Autophagy is a fundamental degradative process, maintaining cellular homeostasis and functions in host defense against intracellular pathogens, including mycobacteria and Salmonella. The thesis... Show moreAutophagy is a fundamental degradative process, maintaining cellular homeostasis and functions in host defense against intracellular pathogens, including mycobacteria and Salmonella. The thesis investigated the function of an regulator of antibacterial autophagy, Damage Regulated Autophagy Modulator 1 (DRAM1) against infection and shows that DRAM1 restricts bacterial growth not only through canonical antibacterial autophagy (xenophagy) but also promotes an autophagy-related pathway, named LC3-associated phagocytosis (LAP). The function of DRAM1 in restricting bacterial proliferation is independent from the recognition of bacteria by xenophagy receptors. Mechanistically, DRAM1 promotes the infection-induced activation of autophagy and LAP as well as the maturation of bacteria-containing vesicles in both pathways. This maturation process, stimulated by DRAM1, involves multiple vesicle fusion steps directing bacteria to lysosomes. Through this maturation process, DRAM1 delivers the cytosolic protein Fau to bacteria-containing vesicles, where it serves as a precursor for antimicrobial peptides. The underlying mechanism may be explained by the discovery of an interaction between DRAM1 and the SNARE protein VTI1B. Overall, the work in this thesis contributes to ongoing research into the potential application of autophagy modulation as a host-directed therapy against infectious diseases. Show less
In this thesis we describe our work regarding the identification of novel biomarkers, host targets and candidate pharmacological compounds for the development of therapies against various... Show moreIn this thesis we describe our work regarding the identification of novel biomarkers, host targets and candidate pharmacological compounds for the development of therapies against various intracellular bacterial infections, focusing primarily on the interplaybetween tuberculosis and diabetes mellitus. We conducted two longitudinal cohort studies in South Africa and Indonesia, and we applied unbiased and selective transcriptomic approaches to identify novel biomarker profiles in tuberculosis patients with concomitant diabetes or hyperglycaemia. Further, we performed experiments with standardized in vitro cellular infection models for drug discovery. We identified potential host targets during Mycobacterium tuberculosis infections and we describe the role of central metabolic pathways bacterial infections in human macrophages. Show less
Every day Tuberculosis (TB) kills approximately three thousand people, a number that is on the rise due to the impact of the current COVID-19 pandemic on essential TB services. The causative agent... Show moreEvery day Tuberculosis (TB) kills approximately three thousand people, a number that is on the rise due to the impact of the current COVID-19 pandemic on essential TB services. The causative agent of TB, Mycobacterium tuberculosis (Mtb), is an ancient pathogen that through its evolution developed complex mechanisms to evade immune surveillance and acquired the ability to establish persistent infection in its hosts. To achieve TB eradication, the discovery of Mtb antigens that effectively correlate with the human response to infection, with the curative host response following TB treatment, and with natural as well as vaccine induced protection is critical. This thesis contributes to this ambitious aim through several findings. First, it uncovers multiple new in vivo expressed Mtb (IVE-TB) antigens by combining Mtb-transcriptomic data with advanced bioinformatics tools and medium throughput cytokine screening. Second, it deepens our understanding of the cellular and humoral immunity to Mtb antigens in latently Mtb infected donors (LTBIs) and TB patients as well as in animal models. Lastly, it demonstrates the feasibility of combining and integrating pre-clinical research of multiple mycobacterial diseases, which are endemic in the same areas and against which vaccines could induce cross-disease protection (i.e., TB and leprosy). Show less
