed for further exploration of the mechanisms of MAIT cell protection. Using three clinically common bacteria; Klebsiella pneumoniae, Staphylococcus aureus, and Escherichia coli, this study established bacterial peritonitis models in mice for MAIT cell research. The three bacteria were all able to produce antigens that stimulate MAIT cells in vitro. For mouse infection models, the optimal dose of K. pneumoniae for inducing productive but nonlethal infection was not established in this study. S. aureus and E. coli peritonitis incurred mild and robust MAIT cell responses in vivo, respectively. Further experiments with E. coli peritonitis showed that MAIT cells accumulated in the intraperitoneal cavity, the spleen and liver, which contributed to bacteria control. The finding suggests possible MAIT cell-based treatments in clinical conditions. While in vitro studies have shown that MAIT cells can be stimulated by various types of cells upon infection, there have been few investigations into how MAIT cells are activated in vivo. In the second project presented in this thesis, the contributions of antigen presenting cells (APCs) in initiating activation in MAIT cells was studied with Francisella tularensis infection. We found that APCs were burdened with bacteria in the early phase of infection. With an ex vivo cellular co-culture assay was specifically optimized for assessing MAIT cell activation induced by the F. tularensis burdened APCs, we found that alveolar macrophages contributed to the best activation of MAIT cells in an MR1 dependent manner. Other cell subsets: monocytes, neutrophils and dendritic cells were also capable of inducing a mild activation. In summary, the present study helped optimize two novel systems for MAIT cell research. With the ex vivo cellular co-culture system, this thesis contributes to understanding how APCs activate MAIT cells in vivo upon bacterial infection. With the in vivo peritonitis models, this study introduces clinically relevant bacteria to the current MAIT cell research in mice, and ultimately to inform and complement clinical research. It is hoped that the insights gained from this study could be of assistance to current research methods and to achieve a better understanding of MAIT cell responses during bacterial infection.
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ItemCharacterising the immune response following nasal colonisation of Staphylococcus aureus( 2019)Staphylococcus aureus (S. aureus) is an opportunistic pathogen found in the nasal passage of 20%~30% of humans. Persistent nasal carriage of S. aureus is a significant risk factor for various diseases, including secondary bacterial pneumonia. To date, there is no vaccine available against S. aureus and the emergence of multidrug resistant strains limit the use of antibiotics as a treatment option. Little research has been undertaken to define the environmental, physiological and immunological changes that cause S. aureus to shift from the upper to the lower respiratory tract and convert from a commensal organism to pathogenic threat. In this project, we have established a nasal colonisation model of S. aureus in C57BL/6 mice and characterised the role of the innate and adaptive immune response in preventing the dissemination of S. aureus from the upper to the lower airways. Using this model, we have found that while both innate and adaptive immune responses are involved in the clearance of S. aureus from the nasal passage, only neutrophils play a critical role in keeping S. aureus confined to the nose and preventing spread into other organs. While the adaptive immune system does not play a role in preventing the spread of nasal S. aureus, it does play an important role bacterial clearance from this site. Moreover, immunisation with heat-killed S. aureus represents an effective approach to limit mice from being nasally colonised with S. aureus. Collectively our research demonstrates that both arms of the immune system can be harnessed to limit the colonisation and dissemination of S. aureus infection.
