The Minimum Information for High Content Screening Microscopy Experiments (MIHCSME) is a metadata model and reusable tabular template for sharing and integrating high content imaging data. It has... Show moreThe Minimum Information for High Content Screening Microscopy Experiments (MIHCSME) is a metadata model and reusable tabular template for sharing and integrating high content imaging data. It has been developed by combining the ISA (Investigations, Studies, Assays) metadata standard with a semantically enriched instantiation of REMBI (Recommended Metadata for Biological Images). The tabular template provides an easy-to-use practical implementation of REMBI, specifically for High Content Screening (HCS) data. In addition, ISA compliance enables broader integration with other types of experimental data, paving the way for visual omics and multi-Omics integration. We show the utility of MIHCSME for HCS data using multiple examples from the Leiden FAIR Cell Observatory, a Euro-Bioimaging flagship node for high content screening and the pilot node for implementing Findable, Accessible, Interoperable and Reusable (FAIR) bioimaging data throughout the Netherlands Bioimaging network. 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
Kusumawardhani, H.; Furtwängler, B.; Blommestijn, M.; Kaltenytė, A.; Poel, J.J.A. van der; Kolk, J.; ... ; Winde, J.H. de 2020
Pseudomonas putida S12 is highly tolerant of organic solvents in saturating concentrations, rendering this microorganism suitable for the industrial production of various aromatic compounds.... Show morePseudomonas putida S12 is highly tolerant of organic solvents in saturating concentrations, rendering this microorganism suitable for the industrial production of various aromatic compounds. Previous studies revealed that P. putida S12 contains the single-copy 583-kbp megaplasmid pTTS12. pTTS12 carries several important operons and gene clusters facilitating P. putida S12 survival and growth in the presence of toxic compounds or other environmental stresses. We wished to revisit and further scrutinize the role of pTTS12 in conferring solvent tolerance. To this end, we cured the megaplasmid from P. putida S12 and conclusively confirmed that the SrpABC efflux pump is the major determinant of solvent tolerance on the megaplasmid pTTS12. In addition, we identified a novel toxin-antitoxin module (proposed gene names slvT and slvA, respectively) encoded on pTTS12 which contributes to the solvent tolerance phenotype and is important for conferring stability to the megaplasmid. Chromosomal introduction of the srp operon in combination with the slvAT gene pair created a solvent tolerance phenotype in non-solvent-tolerant strains, such as P. putida KT2440, Escherichia coli TG1, and E. coli BL21(DE3). Show less
Bacteria switch only intermittently to motile planktonic lifestyles under favorable conditions. Under chronic nutrient deprivation, however, bacteria orchestrate a switch to stationary phase,... Show moreBacteria switch only intermittently to motile planktonic lifestyles under favorable conditions. Under chronic nutrient deprivation, however, bacteria orchestrate a switch to stationary phase, conserving energy by altering metabolism and stopping motility. About two-thirds of bacteria use flagella to swim, but how bacteria deactivate this large molecular machine remains unclear. Here, we describe the previously unreported ejection of polar motors by γ-proteobacteria. We show that these bacteria eject their flagella at the base of the flagellar hook when nutrients are depleted, leaving a relic of a former flagellar motor in the outer membrane. Subtomogram averages of the full motor and relic reveal that this is an active process, as a plug protein appears in the relic, likely to prevent leakage across their outer membrane; furthermore, we show that ejection is triggered only under nutritional depletion and is independent of the filament as a possible mechanosensor. We show that filament ejection is a widespread phenomenon demonstrated by the appearance of relic structures in diverse γ-proteobacteria including Plesiomonas shigelloides, Vibrio cholerae, Vibrio fischeri, Shewanella putrefaciens, and Pseudomonas aeruginosa. While the molecular details remain to be determined, our results demonstrate a novel mechanism for bacteria to halt costly motility when nutrients become scarce. Show less
Kusumawardhani, H.; Hosseini, R.; Winde, J.H. de 2018
The challenge of sustainably producing highly valuable chemical compounds requires specialized microbial cell factories because many of these compounds can be toxic to microbial hosts. Therefore,... Show moreThe challenge of sustainably producing highly valuable chemical compounds requires specialized microbial cell factories because many of these compounds can be toxic to microbial hosts. Therefore, solvent-tolerant bacteria are promising production hosts because of their intrinsic tolerance towards these compounds. Recent studies have helped to elucidate the molecular mechanisms involved in solvent tolerance. Advances in synthetic biological tools will enable further development of streamlined solvent-tolerant production hosts and the transfer of solvent-tolerant traits to established industrial strains. In this review, we outline challenges and opportunities to implement solvent tolerance in bacteria as a desired trait for industrial biotechnology. Show less
