Microbiology & Immunology - Theses
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ItemThe epidemiology of influenza in Northern Australia( 2021)The epidemiology of influenza in Northern Australia may be unique due to the tropical climate, large Indigenous population and wide dispersal of the population in the region. To mitigate the impact of the next influenza pandemic and seasonal influenza epidemics, it is important that the epidemiology of influenza in this area be better understood. Furthermore, estimates of the effectiveness of the influenza vaccine against hospitalisation in Northern Australia and comparisons of vaccine effectiveness in Indigenous and non-Indigenous Australians are lacking. This is despite the Australian Federal Government currently covering the cost of influenza vaccination for all Indigenous Australians aged over 6 months. Chapter 2 of this thesis consists of epidemiological analyses of notified influenza cases in the Northern Territory. Rates of influenza were higher for Indigenous Australians in all age groups through 2007-2016 with the disparity being largest for those in the 55-64 age bracket (rate ratio: 5.56; 95% CI: 4.71, 6.57). Bimodal peaks in influenza activity were seen in the Top End region of the Northern Territory in 3 out of the 10 years studied. Chapter 3 details the first ever use of phylogenetic methods to describe influenza activity in the Northern Territory. Influenza strains in the Northern Territory were shown to undergo regular extinction and are related to strains present in many diverse global regions. A mismatch was seen in the influenza vaccine strain and a circulating influenza B strain during an outbreak in late 2013-early 2014. Chapter 4 consisted of a case-control study employing a test-negative design to examine the effectiveness of the influenza vaccine against hospitalisation in the Northern Territory between 2009-2014 using 1075 cases and 3461 controls. Odds ratios for vaccination in each year were obtained from logistic regression models and meta-analysed using a random-effects model. Overall vaccine effectiveness was estimated at 32% (95% CI: -1%, 54%). Vaccine effectiveness was estimated at 40% (95% CI: -10%, 68%) for non-Indigenous Australians and 23% for Indigenous Australians (95% CI: -16%, 48%). Differences in vaccine effectiveness between Indigenous and non-Indigenous Australians were not statistically significant (p>0.15), but available sample size limited ability to detect a difference. This thesis highlights the burden of influenza upon the Indigenous population of the Northern Territory, the challenges that semi-annual influenza epidemics present in this region, ongoing cycles of importation and extinction of influenza viruses occurring in the Northern Territory and provides evidence that current vaccines have limited effectiveness against hospitalisation in this area. This thesis provides a framework for examining the impact of many infectious diseases in Northern Australia.
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ItemThe role of prion strain in disease progression and pathogenesis( 2021)Prion diseases are a family of invariably fatal, transmissible neurodegenerative diseases. The classic symptoms of prion disease are due to severe, rapid neurological decline hence by the time a diagnosis is made it is too late to commence therapeutic intervention as the patient will have already sustained significant impairments or even have succumbed to disease. Identifying prodromal markers of prion disease will enable diagnosis and treatment to be implemented prior to significant neurodegeneration. The highly variable clinical presentation of prion disease also confounds medical diagnosis and can delay the identification of these fatal, transmissible disorders. This variation has been attributed to the existence of prion strains. The lack of medically relevant models has hampered our understanding of prion strains. The current thesis characterises novel, medically relevant, mouse-adapted, human prion strains with a focus on identifying novel, prodromal disease signs. Human-derived sporadic Creutzfeldt–Jakob disease strains have undergone serial passaging to enable their adaptation to murine hosts. Three sporadic Creutzfeldt–Jakob disease strains, T1MM, T2MM and T3MM (Collinge classification system) were used to generate mouse-adapted models MU-01, MU-02 and MU-03 respectively. Characterisation of these strains in contrast to the mainstay murine-adapted prion strain of Australian researchers, M1000, was performed to enable comparison of the clinical profile, neuropathology and biochemical features of these strains. Several novel and potentially prodromal clinical signs were identified and underwent further investigation. The mouse-adapted prion strains exhibited high circulating cholesterol levels, dilated cardiomyopathy and increased appetites leading to weight gain. Weight gain was the first sign of disease observed in MU-03 inoculated mice and presented markedly earlier than classic signs of prion disease neurological decline. These findings led to the examination of the patient database held by the Australian National Creutzfeldt-Jakob Disease Registry to determine if these signs are phenotypical of human prion disease. There were significantly