In individuals with tuberculosis-infection – until recently referred to as latent tuberculosis infection – the risk of progression to active tuberculosis (reactivation) varies strongly. Among those... Show moreIn individuals with tuberculosis-infection – until recently referred to as latent tuberculosis infection – the risk of progression to active tuberculosis (reactivation) varies strongly. Among those at increased risk of reactivation are patients with an impaired immune system, e.g. due to immunosuppressive therapy. Therefore, prior to planned immunosuppression, patients are screened for tuberculosis-infection and subsequently treated in case of infection. Current screening methods include the Mantoux test, Interferon-γ release assays (i.e., the QuantiFERON-TB Gold Plus and T-SPOT.TB) and chest X-ray. However, despite screening, cases of reactivation continue to occur – in part due to the lack of a gold standard test for tuberculosis-infection. Therefore, the aims of this thesis were to increase the diagnostic sensitivity for tuberculosis-infection prior to immunosuppression. Using various (novel) methods we showed that approximately two-thirds of all QuantiFERON-TB Gold Plus results just below the manufacturer’s cut-off (in the borderline range) are caused by Mycobacterium tuberculosis-infection, which now warrants preventive treatment in patients with such a result. Furthermore, we quantified the diagnostic accuracy of chest X-ray for tuberculosis-infection and showed that using a novel ultra-low dose CT scanning technique, sensitivity for tuberculosis-infection could be significantly increased by three-fold compared to chest X-ray. Show less
New drugs for use as tuberculosis (TB) treatment are needed due to the constrains of classical antibiotics against TB and the rise of antibiotic-resistant strains, making TB a harder and harder... Show moreNew drugs for use as tuberculosis (TB) treatment are needed due to the constrains of classical antibiotics against TB and the rise of antibiotic-resistant strains, making TB a harder and harder disease to treat. This thesis is focused on using the in vivo whole animalzebrafish embryo model for TB to evaluate potential anti-TB host-directed therapeutics (HDTs) arising from in vitro screens. Although in vitro screens for HDTs using cellular models can be performed at high throughput, a limiting step is the validation in whole animal models and translation of results to clinical applications. Due to the complex infection dynamics of mycobacteria, the use of whole animal models is indispensable in research into TB and the zebrafish model has contributed key findings about host-pathogen dynamics during mycobacterial infection. One of the most promising host targets of HDTs is autophagy, which is recognized as an important host-protective pathway. Boosting autophagy levels using HDTs could be a way to overcome the pathogen’s autophagy evasion strategies and could therefore be a promising therapeutic route. For this thesis we took advantage of the possibilities of the zebrafish embryo model for TB and the zebrafish toolkit to study several autophagy-modulating HDTs as potential anti-TB drugs. Show less
The research described in this thesis has, using the zebrafish as a model system, shed new light on the intricate relationship between TB and DM2, in particular on the role of leptin, SHP-1 and... Show moreThe research described in this thesis has, using the zebrafish as a model system, shed new light on the intricate relationship between TB and DM2, in particular on the role of leptin, SHP-1 and glucocorticoids.Leptin plays an important role during TB infection and has a huge impact on insulin sensitivity in zebrafish larvae. Similarly to what has been observed in the murine model, leptin deficiency in zebrafish increased the bacterial burden and mortality during the infection, leading to hyperglycemia and the development of insulin resistance. In addition, a novel SHP-1/SHP-2 inhibitor, NSC-87877, was shown to represent a promising anti-diabetic drug that can be used for further DM2 research, as it is able to rescue the phenotype of the leptin-deficient zebrafish and to restore glucose transport to the tissues. In contrast to metformin, NSC-87877 can act at very early developmental stages and inhibits the function of SHP-1 and factors that underlay impaired glucose metabolism, whereas metformin is mostly known to improve insulin sensitivity. Additionally, treatment with the glucocorticoid beclomethasone attenuates the metabolic changes associated with the infection, and transcriptional alterations induced by beclomethasone treatment suggest that genes involved in glucose metabolism, insulin and leptin signaling all play an important role in the modulation of the metabolism.Our data show that zebrafish larvae represent an interesting model system to investigate the complex pathology of TB, and the studies described in this thesis in which this model has been used have provided novel insights into the molecular mechanisms underlying wasting syndrome and the possibilities for adjunctive glucocorticoid therapy to alleviate this metabolic state. Show less
