Infection of zebrafish embryos with live fluorescent Streptococcus pneumoniae as a real-time pneumococcal meningitis model

Background: Streptococcus pneumoniae is one of the most important causes of bacterial meningitis, an infection where unfavourable outcome is driven by bacterial and host-derived toxins. In this study, we developed and characterized a pneumococcal meningitis model in zebrafish embryos that allows for real-time investigation of early host-microbe interaction. Methods: Zebrafish embryos were infected in the caudal vein or hindbrain ventricle with green fluorescent wild-type S. pneumoniae D39 or a pneumolysin-deficient mutant. The kdrl:mCherry transgenic zebrafish line was used to visualize the blood vessels, whereas phagocytic cells were visualized by staining with far red anti-L-plastin or in mpx:GFP/mpeg1:mCherry zebrafish, that have green fluorescent neutrophils and red fluorescent macrophages. Imaging was performed by fluorescence confocal and time-lapse microscopy. Results: After infection by caudal vein, we saw focal clogging of the pneumococci in the blood vessels and migration of bacteria through the blood-brain barrier into the subarachnoid space and brain tissue. Infection with pneumolysin-deficient S. pneumoniae in the hindbrain ventricle showed attenuated growth and migration through the brain as compared to the wild-type strain. Time-lapse and confocal imaging revealed that the initial innate immune response to S. pneumoniae in the subarachnoid space mainly consisted of neutrophils and that pneumolysin-mediated cytolytic activity caused a marked reduction of phagocytes. Conclusions: This new meningitis model permits detailed analysis and visualization of host-microbe interaction in pneumococcal meningitis in real time and is a very promising tool to further our insights in the pathogenesis of pneumococcal meningitis

A Novel Extracytoplasmic Function (ECF) Sigma Factor Regulates Virulence in Pseudomonas aeruginosa

Next to the two-component and quorum sensing systems, cell-surface signaling (CSS) has been recently identified as an important regulatory system in Pseudomonas aeruginosa. CSS systems sense signals from outside the cell and transmit them into the cytoplasm. They generally consist of a TonB-dependent outer membrane receptor, a sigma factor regulator (or anti-sigma factor) in the cytoplasmic membrane, and an extracytoplasmic function (ECF) sigma factor. Upon perception of the extracellular signal by the receptor the ECF sigma factor is activated and promotes the transcription of a specific set of gene(s). Although most P. aeruginosa CSS systems are involved in the regulation of iron uptake, we have identified a novel system involved in the regulation of virulence. This CSS system, which has been designated PUMA3, has a number of unusual characteristics. The most obvious difference is the receptor component which is considerably smaller than that of other CSS outer membrane receptors and lacks a β-barrel domain. Homology modeling of PA0674 shows that this receptor is predicted to be a bilobal protein, with an N-terminal domain that resembles the N-terminal periplasmic signaling domain of CSS receptors, and a C-terminal domain that resembles the periplasmic C-terminal domains of the TolA/TonB proteins. Furthermore, the sigma factor regulator both inhibits the function of the ECF sigma factor and is required for its activity. By microarray analysis we show that PUMA3 regulates the expression of a number of genes encoding potential virulence factors, including a two-partner secretion (TPS) system. Using zebrafish (Danio rerio) embryos as a host we have demonstrated that the P. aeruginosa PUMA3-induced strain is more virulent than the wild-type. PUMA3 represents the first CSS system dedicated to the transcriptional activation of virulence functions in a human pathogen

MyD88 Innate Immune Function in a Zebrafish Embryo Infection Model

Innate immunity signaling mechanisms during vertebrate embryogenesis are largely unknown. To study Toll-like receptor (TLR) signaling function in the zebrafish embryo model, we designed an experimental setup for antisense morpholino knockdown under conditions of bacterial infection. Clearance of Salmonella enterica serovar Typhimurium Ra bacteria was significantly impaired after knockdown of myeloid differentiation factor 88 (MyD88), a common adaptor protein in TLR and interleukin-1 receptor signaling. Thereby, we demonstrate for the first time that the innate immune response of the developing embryo involves MyD88-dependent signaling, which further establishes the zebrafish embryo as a model for the study of vertebrate innate immunity