more prion disease cases who were identified as displaying overeating, weight gain and hypercholesterolemia when compared to non-prion cases afflicted with other neurological illnesses. The inverse was found when examining the presence of prion-like cardiomyopathy, which was significantly underrepresented in prion disease cases. This may reflect prion disease patients succumbing to prion-associated cardiomyopathy prior to fulfilling the clinical criteria for referral to the Australian National Creutzfeldt-Jakob Disease Registry. Rapid obesity of an unknown origin may be an early indication of prion disease and has the potential to aid early identification and treatment of prion disease prior to the classic phase of irreversible, rapid decline. High circulating cholesterol levels may prove to be supportive in a diagnosis of prion disease and the results of the current study support further investigation into the use of statin treatment in patients with prion disease. Further work is required to determine the prevalence and relevance of prion-associated cardiomyopathy in patients as well as any transmission risk it may pose during cardiac surgery. The newly, characterised human-derived, mouse-adapted prion strain models are well placed to be valuable resources for understanding these newly identified disease signs and enabling the development of strategies to improve outcomes for patients.
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ItemInactivation Mechanisms of Therapeutic CD8+ T Cells against a non-Hodgkin B-cell Lymphoma Mouse Model( 2021)Clinical and animal studies have demonstrated the capability of innate or adaptive components of the immune system to eliminate tumour cells. Cytotoxic CD8 T lymphocytes (CTLs) are the main population of the adaptive immune system involved in tumour cells elimination. In clinical studies, autologous tumour associated antigen (TAA)-specific CTLs have been adoptively transferred into patients to eliminate tumour cells expressing the cognate antigen, an approach known as Adoptive Cell Therapy (ACT). In many conditions, these CTLs are functionally impaired. Similar observations have been made in mouse models, including our own. We found that anti-ovalbumin (OVA) OT-I CTL injected into mice were capable of eliminating non-Hodgkin B cell lymphoma cells that express OVA as a model TAA. However, this ACT failed in mice harbouring a large tumour burden because many of the OT-I CTL were eliminated soon after ACT, and even though the surviving ones expanded, they remained functionally impaired. These observations recapitulate successful vs failed outcomes of ACT in the clinic. So far little is known about the extrinsic and intrinsic mechanisms that determine these outcomes. My results show that the mechanisms of CTL inactivation we observe are most likely physiological responses to large target cell burdens and provide experimental system to further understand the mechanisms of inactivation and approaches for the generation of more effective CTL for ACT.
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ItemNo Preview AvailableGlobal 3’-UTR Length Changes Form a Novel Layer of Regulation in the Macrophage Response to Interferon-Beta( 2021)Interferon signaling is one of the most important mechanisms shaping innate immune responses and needs to be tightly regulated to successfully fight infections and modulate immune responses while avoiding toxicity. Type I interferons (IFNs) have been shown to induce multiple transcriptional, translational and metabolic changes. The global response of murine and human macrophages to IFNb stimulation was characterized in this study using multi- omics strategies. These analyses gave insight into a complex regulatory network of transcripts, proteins and metabolites that results in global reprogramming of the cell. Post-transcriptional gene regulation is an important component of this network and is centered around 3’-untranslated regions (3’-UTRs), regions heavily targeted by miRNAs and harboring binding sites for many RNA-binding proteins. Poly-A-tail sequencing (PAT-seq) experiments revealed that many transcripts expressed shortened 3’-UTRs in response to IFNb, a result of changed alternative polyadenylation (APA) patterns. APA and changed 3’-UTR lengths are emerging fields of broad importance in physiological and pathological processes that are only starting to be explored. Differences in APA patterns and their regulation have not previously been studied in context of IFN. Recent publications have described a scaffold-like role for 3’-UTRs that facilitates the formation of different protein complexes depending on 3’-UTR length, which can affect localization and function. This unique regulatory mechanism was investigated for two IFNb-regulated transcripts with shortened 3’-UTRs, EIF4EBP2 and MAVS. 3’-UTR-dependent protein-protein interactions were identified by mass spectrometry using tagged overexpression constructs encoding the different transcript isoforms. This study describes a new aspect of IFN signaling and a novel layer of regulation through genes that are not part of the typical and well-characterized IFN transcriptional response. It shows how differential expression of distinct 3’-UTR transcript isoforms influences macrophage innate immune responses.