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ItemImmunoglobulin allotypes, antibody-dependent neutrophil phagocytosis: impact on HIV and influenza( 2019)Antibodies (Abs) are highly functional immune proteins. Abs can deactivate pathogens through neutralization and mediating various Fc receptor (FcR) functions. Two common FcR-mediated functions are Ab-dependent cellular cytotoxicity (ADCC) and Ab-dependent (cellular) phagocytosis (ADCP or ADP). These functions are triggered by binding of the Fc region of Abs to FcRs on innate immune cell surface. Studies including analyses of the moderately protective human phase III RV144 HIV vaccine trial suggest that antibodies with the ability to mediate FcR functions may be important in HIV protection and control. IgG, the most abundant Ab isotype, has different subclasses including IgG1, IgG2, IgG3, and IgG4. The IgG1 and IgG3 subclasses when complexed with antigens, bind efficiently to all Fcγ receptors (FcγRs), thus initiating FcR-mediated functions (the FcR-mediated functions discussed in this thesis are all FcγR-mediated functions). There are several allelic variations (termed allotypes) of IgG1 in the constant region including G1m1 and G1m3. The prevalence of allotypes differs among ethnic groups. There is growing evidence that IgG1 allotypes differentially influence IgG subclass levels, FcR binding, and FcR-mediated functions. However, the understanding of the influence of IgG1 allotype on FcR-mediated functions is still limited at present. This thesis focuses primarily on the investigation of the impact of IgG1 allotypes on vaccine-induced immune responses. The first chapter provides the literature review of the whole study, ranging from the general humoral immunity to specific FcR-mediated functions. It also introduces IgG1 allotypes and the importance of FcR functions in infectious diseases including Human Immunodeficiency Virus (HIV) and Influenza. The second chapter focuses on the material and methods utilized in these studies. Information on the subjects who provided samples are also described in chapter two. The third chapter addresses two approaches used to determine the IgG1 allotypes. Normally the IgG1 allotypes are determined by sequencing as the DNA sequence difference is the intrinsic difference among different IgG1 allotype carriers. This study optimized another ELISA-based approach of IgG1 allotyping on protein levels utilizing only purified IgG or plasma to identify allotypes. This approach makes it possible to allotyp subjects when genomic DNA is unavailable. Furthermore, this study validates that sequencing allotyping and ELISA allotyping for the G1m1 and G1m3 allotypes are consistent with each other. In the fourth chapter, a neutrophil-like HL-60 cell line was developed as a model to study neutrophil responses to Fc-functional HIV-specific antibodies. Neutrophils are innate immune cells that express a range of FcRs and can trigger various FcR-mediated functions including Ab-dependent cellular phagocytosis (ADCP). However, the role of neutrophil FcR-mediated functions in HIV control is not well explored, largely due to the difficulty in working with primary neutrophils as they die rapidly (half-life 6-8 hours) ex vivo. In this chapter we cultured HL-60 cells for 5 days with DMSO such that they expressed a range of neutrophil-like cell markers. We then successfully used these neutrophil-like HL-60 cells to measure neutrophil FcR-mediated ADCP against HIV. The fifth chapter explored the influence of IgG1 allotypes in response to Influenza vaccination. IgG subclass levels, IgG-FcR binding, ADCC and ADCP (using the HL-60 method generated above) were all evaluated. We found that IgG1 allotypes are associated with antigen-specific IgG subclass levels upon Influenza vaccination. However, no functional assay difference regarding ADCC and ADCP was found, implying substantial complexity in allotypic effects on FcR-mediated functions. The final chapter covers the overall discussion and conclusion of the study. In summary, this project developed useful methods for IgG1 allotyping and Fc-functional assays of a neutrophil-like cell line. We utilized these methodologies to provide insight into the role of IgG1 allotypes upon vaccine-induced immune responses. This work could ultimately have implications for the development of global vaccinations against infectious diseases including HIV, where antigen-specific IgG subclasses contribute to protection.
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ItemCharacterization of roles of IRF8 in dendritic cell development and function( 2018)Dendritic cells (DC) are an essential component of immune system. Antigen presentation by DCs initiates T cell responses and builds up defense against pathogens. There are two major types of DC, conventional DCs (cDCs) and plasmacytoid DCs (pDCs), both of which derive from the same progenitor. The cDCs can be further classified by surface marker CD11b and CD8. While cDCs function as the major antigen presenting cells, pDCs are more efficient in type I IFN production. Regarding the functional difference, it is important to comprehend factors that control the production of these two types of DC. It has been reported that IRF8 mutation in human causes selective loss of DC subsets. Although IRF8 has been identified as key regulator in DC transcription network, study addressing the function of IRF8 in DC development and function remains incomplete. In this project, we show that the generation of pDCs and cDC1s depends largely but not entirely on IRF8. Upon germline deletion of IRF8, normal function of pDCs was disrupted while the antigen presentation of cDC1s was complemented by other mechanisms.