In this thesis, I study 1) metabolic alterations in tuberculosis related to wasting syndrome in human patients as well as in rodent and fish animal models. 2) effects of the mutation of the leptin... Show moreIn this thesis, I study 1) metabolic alterations in tuberculosis related to wasting syndrome in human patients as well as in rodent and fish animal models. 2) effects of the mutation of the leptin gene on cachexia and diabetes in rodent and zebrafish animal models. 3) how tuberculosis infection and resulting metabolic reprogramming are dependent on leptin signaling in mice and zebrafish larvae. Show less
In this thesis several aspects of complement proteins are described, from circulating levels in blood to their intracellular presence and from autoimmunity to the infectious disease tuberculosis.... Show moreIn this thesis several aspects of complement proteins are described, from circulating levels in blood to their intracellular presence and from autoimmunity to the infectious disease tuberculosis. We explored the local production of complement and we describe in Chapter 2 the production of C1q by chondrocytes. Additionally, studies addressing the potential intracellular C3 role are described in Chapter 3. The potential role of the complement system as biomarker was investigated by addressing the presence and concentrations of C1q in serum of patients with active tuberculosis and controls in Chapter 4. Like C1q, we also investigated the expression and concentration of the natural inhibitor C1-INH in Chapter 5. C1q protein was further analysed as biomarker for tuberculosis in experimental non-human primate models in Chapter 6. In this thesis, a newly identified case of a lupus patient is described with a complex medical history and a compound heterozygous deficiency of C1q in Chapter 7. To better comprehend a possible role of a prominent post-translational modification associated rheumatic disease, carbamylation, the interaction between carbamylated IgG was investigated in relation to the ability to activate the complement system. These studies are described in Chapter 8. Show less
Diabetes mellitus type 2 (DM) is a major risk factor for developing active tuberculosis (TB) disease, yet the causal mechanisms driving this association remain largely elusive. As the incidence of... Show moreDiabetes mellitus type 2 (DM) is a major risk factor for developing active tuberculosis (TB) disease, yet the causal mechanisms driving this association remain largely elusive. As the incidence of DM is rising, especially in TB endemic countries, it is important to identify the relevant immunological and metabolic processes that underlie TB-DM comorbidity, because such insights will facilitate optimal treatment, diagnosis and prevention. In this thesis, we have started to unravel key factors underlying the association between TBand DM using two approaches. Firstly, we identified and analyzed human macrophage subsets and studied the interactions between these human cells and a major pathogen, Mycobacterium tuberculosis (Mtb), and the specific metabolic changes involved using well-controlled in vitro systems. Next, we employed metabolomics to determine the impact of concurrent TB-DM on circulating metabolites in patient cohorts ex vivo. In this thesis we present evidence derived from in vitro experiments and from ex vivo observational data which collectively suggest a pathogenic role of atherogenic lipid species during TB development. Show less
The zebrafish is a promising vertebrate model organism in early drug discovery and development. Translation of pharmacological findings to higher vertebrates requires quantification of the... Show moreThe zebrafish is a promising vertebrate model organism in early drug discovery and development. Translation of pharmacological findings to higher vertebrates requires quantification of the underlying pharmacological and (patho)physiological processes. In this thesis, we therefore developed and integrated innovative experimental and computational methods for the successful quantification of 1) the internal exposure over time after waterborne drug treatment, 2) disease dynamics and drug-induced changes therein, and 3) between-species differences in disease mechanisms. The state-of-the-art methods that we developed included nanoscale blood sampling, sensitive LC-MS/MS methods for drugs and their isomers and metabolites, and three-dimensional microscopy, integrated with non-linear mixed effects modelling to quantify the pharmacological processes in this small vertebrate. This multidisciplinarity enabled quantification of internal drug exposure-response relationships, contributed to positioning the zebrafish in the preclinical drug development pipeline, and inspired continuous collaborations between experimental and computational scientists. Show less