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ItemInvestigating the ability of RIG-I agonists to provide protection in mouse and ferret models of respiratory virus infection( 2021)Respiratory infections caused by influenza A virus (IAV) or respiratory syncytial virus (RSV) lead to substantial morbidity and mortality. Treatment options are limited and there is urgent need for the development of efficient therapeutic and prophylactic treatments. Pattern recognition receptors (PRRs) such as the cytoplasmic helicase retinoic-acid-inducible gene I (RIG-I) are part of the innate immune system. RIG-I can be activated by recognition of viral nucleic acids, leading to downstream activation of interferon-stimulated genes (ISGs) and restriction of viral replication. We have used synthetic RNA oligonucleotides to stimulate RIG-I to inhibit replication of respiratory viruses using in vitro and in vivo models of infection. Our in vitro approaches used airway cell lines from humans, mice and ferrets and investigated the effects of RIG-I agonist pre-treatment on subsequent infection with either IAV or RSV. Prophylactic RIG-I agonist treatment induced multiple ISGs and inhibited infection and growth of respiratory viruses in cell lines from each of the different species. In vivo, we utilised mouse and ferret models to study the antiviral potential of RIG-I agonists against IAV and RSV. In mice, we compared animals which do or do not express a functional Mx1 protein and found that a single prophylactic treatment with RIG-I agonist via the intravenous route resulted in ISG induction in the lungs and this correlated with reduced IAV replication. Of interest, these effects were particularly potent and long-lasting in mice expressing a functional Mx1 confirming an important role of Mx1 for RIG-I agonist-induced protection against IAV. In a mouse model of RSV, we found that a single prophylactic treatment with RIG-I agonist resulted in reduced replication of virus in the lung, as observed using bioluminescence imaging of luciferase-labelled RSV as well as plaque assay for infectious virus. Thus, our studies in mouse models indicate that a single pre-treatment with RIG-I agonists resulted in potent inhibition of two very different respiratory viruses. In ferrets, after establishing assays to monitor ISG induction in the blood and in airway tissues, we confirmed that a single intravenous injection of RIG-I agonist induced ISG induction in both peripheral blood mononuclear cells (PBMCs) and the lungs. Moreover, a single treatment prior to infection also resulted in reduced replication of both IAV and RSV in ferret lungs, although this treatment had only negligible effects on virus replication in the nasal tissues. A single treatment to animals with an established IAV infection also resulted in reduced virus titres in the lungs, suggesting its potential as a therapeutic antiviral agent. Myxoma (Mx) proteins are ISGs with potent antiviral effects against IAV. While human and mouse Mx proteins have been studied in detail, ferret Mx proteins have not been characterised. Therefore, we generated different experimental approaches to assess the induction of three endogenous ferret Mx (two splice variants of Mx1 as well as Mx2) in a ferret cell line, as well as in vitro overexpression systems to assess the cellular localisation and antiviral functions of each ferret Mx. Our findings indicate that each ferret Mx localises to the cytoplasm and that particular proteins exhibit antiviral functions against IAV, but not RSV. However, further studies are required to clearly define the antiviral activity of ferret Mx, since our preliminary results indicate that ferret Mx proteins display different antiviral activity following overexpression in human or in ferret cells. Together, studies described in this thesis demonstrate the potential of RIG-I agonists as antiviral treatments against diverse respiratory viruses both in vitro and in vivo and represent an important step towards the development of novel antiviral treatments in humans.