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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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ItemControl of virulence expression in enterotoxigenic Escherichia coli by the master regulator Rns, and targeting Rns for inhibition of bacterial virulence( 2016)Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhoea, particularly for children in developing countries, and is the most common cause of travellers' diarrhoea. The ETEC pathotype is defined by the presence of at least one colonisation factor (CF) and either or both of two toxins, the heat-labile (LT) and the heat-stable (ST) enterotoxins. In addition, a variety of accessory virulence factors have been identified that aid in pathogenesis, but are not present in every strain of ETEC. CFs are antigenically diverse and immunologically distinct, but many are transcriptionally controlled by a conserved AraC-like regulator, Rns. Rns activates transcription of its target genes by binding to curved, AT-rich regions of DNA in the target promoter, releasing the global gene silencer H-NS, and allowing RNA-polymerase to transcribe the target gene. Rns is part of a family of AraC-like virulence regulators which also includes the master virulence regulators ToxT and RegA, from Vibrio cholerae and Citrobacter rodentium respectively. These are master virulence regulators that control the expression of an assortment of virulence genes. To date the complete Rns regulon has not been identified, and more gene targets with an impact of ETEC virulence may yet be unidentified. In order to identify all of the gene targets of Rns, mRNA-sequencing was performed on ETEC strain H10407 and its isogenic rns negative mutant. This experiment identified four potential new virulence targets that required the presence of Rns for full expression. These are the agn43, etpB, yghJ and rtr genes which encode two autotransporter adhesins, a mucinase and the AraC-family negative regulator (ANR) respectively. Results from further transcriptional assays suggest that Rns directly activates the expression of etpB, yghJ and rtr but indirectly activates the expression of agn43. With antimicrobial resistance an increasing problem, alternative methods of bacterial control are urgently required. Rns is absolutely required for the expression of CFs, an essential virulence factor of ETEC, and this makes it an attractive drug target. With the aim of identifying an inhibitor of Rns activity, and therefore of ETEC-induced diarrhoea, chemical compounds from two sources (those from a commercial small molecule library as well as those from structure based and computer-aided drug design) were screened for their ability to inhibit Rns activity. Several hit compounds were identified and the IC50 determined.
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ItemThe influence of BCG vaccination on the human immune response to Plasmodium falciparum( 2016)A recent WHO review emphasises the importance of understanding the heterologous immunomodulatory effects of the Bacillus Calmette–Guérin (BCG) vaccine1, which is administered to protect against tuberculosis (TB). An increasing number of studies demonstrate that BCG has protective effects against infections unrelated to TB. Malaria is one of the most important infectious diseases, leading to millions of deaths worldwide2. Of the five malaria-causing species, Plasmodium falciparum causes nearly all malaria deaths. In mice, BCG vaccination has been shown to reduce parasitaemia and mortality following malaria infection3. My study investigated the effects of BCG vaccination on the immune response to malaria in humans. In vitro models are commonly used to explore and understand the immunopathogenesis of malaria. However, interpretation of the results from these studies and comparison between them is hampered by variation in the methods and protocols used. The first part of my thesis therefore comprises a systematic review of studies investigating cytokine production following in vitro stimulation of human blood or its components with P. falciparum-infected erythrocytes (PfIE). In this review, I identified 2,085 publications, of which 54 met the inclusion criteria. There was considerable heterogeneity between studies, including the use of different P. falciparum lines, choice of human sample type, incubation time with parasites, cytokines measured and their detection method. Amongst the studies, 27 different cytokines were measured, of which the most commonly tested were IFN-ϒ, TNF-α, IL-10, IL-2 and IL-6. Despite this heterogeneity, I identified a number of cytokines that were consistently produced in vitro and these were included in my laboratory-based study. The second part of my thesis describes a laboratory-based study aimed to investigate the effects of BCG vaccination on the immune response to malaria in blood samples from infants recruited from two trials. In the first participant group, neonates were randomised to BCG vaccination (n=66) or no BCG vaccination (n=43) at birth and blood samples were collected 7 days later. The second participant group included 12 infants younger than 1 year requiring BCG vaccination before travelling overseas. Blood samples were collected prior to and 7 days after BCG vaccination. In both studies, whole blood samples were stimulated with PfIE, uninfected erythrocytes (UE), E. coli and culture media alone for 24 hours. Cytokine concentrations in the supernatants were measured by ELISA (IL-10 and IFN-ᵧ) and multiplex bead assay (TNF-α, IL-1β, IL-6, GM-CSF, MIP-1α, MCP-1, IL-8, MIG, IP-10). In the first study group, mean IP-10 (p=0.11, unpaired t-test) and MIG (p=0.14) levels were higher after incubation with PfIE in samples from BCG-vaccinated infants compared to controls. In the second group, mean IL-10 levels decreased (p=0.07, paired t-test), and IL-1β (p=0.08) and IFN-ᵧ (p=0.09) increased after BCG vaccination. None of these differences reached statistically significant. In conclusion, under the experimental conditions used in this study, there was no definitive evidence to support the hypothesis that BCG influences the in vitro cytokine response to P. falciparum.
