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
Tuberculosis (TB) is the most prevalent bacterial infectious disease in the world, caused by the pathogen Mycobacterium tuberculosis (Mtb). In this study, we have used Mycobacterium marinum (Mm)... Show moreTuberculosis (TB) is the most prevalent bacterial infectious disease in the world, caused by the pathogen Mycobacterium tuberculosis (Mtb). In this study, we have used Mycobacterium marinum (Mm) infection in zebrafish larvae as an animal model for this disease to study the role of the myeloid differentiation factor 88 (Myd88), the key adapter protein of Toll-like receptors. Previously, Myd88 has been shown to enhance innate immune responses against bacterial infections, and in the present study, we have investigated the effect of Myd88 deficiency on the granuloma morphology and the intracellular distribution of bacteria during Mm infection. Our results show that granulomas formed in the tail fin from myd88 mutant larvae have a more compact structure and contain a reduced number of leukocytes compared to the granulomas observed in wild-type larvae. These morphological differences were associated with an increased bacterial burden in the myd88 mutant. Electron microscopy analysis showed that the majority of Mm in the myd88 mutant are located extracellularly, whereas in the wild type, most bacteria were intracellular. In the myd88 mutant, intracellular bacteria were mainly present in compartments that were not electron-dense, suggesting that these compartments had not undergone fusion with a lysosome. In contrast, approximately half of the intracellular bacteria in wild-type larvae were found in electron-dense compartments. These observations in a zebrafish model for tuberculosis suggest a role for Myd88-dependent signalling in two important phenomena that limit mycobacterial growth in the infected tissue. It reduces the number of leukocytes at the site of infection and the acidification of bacteria-containing compartments inside these cells. 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