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ItemStudy of innate immune responses to neurotropic virus infection( 2021)Viral encephalomyelitis is one of the leading causes of morbidity and mortality associated with viral infections. The alphaviruses are an important group of viruses that cause disease in humans and animals. Experimental studies in rodents using Semliki Forest virus (SFV), a prototypical alphavirus, have shown that the outcome of brain infection is age dependent. For this infection and several others, maturation of the central nervous system is associated with less severe encephalitis. However, the age-dependent susceptibility mechanisms to virus encephalitis are still unclear. In this thesis, I examined the age-dependent susceptibility to SFV infection in immature and mature primary cortical neurons and the role of type I IFN in SFV infected neurons. Both immature and mature neurons could be infected with SFV, but infection was greater in immature neurons than in mature neurons. Relative to immature neurons, differentiated mature neurons showed reduced virus replication and virus production. Notably, in response to the SFV infection, mature neurons produced IFN-beta whereas immature neurons failed to produce IFN-beta. I then explored whether IFN-beta could protect primary neuronal cultures against SFV infection. Immature neurons responded to IFN-beta pre-treatment however, pre-treatment with IFN-beta in immature neurons sets up only a transient antiviral state which is lost as the infection progresses. In conclusion, mature neurons produce IFN-beta in response to SFV infection whereas, immature neurons do not. However, immature neurons can respond to IFN-beta transiently curtailing virus replication. I further investigated the role of type I IFN in SFV infected neurons, and the results demonstrated that in its absence, mature neurons are more susceptible to SFV infection. Immature neurons from both WT and IFNAR1-/- mice exhibited the same characteristics in terms of infection rates and viral loads. These findings show that the susceptibility of immature neurons to infection is independent of the type I IFN response. In another approach to investigate the mechanisms, including the difference in IFN responses, underlying the age-dependent differential susceptibility of neurons to SFV infection, we undertook a transcriptomic approach. The transcriptomic profile of immature neurons is very different from mature neurons. The differential gene expression analysis and subsequent pathway activation and upstream regulatory analysis confirmed that mature neurons are equipped to curtail infection and immature neurons lack the key innate immune mechanisms to respond to infection. Additionally, this thesis also investigated the capacity of lyssaviruses P protein to inhibit interferon induction. There is a diversity among different lyssaviruses in their ability to inhibit IFN induction. I characterised an important P protein sequence that strongly inhibits RIG-I signalling and IFN induction. Together, these results highlight the importance of type I IFN in neurotropic virus infection. The findings presented in this thesis have shown that neuronal maturation and innate immunity are linked. Collectively, maturation of neurons is associated with increased levels of components of the innate immune system, contributing to the development of a functioning innate immune system that has the ability to restrict SFV replication in mature neurons.