Tuberculosis (TB) continues to remain a major public health problem globally. WHO’s End TB Strategy has set a goal to end the epidemic with ambitious targets for 2035. About 27% of the global TB... Show moreTuberculosis (TB) continues to remain a major public health problem globally. WHO’s End TB Strategy has set a goal to end the epidemic with ambitious targets for 2035. About 27% of the global TB cases occur in India which poses a challenge to global TB control. This is mainly because majority of patients in India approach the private sector, which is usually diverse, disorganized, unregulated and often disconnected from the national TB control programme (NTP). The quality of care is not consistent across the private sector and completion of treatment is not ensured. Non-standard treatment leads to the emergence of drug-resistant TB. Public-private collaborations in India have shown promising results in improving the situation. This research analyzed the manner and conditions for private sector engagement to increase the chances of ending TB. For this, India will have to improve and scale up public-private partnerships. This will need heavy investments and adoption of newer and innovative approaches, tools and technology aiming Universal Health Coverage. This research used selected publications and reviewed relevant research in the background of the global approaches for ending TB by 2035. The findings originate from research done mostly in India which is relevant for other developing countries. Show less
Antibiotic resistance is an increasing problem in the battle against (bacterial) infectious diseases. The emergence of drug-resistant Mycobacterium tuberculosis (Mtb) threatens to render... Show moreAntibiotic resistance is an increasing problem in the battle against (bacterial) infectious diseases. The emergence of drug-resistant Mycobacterium tuberculosis (Mtb) threatens to render tuberculosis (TB) untreatable. Efforts to develop novel antibiotics have so far been unsuccessful, calling for additional approaches for treatment of bacterial infections. Intracellular pathogens like Mtb and Salmonella can survive in the host by manipulating host cell signaling. This provides opportunities for novel therapeutic strategies by targeting the host, rather than the bacterium (host-directed therapy). In this thesis we report the development and application of novel (in vitro and in vivo) methods for identifying host genes and proteins involved in host control of intracellular bacteria, as well as chemical compounds that target host molecules as a basis for drug development for host-directed therapies. As a result, we report the identification of RTK inhibitors, the novel kinase inhibitor 97i, the human kinase family PCTAIRE and the host protein DRAM1 as promising leads for further drug development for host-directed therapeutic strategies for intracellular bacterial infections. Show less
The effective treatment of tuberculosis (TB) remains a major challenge to global health. Drug-resistant Mycobacterium tuberculosis (Mtb) strains and co-infection with HIV further increase the... Show moreThe effective treatment of tuberculosis (TB) remains a major challenge to global health. Drug-resistant Mycobacterium tuberculosis (Mtb) strains and co-infection with HIV further increase the difficulty of controlling TB. Thus, under the current situation, it is essential to develop effective treatment strategies for Mtb infections. Autophagy is a lysosomal degradation process and substantial experimental evidence has demonstrated that autophagy is an important host immune defense mechanism against mycobacterial infection. However, the development of effective therapies requires a better understanding of the interaction between the host and invading pathogens to identify host processes that can be targeted. A useful tool for such studies is the zebrafish model for TB. Zebrafish can be infected with Mycobacterium marinum (Mm), which is closely related to Mtb and causes similar disease characteristics. Taking advantage of the zebrafish TB model, this thesis presents new in vivo evidence for the important function of autophagy to inhibit mycobacterial proliferation inside macrophages. Furthermore, this study supports that stimulating the innate host defense processes that are dependent on the autophagy modulator, Dram1, and the selective autophagy receptors, p62 and Optineurin, could be a useful strategy to explore for adjunctive treatment of antibiotic-resistant TB infections. Show less
