Tuberculosis (TB) is associated with 1.5 million deaths annually. There is a need exists to optimize both current as well as novel antibiotic combination treatment strategies to improve the... Show moreTuberculosis (TB) is associated with 1.5 million deaths annually. There is a need exists to optimize both current as well as novel antibiotic combination treatment strategies to improve the effectiveness and safety of treatments against TB. This PhD thesis has described how various quantitative pharmacology modeling approaches can contribute to the further development and optimization of both existing and novel therapies and treatment strategies against TB. Show less
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
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
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
Regulatory processes are responsible for the organization, division and death of cells in multicellular organisms such as humans. Additionally, cells are highly regulated internally, able to... Show moreRegulatory processes are responsible for the organization, division and death of cells in multicellular organisms such as humans. Additionally, cells are highly regulated internally, able to survive and respond in vastly different micro-environments. Many types of interactions of cells with their environment can be distinguished, and need to be controlled in experiments aimed at unravelling and predicting cellular behavior in vivo. The in vivo microenvironment is mimicked by exposing cells to complex and changing environments. To describe the stochastic differences between cells and the local experimental conditions in sufficient detail and to obtain statistically relevant results, high-throughput experimentation is required. In this thesis four new research methods are developed, aimed at a deeper understanding of cellular regulation in vivo. Show less
Tuberculosis (TB) is an infectious disease, caused by Mycobacterium tuberculosis. MTB infection does not necessarily progress to TB. Only 5-10% of exposed individuals develop clinical signs and... Show moreTuberculosis (TB) is an infectious disease, caused by Mycobacterium tuberculosis. MTB infection does not necessarily progress to TB. Only 5-10% of exposed individuals develop clinical signs and symptoms of TB. Given the impact of mycobacterial exposure and the immunoregulatory consequences for host immunity, it is important to study the integrity and the regulation of immune responses and their downstream signaling pathways in TB endemic areas. Indonesia is a highly TB-burdened country and ranks third globally in TB cases. This thesis, consisting of six studies, explored variation in host immune responses to TB and their genetic background, and variation in clinical presentation. (1) MTB-specific stimulation of IFN-_ production as well as IFN-_ receptor signaling was significantly down-regulated during active TB, correlated with disease severity and __activity. (2) Concentrations of plasma granulysin of active TB patients were found to be low during acute disease. (3) Several genetic markers have been identified to affect susceptibility to TB (IL12B, IL12RB1, IFNG and IFNGR1) and (4) TLR8, DC-SIGN, complement component and scavenger receptor. (5) NRAMP polymorphisms were, however, not associated with susceptibility to TB. (6) TB with concomitant type2 DM presented more symptoms; screening fasting blood glucose in TB patients is clinically important. Show less