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ItemRole of IFN-y induced genes in cell autonomous defence against Legionellae( 2021)L. pneumophila and L. longbeachae are ubiquitous environmental bacteria that can cause a severe pneumonia, known as Legionnaires’ Disease, when contaminated aerosols are inhaled by susceptible humans. The co-evolution of the bacteria with their environmental hosts has equipped the bacteria with the ability to subvert the cell intrinsic host defence mechanisms in human cells, thereby allowing the pathogens to survive and replicate within the lung macrophages. During infection, Legionella establishes an intracellular niche, known as the Legionella containing vacuole (LCV). The biogenesis of the LCV is dependent on the defective organelle trafficking/intracellular multiplication (Dot/Icm) type IV secretion system, which translocates a large arsenal of bacterial effector proteins into the host cell. These effectors are known to modulate host metabolism and cell-autonomous defence, protect the integrity of the LCV and allowing the bacteria to acquire nutrients from the host to ensure intracellular survival and enable intracellular replication of the pathogen. It is known that during Legionella lung infection, the mammalian host mounts a robust inflammatory response, producing cytokines, such as TNF-alpha, IL-1-alpha, IL-6, IL-12 as well as type I interferons and IFN-gamma, which usually leads to the restriction of intracellular replication and culminates in the clearance of the infection. It was previously shown that IFN-gamma is crucial for host defence against Legionella in mice, since disruption of IFN-gamma signalling or IFN-gamma deficiency results in a high replication of Legionella in the lung as well as a failure to clear the infection from the host, despite the activity of other inflammatory cytokines. Exposure of cells to interferons (IFN), including IFN-gamma, results in the induction of a network of genes that combat infection, leading to so-called IFN-mediated cell-autonomous defence. This network is finely-tuned to balance efficient pathogen control while preventing collateral tissue damage. However, which interferon-induced genes and through what mechanism this strikingly potent restriction is mediated remains elusive for Legionella. In this study, we shed some light on the mode of action of the IFN-gamma induced host defence against Legionella. We identified a new mechanism of host defence mediated by interferon stimulated genes (ISGs), that results in the disruption of effector translocation into host cells by the Dot/Icm secretion system. We demonstrated that this mechanism is uniquely triggered by interferon signalling and is independent of well-known host defence mechanisms such as host cell death, direct bactericidal activities, inflammasome activation as well as proteasome and autophagy-mediated degradation. By utilising mRNA sequencing of IFN-gamma and type I interferon-stimulated macrophages, we identified possible factors that mediate this inhibition: ISG15 and PARPs. These proteins have not previously been implicated in Legionella host defence and represent a unique opportunity to increase our knowledge of interferon mediated cell-autonomous host defence. Currently, more than 65 Legionella species are known and roughly half of them have been clinically associated with infection, frequently in immune compromised patients. After L. pneumophila, L. longbeachae is the second most common causative agent of Legionnaire’s Disease worldwide and is the leading causative agent in Australia and south-east Asia. Despite this, knowledge about the pathogenesis of L. longbeachae is minimal. Therefore, during this study, we also aimed to provide new insights into the pathogenesis of L. longbeachae infection and characterise the impact of IFN-gamma on immune control. We observed unique features of L. longbeachae infection in comparison to L. pneumophila, such as the ability to survive within a wider range of lung phagocytes, dampening of the cytokine response of the host and translocation of effectors into all lung phagocytes tested. These unique features may enable L. longbeachae to subvert the host defence more efficiently than L. pneumophila and thus replicate to higher numbers. Furthermore, we were able to show that IFN-gamma is crucial for host defence against L. longbeachae in vivo, with neutrophils and monocyte derived cells dependent on IFN-gamma signalling to mediate their bactericidal properties. In addition, we were able to demonstrate that IFN-gamma stimulation restricts L. longbeachae Dot/Icm secretion system effector translocation into host cells. Overall, this study substantiates the importance of IFN-gamma in host defence against Legionella and supports the need to broaden research efforts to non-L. pneumophila species. Investigation and deeper understanding of critical host defence mechanisms can be used as a starting point to develop anti-infective agents against pathogens targeting the process of effector translocation or effector mediated manipulation of host function and cell-autonomous defence.