This thesis describes the structural and biochemical characterization of the β-lactamase BlaC from Mycobacterium tuberculosis (Mtb), and the Alr and YlmE proteins from Streptomyces coelicolor A3(2)... Show moreThis thesis describes the structural and biochemical characterization of the β-lactamase BlaC from Mycobacterium tuberculosis (Mtb), and the Alr and YlmE proteins from Streptomyces coelicolor A3(2).Mtb is the main cause of tuberculosis. The inherent production of BlaC by Mtb makes the antibiotic treatment of tuberculosis particularly difficult because BlaC renders Mtb naturally resistant to β-lactam antibiotics. One possible way to circumvent this BlaC-mediated resistance is the co-administration of β-lactamase inhibitors, thus preventing antibiotics’ hydrolysis. The crystal structure of BlaC was determined in complex with the β-lactamase inhibitors clavulanic acid, sulbactam, tazobactam, and avibactam, and new BlaC-inhibitors covalent adducts were visualized. The affinity of BlaC for the inhibitors was further studied using catalytically inactive mutants of the enzyme.In parallel, the Alr and YlmE proteins from S. coelicolor A3(2) were studied. Alr and YlmE are putatively involved in the racemization of L-Ala into D-Ala. The latter is an essential peptidoglycan building block, and ensures cell wall compaction and bacterial survival. The structural and biochemical characterization of the heterologous, purified Alr and YlmE proteins showed that while Alr is indeed involved in Ala racemization, YlmE is not. Our findings revealed a possible new, surprising role for YlmE in nucleic acid binding. Show less
In this thesis I studied the functions of the zebrafish orthologs of the human TLR5 and TLR2 genes that were shown to be responsible for recognition of bacterial flagellin and a broad spectrum... Show moreIn this thesis I studied the functions of the zebrafish orthologs of the human TLR5 and TLR2 genes that were shown to be responsible for recognition of bacterial flagellin and a broad spectrum of bacterial cell wall components, respectively. One of the focal points of this thesis is the difference at the transcriptomic level of the downstream pathway of the TLR5 and TLR2 receptors and the roles of TLR signaling in host innate immune responses to infection by Mycobacterium marinum, a close relative to Mycobacterium tuberculosis and a natural pathogen of zebrafish. The new possibilities for analysis of transcriptomes using RNA deep sequencing make it highly attractive to analyze the responses of an entire test animal model at the system biology level. Furthermore, we used genetic knockdown and knockout tools to further analyze the function of TLR5 and TLR2 and downstream signaling partners in innate immunity, infectious disease and insulin resistance. Show less
Mycobacterium tuberculosis, the agent of TB, is one of the deadliest human pathogens, infecting one third of the global population. Establishment of infection by mycobacteria relies on complex... Show moreMycobacterium tuberculosis, the agent of TB, is one of the deadliest human pathogens, infecting one third of the global population. Establishment of infection by mycobacteria relies on complex interactions with host innate immune cells, especially macrophages. Once engulfed by macrophages, mycobacteria “usurp” the host cell machineries to facilitate dissemination and to establish an intracellular niche for survival and replication. To investigate how mycobacteria force the immune cells to support infection, we explored the chemokine pathway, best known for its capability to induce cell migration. To dissect the interplay between immune cells and the pathogen, we modelled human TB using the zebrafish-Mycobacterium marinum natural host-pathogen pair, which is attractive for the excellent optical accessibility of the zebrafish larvae and the possibility to apply genetic tools to impair the chemokine signaling. We show that depletion of either CXCR3 or CXCR4 axes are beneficial to the host. Exploitation of CXCR3 signaling leads to macrophage recruitment and to transcriptional changes in macrophages that make them more permissive for mycobacterial intracellular persistence. Activating CXCR4 signaling triggers instead vascularization of the nascent tuberculous granulomas, which in turn supports expansion of the infection. Therefore, inhibitions of these pathways represent promising host-directed therapeutic avenues to counteract mycobacterial diseases. Show less