Modeling tuberculous meningitis in zebrafish using Mycobacterium marinum

Tuberculous meningitis (TBM) is one of the most severe extrapulmonary manifestations of tuberculosis, with a high morbidity and mortality. Characteristic pathological features of TBM are Rich foci, i.e. brain- and spinal-cord-specific granulomas formed after hematogenous spread of pulmonary tuberculosis. Little is known about the early pathogenesis of TBM and the role of Rich foci. We have adapted the zebrafish model of Mycobacterium marinum infection (zebrafish-M. marinum model) to study TBM. First, we analyzed whether TBM occurs in adult zebrafish and showed that intraperitoneal infection resulted in granuloma formation in the meninges in 20% of the cases, with occasional brain parenchyma involvement. In zebrafish embryos, bacterial infiltration and clustering of infected phagocytes was observed after infection at three different inoculation sites: parenchyma, hindbrain ventricle and caudal vein. Infection via the bloodstream resulted in the formation of early granulomas in brain tissue in 70% of the cases. In these zebrafish embryos, infiltrates were located in the proximity of blood vessels. Interestingly, no differences were observed when embryos were infected before or after early formation of the blood-brain barrier (BBB), indicating that bacteria are able to cross this barrier with relatively high efficiency. In agreement with this observation, infected zebrafish larvae also showed infiltration of the brain tissue. Upon infection of embryos with an M. marinum ESX-1 mutant, only small clusters and scattered isolated phagocytes with high bacterial loads were present in the brain tissue. In conclusion, our adapted zebrafish-M. marinum infection model for studying granuloma formation in the brain will allow for the detailed analysis of both bacterial and host factors involved in TBM. It will help solve longstanding questions on the role of Rich foci and potentially contribute to the development of better diagnostic tools and therapeutics

In vitro cell migration of PaTu-T cells.

<p>A scratch (wound healing) assay was performed with PaTu-T, PaTu-T/Gal-4 and PaTu-T/mock cells. PaTu-T/mock and PaTu-T/Gal-4 cells were seeded on a 24 well plate and scratched on the surface with a 200-µl pipette tip. Relative values were set at 100% of the gap width at the time of the scratch. <b>A)</b> Representative photographs at time points 0, 6, 19 and 24 hours after the wound (scratch) for all conditions are depicted. <b>B)</b> Histogram representation of data analyzed from photographs taken at 0 h; 6 h, 19 h and 24 h after the scratch. Measurements were done in duplicate in 3 separate experiments, and data are depicted as average gap width ± SEM. (* p≤0.05 and ** p≤0.01, using one way ANOVA Tukey t tests).</p

Gal-4 mRNA expression in pancreatic cancer cell lines.

<p>Gal-4 mRNA expression of normal human pancreatic duct epithelial-like cell line (hTERT-HPNE) and 9 different human pancreatic cancer cell lines was analyzed by quantitative real-time PCR and depicted as the relative amount of Gal-4 transcripts (± SEM) compared to the expression of the endogenous reference gene <i>GAPDH</i>. (*** p≤0.001 versus all other cell lines, using one way ANOVA with post Dunnett two sided t tests).</p

Gal-4 and Gal-4 binding sites in PaTu-S and PaTu-T cells.