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ItemHIV persistence, inflammation and the gut( 2021)Human Immunodeficiency Virus (HIV) remains incurable despite antiretroviral therapy (ART) due to its persistence as integrated provirus within long-lived and proliferating CD4+ T cells. Following initial infection, it replicates efficiently within and depletes Th17 cells which are enriched within the gut and play an important role in maintaining gut barrier integrity. This depletion leads to gut barrier permeability, bacterial translocation across the gut wall and local and systemic inflammation. These findings do not completely resolve on ART and may contribute to HIV persistence by promoting proliferation of infected CD4+ T cells and/or exhaustion of an effective immune response against HIV. Integrated provirus is enriched within Th17 cells in people living with HIV on ART. Th17 cells express CCR6 which promotes their migration towards CCL20 secreted by gut epithelium. Our laboratory has previously shown that CCL20 can promote HIV latency establishment in vitro likely through depolymerisation of the cortical actin barrier of resting CD4+ T cells allowing HIV to traverse this barrier and migrate to the nucleus. It is unknown to what extent CCL20-mediated gut migration or actin depolymerisation might contribute to HIV persistence in vivo. We compared the half-maximal effective concentration (EC50) of CCL20 for HIV latency establishment within resting memory CD4+ T cells with that of AKT phosphorylation, polarisation (a surrogate for actin depolymerisation) and migration of these cells. We reasoned that if the EC50 for HIV latency establishment were considerably lower than the EC50 for any of these cellular processes then that cellular process may not be required for CCL20-induced HIV latency establishment. We found progressively increasing EC50s for AKT phosphorylation, polarisation and migration; however, we were unable to demonstrate an effect of CCL20 on latency establishment. This was independent of viral tropism. Discrepancy with our laboratory’s previous findings may have been due to a shorter duration of exposure to chemokine in our experiments and/or a shorter resting time between cell sorting and infection which may have influenced cellular activation state and hence permissiveness to infection independent of chemokine. Vitamin D3 is a steroid hormone with pleiotropic effects on the immune system including reductions in CD4+ and CD8+ T cell activation, proliferation and exhaustion and reductions in frequency of Th17 and Th1 cells, both of which are important HIV reservoirs. It has also been shown in animal models to promote gut barrier integrity and in humans to reduce gut dysbiosis. We hypothesised that vitamin D3 may be able to deplete the HIV reservoir through these systemic and gut anti-inflammatory effects. We performed a randomised placebo-controlled trial evaluating the effect of 10,000 international units vitamin D3 per day for 24 weeks on markers of HIV persistence and immunology. Participants were followed for an additional 12 weeks post treatment. The primary endpoint was the difference between treatment arms in the change in frequency of total HIV DNA within CD4+ T cells from baseline to week 24. We found no effect of vitamin D3 on the primary endpoint. However, we found an increase at week 12 and a decrease at week 36 in frequency of total HIV DNA in the vitamin D3 arm relative to placebo. Importantly, 25-hydroxyvitamin D levels were still elevated at week 36 in the vitamin D3 arm relative to the placebo arm likely due to its long half-life. We also found a shift away from more differentiated subsets towards central memory CD4+ and CD8+ T cells at all time points including a reduction in frequency of effector memory CD4+ T cells at week 36. Other findings included a reduction in frequency of Th1 cells and levels of monocyte activation as expected but a paradoxical increase in frequency of Th17 cells and activated CD8+ T cells and NK cells in the vitamin D3 arm relative to the placebo arm. We hypothesise that the decrease in total HIV DNA frequency at week 36 may be due to the reduction in frequency of effector memory CD4+ T cells, known to be enriched in HIV DNA, which in turn may reflect the known anti-proliferative activity of vitamin D3. Increases in CD8+ T cell and NK cell activation may also have contributed to depletion of the HIV reservoir at week 36. Our findings support larger clinical trials which could incorporate an analytical treatment interruption to determine whether vitamin D3 is able to exert a clinically meaningful impact on the HIV reservoir.