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ItemPhenotypic and genomic comparisons of community-associated Staphylococcus aureus Clone ST93( 2015)Staphylococcus aureus is an opportunistic bacterial pathogen that primarily colonises the anterior nares of 30-50% of individuals at any one time, without causing disease. S. aureus does however cause a wide range of diseases including skin and soft tissue infections, pneumonia, osteoarticular infections and bacteraemia or septicaemia, frequently resulting in hospitalisation and a high mortality rate for invasive disease. S. aureus strains have acquired resistance to many classes of antibiotics, most importantly resistance to beta-lactam antibiotics (methicillin-resistant S. aureus, MRSA). While traditionally a health care associated issue, community-acquired MRSA (CA-MRSA) clones are increasing worldwide resulting in severe, antibiotic resistant infections occurring in patients without healthcare contact. The ST93 clone of CA-MRSA is essentially unique to Australia, and has been associated with severe, invasive S. aureus infections in otherwise healthy individuals. ST93 CA-MRSA is also the most common CA-MRSA clone in Australia. The overall aims of this study were to determine the virulence characteristics of a collection of ST93 S. aureus isolates from around Australia, and to uncover the molecular determinants of virulence in ST93 S. aureus. Fifty-eight ST93 isolates were assessed for virulence using the Galleria mellonella invertebrate virulence model and by measuring expression of key virulence factors. Whole genome sequencing and genomic analysis of all isolates was used to uncover genetic differences that might account for differences in virulence characteristics. In this study ~50% of isolates (n=28) were avirulent when compared to a virulent reference ST93 isolate JKD6159 using the G. mellonella model. This study also revealed that the G. mellonella model does not respond to exotoxin expression of isolates with no significant differences in G. mellonella mortality between culture supernatant of virulent and avirulent isolates used in the G. mellonella model. The expression levels of PSMα3 and δ-toxin varied significantly amongst the isolate collection, and the concentration of δ-toxin was found not to correlate with that of PSMα3, indicating that systems other than the quorum-sensing agr system must be controlling the expression of PSMα3 in ST93 S. aureus.
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ItemCharacterisation of eukaryotic-like proteins in Legionella longbeachae( 2015)Legionella longbeachae is the main causative agent of sporadic Legionnaire’s disease within Australia, New Zealand and Thailand. Unlike the typically aquatic L. pneumophila, L. longbeachae replicates within soil protozoa and is commonly found in commercial potting mix. However, its parasitic lifestyle has equipped it with the ability to replicate within amoeboid human lung macrophages giving rise to legionelloses. L. longbeachae also encodes a defect in organelle trafficking/ intracellular multiplication (Dot/Icm) type IV secretion system (T4SS), which is presumed to translocate a myriad of eukaryotic-like effectors into host cells. Previously, a Hidden Markov Model (HMM) was used to identify LncP, a L. pneumophila mitochondrial carrier family (MCF) protein and Dot/Icm effector. LncP localised to mitochondria in macrophages and appeared to influence the transport of purine nucleotides across the mitochondrial inner membrane. This led to the identification of three putative MCF proteins, LmpA, LmpB and LmpC, encoded by L. longbeachae. Construction of reporter fusion proteins expressing a calmodulin-dependent adenylate cylase (Cya) from Bordetella pertussis at the amino termini of these proteins demonstrated their translocation by the Dot/Icm T4SS of the surrogate L. pneumophila as well as L. longbeachae. Immunofluorescence microscopy revealed localisation of fusion proteins of these effectors to mitochondria of host cells when expressed ectopically, as well as when translocated by L. pneumophila or L. longbeachae. While limited insight was gained into potential substrates of LmpA or LmpC, complementation of yeast strains deleted for known MCF proteins of S. cerevisiae suggested that LmpB also transports purine nucleotides but differs significantly from LncP in its biochemical activity. Further characterisation could be sought in the future through infection and yeast mitochondrial import experiments.