<p>Detection of endogenous Gal-4, and Gal-4 ligands, in PaTu-S and PaTu-T cells by flow cytometry. A histogram of one representative experiment is depicted for each condition of least two independent experiments. <b>A)</b> Dot plots of Gal-4 staining of permeabilized PaTu-S and PaTu-T cells. Gal-4 was detected at 4°C with anti-hGal-4 Abs in fixed permeabilized cells. Secondary Abs staining without anti-hGal-4 Abs was used as background autofluorescence control. <b>B)</b> Presence of endogenous bound Gal-4 to the surface of PaTu-S and PaTu-T after washing the cells with 500 mM lactose prior to Gal-4 staining. The presence of Gal-4 was established by FACS analysis using anti-hGal-4 Abs at 4°C. Endogenous Gal-4 bound to the surface is shown by a black line. <b>C)</b> The presence of Gal-4 binding sites on PaTu-S and PaTu-T cells was determined after washing the cells with 500 mM lactose prior to Gal-4 staining. The binding of externally added recombinant (rec) hGal-4 (5 µg/ml, black line) was investigated. Binding of rec hGal-4 to the surface could be inhibited by adding lactose (dark field). Background staining with secondary Abs is depicted as light grey fields in B and C.</p

Immunocytochemical localization of Gal-4 in PaTu-S cells.

<p>Photographs of representative ICC analysis of the cellular localization of Gal-4 in PaTu-S cells. Gal-4 was detected using Alexa-labeled anti-Gal-4 Abs (green), Actin was stained using Phalloidin (red) and nucleus staining obtained using HOESCHS (blue); the third panel shows the merging of the different stainings. Bar = 25 µm.</p

Identification and High-Resolution Imaging of alpha-Tocopherol from Human Cells to Whole Animals by TOF-SIMS Tandem Mass Spectrometry

A unique method for identification of biomolecular components in different biological specimens, while preserving the capability for high speed 2D and 3D molecular imaging, is employed to investigate cellular response to oxidative stress. The employed method enables observing the distribution of the antioxidant α-tocopherol and other molecules in cellular structures via time-of-flight secondary ion mass spectrometry (TOF-SIMS (MS1)) imaging in parallel with tandem mass spectrometry (MS2) imaging, collected simultaneously. The described method is employed to examine a network formed by neuronal cells differentiated from human induced pluripotent stem cells (iPSCs), a model for investigating human neurons in vitro. The antioxidant α-tocopherol is identified in situ within different cellular layers utilizing a 3D TOF-SIMS tandem MS imaging analysis. As oxidative stress also plays an important role in mediating inflammation, the study was expanded to whole body tissue sections of M. marinum-infected zebrafish, a model organism for tuberculosis. The TOF-SIMS tandem MS imaging results reveal an increased presence of α-tocopherol in response to the pathogen

Mycobacterium marinum Strains Can Be Divided into Two Distinct Types Based on Genetic Diversity and Virulence

Mycobacterium marinum causes a systemic tuberculosis-like disease in a large number of poikilothermic animals and is used as a model for mycobacterial pathogenesis. In the present study, we infected zebra fish (Danio rerio) with different strains of M. marinum to determine the variation in pathogenicity. Depending on the M. marinum isolate, the fish developed an acute or chronic disease. Acute disease was characterized by uncontrolled growth of the pathogen and death of all animals within 16 days, whereas chronic disease was characterized by granuloma formation in different organs and survival of the animals for at least 4 to 8 weeks. Genetic analysis of the isolates by amplified fragment length polymorphism showed that M. marinum strains could be divided in two clusters. Cluster I contained predominantly strains isolated from humans with fish tank granuloma, whereas the majority of the cluster II strains were isolated from poikilothermic species. Acute disease progression was noted only with strains belonging to cluster I, whereas all chronic-disease-causing isolates belonged to cluster II. This difference in virulence was also observed in vitro: cluster I isolate Mma20 was able to infect and survive more efficiently in the human macrophage THP-1 and the carp leukocyte CLC cell lines than was the cluster II isolate Mma11. We conclude that strain characteristics play an important role in the pathogenicity of M. marinum. In addition, the correlation between genetic variation and host origin suggests that cluster I isolates are more pathogenic for humans