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ItemThe regulation of MR1 cell surface expression and antigen presentation( 2021)Major histocompatibility complex class I-related protein 1 (MR1) presents vitamin B metabolites that are synthesised by wide range of microbes. Presentation of MR1-metabolite complexes by cells which have encountered bacteria activate the highly abundant innate-like T cells, mucosal associated invariant T (MAIT) cells, which then secrete inflammatory cytokines and perform cytotoxic activity to clear the infection. However, the molecular mechanism that regulates MR1 surface expression remains unclear. In this thesis, I revisited some of the mechanisms regulating MR1 surface expression proposed by previous studies. I found that while a small amount of MR1 associates with invariant chain (Ii), it does not require it for its surface expression nor presentation of antigen (Ag) from intracellular bacteria. Meanwhile, a small degree of modulation of MR1 surface expression by TLR signalling was observed in different primary cell types and cell lines. To characterise the molecular mechanism regulating MR1 surface expression and endocytosis, I employed several experimental approaches. I found that co-immunoprecipitation was not robust enough for the detection of regulatory proteins that interacts with MR1 at the cytoplasmic tail and trialled a genetically incorporated crosslinker that had showed promise. Ultimately, I employed a genome wide CRISPR/Cas9 knockout library screen which revealed that MR1 endocytosis requires alpha subunit (encoded by AP2A1) of the endocytic adaptor protein complex 2 (AP2). I found that AP2A1 interacts with the MR1 cytoplasmic tail and mediates endocytosis of MR1-antigen complexes. I further explored how AP2A1 regulates MR1 cellular behaviour by knocking out AP2A1 from two cell lines using CRISPR/Cas9. These cells showed significantly reduced MR1 internalisation rate, resulting in a higher surface expression level. Impaired MR1 internalisation in AP2A1-deleted cells leads to the accumulation of MR1-ligand complex on the cell surface, in turn causing prolonged activation of MAIT cells even 24 hours after metabolite Ag exposure. Meanwhile, I also found that recycling pathway is not critical for MR1 Ag uptake and presentation. Next, I explored the sorting signal of MR1 at the cytoplasmic tail likely recognised by AP2. I found that MR1 half-life was regulated by a tetrapeptide tyrosine-based motif at the cytoplasmic tail highly conserved in mammals. This motif lacks a feature of canonical tyrosine-based motifs; and when mutated to be canonical motif it accelerated the MR1 internalisation and degradation. In contrast, ablating MR1’s tyrosine residue, MR1 internalisation was impaired. Together, it suggested that MR1 Ag presentation was regulated using a unique non-canonical sorting motif, that allows appropriate MAIT cell activation. While our understanding of the regulatory mechanism of MR1 intracellular trafficking is ongoing, my findings reveal a novel insight into the unique regulation of MR1 surface expression which has important implications in the context of MAIT cell biology.
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ItemIdentification and characterization of proteins and mechanisms involved in the uptake and traffic of vitamin B related antigens( 2021)Major histocompatibility complex, class I-related (MR1) presents Vitamin B-related antigens (VitBAg) at the cell surface to activate mucosal associated invariant T (MAIT) cells, directing homeostasis and immune responses. Although previous work has suggested endocytosis as a participant in MR1 presentation, how these antigens are captured by the cell is currently unknown. It is likely that MR1 ligands are uptake as metabolites for they have several structural similarities with molecules known to be transported through solute carrier (SLC) transporters. Here, we shown that flavins are pathway-specific inhibitors of MR1-5-OP-RU, and do not inhibit MR1-Ac-6-FP upregulation. We revealed that 5-OP-RU, ribityl lumazine (RL) and bacterial VitBAg, but not folate derived ligands, enter the cell through SLC52A family of riboflavin transporters, as their expression increases MR1 presentation and MAIT cell activation in a riboflavin modulated manner. In contrast, knock-outs models SLC52A family drastically reduce the incorporation of RLs but do not abolish the capacity to present 5-OP-RU through MR1. In fact, pathway specific inhibitors of MR1-5-OP-RU and MR1-Ac-6-FP extend to nucleosides, nucleobases and other drugs, arguing for the contribution of more SLC transporters in their uptake. Likewise, MR1 presentation during infection is increased by ligand-producing bacteria located in the cytosol, stating a cytosolic step to reach empty MR1 molecules. Finally, we showed that 5-OP-RU alters the metabolome of cells like LPS, leading to changes in their transcriptome profile. Our results unveil a new route for 5-OP-RU, RL and bacterial VitBAg uptake through SLC52A transporters, contributing to their capture and modulating MR1 presentation, together with a new potential role of 5-OP-RU as a pathogen-associated molecular patterns (PAMPs) molecule.