Organic solvent-tolerant bacteria are outstanding and versatile hosts for the bio-based production of a broad range of generally toxic aromatic compounds. The energetically costly solvent tolerance... Show moreOrganic solvent-tolerant bacteria are outstanding and versatile hosts for the bio-based production of a broad range of generally toxic aromatic compounds. The energetically costly solvent tolerance mechanisms are subject to multiple levels of regulation, involving among other mobile genetic elements. The genome of the solvent-tolerant Pseudomonas putida S12 contains many such mobile elements that play a major role in the regulation and adaptation to various stress conditions, including the regulation of expression of the solvent efflux pump SrpABC. We recently sequenced the genome of P. putida S12. Detailed annotation identified a threefold higher copy number of the mobile element ISS12 in contrast to earlier observations. In this study, we describe the mobile genetic elements and elaborate on the role of ISS12 in the establishment and maintenance of solvent tolerance in P. putida. We identified three different variants of ISS12 of which a single variant exhibits a high translocation rate. One copy of this variant caused a loss of solvent tolerance in the sequenced strain by disruption of srpA. Solvent tolerance could be restored by applying selective pressure, leading to a clean excision of the mobile element. Show less
In this thesis the zebrafish tail fin infection model is presented, which enables the study of a complex immune response towards (myco)bacterial infection using a combination of light and electron... Show moreIn this thesis the zebrafish tail fin infection model is presented, which enables the study of a complex immune response towards (myco)bacterial infection using a combination of light and electron microscopy. The induction of autophagy upon a mycobacterial infection as an important innate immune response was visualized using correlative light and electron microscopy. Studying the role of leukocyte dynamics and function during the course of infection provided new insights into the complex host-pathogen interactions. Using a myd88 mutant zebrafish line it was shown that the recruitment of leukocytes towards the site of infection and subsequent phagocytosis of bacteria is dependent on MyD88-mediated signaling. With the advancement of medical translational studies using zebrafish disease models, the tail fin infection model may 104 5 provide new opportunities to develop novel therapies against pathogenic infections like tuberculosis. Show less
Vaart, M. van der; Korbee, C.J.; Lamers, G.E.M.; Tengeler, A.C.; Hosseini, R.; Haks, M.C.; ... ; Meijer, A.H. 2014
High-resolution imaging of autophagy has been used intensively in cell culture studies, but so far it has been difficult to visualize this process in detail in whole animal models. In this study we... Show moreHigh-resolution imaging of autophagy has been used intensively in cell culture studies, but so far it has been difficult to visualize this process in detail in whole animal models. In this study we present a versatile method for high-resolution imaging of microbial infection in zebrafish larvae by injecting pathogens into the tail fin. This allows visualization of autophagic compartments by light and electron microscopy, which makes it possible to correlate images acquired by the 2 techniques. Using this method we have studied the autophagy response against Mycobacterium marinum infection. We show that mycobacteria during the progress of infection are frequently associated with GFP-Lc3-positive vesicles, and that 2 types of GFP-Lc3-positive vesicles were observed. The majority of these vesicles were approximately 1 μm in size and in close vicinity of bacteria, and a smaller number of GFP-Lc3-positive vesicles was larger in size and were observed to contain bacteria. Quantitative data showed that these larger vesicles occurred significantly more in leukocytes than in other cell types, and that approximately 70% of these vesicles were positive for a lysosomal marker. Using electron microscopy, it was found that approximately 5% of intracellular bacteria were present in autophagic vacuoles and that the remaining intracellular bacteria were present in phagosomes, lysosomes, free inside the cytoplasm or occurred as large aggregates. Based on correlation of light and electron microscopy images, it was shown that GFP-Lc3-positive vesicles displayed autophagic morphology. This study provides a new approach for injection of pathogens into the tail fin, which allows combined light and electron microscopy imaging in vivo and opens new research directions for studying autophagy process related to infectious diseases. Show less
Vaart, M. van der; Korbee, C.J.; Lamers, G.E.M.; Tengeler, A.C.; Hosseini, R.; Haks, M.C.; ... ; Meijer, A.H. 2014