There is no effective vaccine against tuberculosis (TB). The only available TB-vaccine, M. bovis BCG, induces only limited, and highly variable protection. TB-vaccine efficacy would have to include... Show moreThere is no effective vaccine against tuberculosis (TB). The only available TB-vaccine, M. bovis BCG, induces only limited, and highly variable protection. TB-vaccine efficacy would have to include protection against active pulmonary TB, since this is the transmissible form of the disease, in the adult population; an effective vaccine would have an enormous impact on the TB-epidemic. This thesis has aimed to characterize the M. bovis BCG-reactive human T-cell response, in order to identify cellular responses that may account for the variable and poorly understood protective efficacy of BCG-vaccination. The studies presented in this thesis describe three BCG-induced cellular immune responses in adults: (i) the induction of CD8+ regulatory T-cells (Tregs), that suppress immunity partly via the ectoenzyme CD39, (ii) a dichotomous pro-inflammatory response, consisting of either induction of polyfunctional CD4+ T-cells in vaccinees with high skin inflammation of the vaccine lesion, or virtually no induction of cytokines with concomitant induction of CD8+ Tregs in vaccinees with low skin inflammation, and (iii) induction of inhibitory KLRG1+ CD8+ T-cells. This network of inter-related and partly opposing regulatory, pro-inflammatory and inhibitory immune responses may impact vaccine-induced protective immunity against TB and this could assist in guiding future TB-vaccine design. Show less
Currently, only one tuberculosis (TB) vaccine is available: Mycobacterium bovis Bacille Calmette-Gu_rin (BCG). This vaccine induces highly variable protection against pulmonary TB, the most common... Show moreCurrently, only one tuberculosis (TB) vaccine is available: Mycobacterium bovis Bacille Calmette-Gu_rin (BCG). This vaccine induces highly variable protection against pulmonary TB, the most common and contagious form of TB. There is an urgent need for an effective TB vaccine which is safe also in the immunocompromised host. The main focus of this thesis was to identify Mycobacterium tuberculosis (Mtb) infection phase related antigens and to evaluate these as potential antigens for TB vaccines. The studies presented in this thesis describe: (i) the immunogenic potential of two previously described sets of antigens; resuscitation promoting factor (Rpf) and dormancy regulon encoded (DosR) antigens, (ii) the identification and immunogenicity of a third set of antigens known as in vivo expressed Mtb (IVE-TB) antigens, (iii) the protective value of IVE-TB antigen Rv2034 and (iv) the analysis of Rv2034-specific T cell r esponses at the clonal level. Together, these data illustrate the vaccine potential of infection phase related antigens. Show less
This thesis focuses on the recognition of pathogenic bacteria and the defense mechanisms that are activated during the innate immune response to infection. Detection of pathogens, such as bacteria,... Show moreThis thesis focuses on the recognition of pathogenic bacteria and the defense mechanisms that are activated during the innate immune response to infection. Detection of pathogens, such as bacteria, viruses, and parasites, depends on receptors that bind to evolutionary conserved structures on their surface. The most extensively studied class of immune receptors is the Toll-like receptor (TLR) family, which signals via adaptor molecules such as myeloid differentiation factor 88 (MyD88) to initiate gene expression and activate the appropriate response upon recognition of a pathogen. We have used the zebrafish as a model organism to study how MyD88 orchestrates the immune response against intracellular bacterial pathogens like Mycobacterium marinum, the causative agent of tuberculosis disease (TB) in fish. We found that several defense mechanisms against TB are highly dependent on MyD88, including autophagy, cytokine and chemokine production, and the generation of microbe killing radicals. These findings in the zebrafish model will hopefully aid in the development of new therapeutic strategies against multi-drug resistant tuberculosis infections. Show less
Type I immune responses play an essential role in the control of mycobacterial infections. Mutations in the genes involved in the type I cytokine pathway were found in patients with Mendelian... Show moreType I immune responses play an essential role in the control of mycobacterial infections. Mutations in the genes involved in the type I cytokine pathway were found in patients with Mendelian susceptibility to mycobacterial diseases. These patients are highly susceptible to infections with non-tuberculous mycobacteria (NTM), which are usually poorly pathogenic. The first part of this thesis focuses on the relation between the genotype and phenotype in the cause of an impaired immunity leading to the susceptibility to NTM infections. The role of genetic factors in the control of infections with more virulent tuberculous mycobacteria is less evident. The second part of this thesis focuses on putative non-genetic causes of an impaired immunity in the control of tuberculous mycobacterial diseases. In tuberculosis patients type I immune responses regulated by interferon-_ are also repressed. Virally induced interferons, other than interferon-_, may be involved in this repression, thereby influencing the immunopathogenesis of tuberculosis. Show less
