Transcriptome analysis of the zebrafish embryonic host response to Edwardsiella tarda infection using a static immersion systems [experiment A]
ABSTRACT: In this study we analyzed the zebrafish embryonic host response induced by E. tarda (FL6-60) immersion. The E. tarda induced transcriptome profile was compared to those induced by either E. coli or Pseudomonas aeruginosa immersion using the same experimental setup. All infection experiments were performed using mixed egg clutches of Albino strain zebrafish. At 24 hpf embryos were dechorionated using 2mg/ml pronase and left to recover for one hour in egg water. Subsequently embryos were immersed in a bacterial suspension (E. tarda (1E8 CFU/ml), E. coli (1E8 CFU/ml), Pseudomonas aeruginosa PAO1 (1E9 CFU/ml) or P. aeruginosa PA14 (1E9 CFU/ml)) and incubated for 5 hours at 28 ˚C. After the incubation period the embryos were snap-frozen in liquid nitrogen. All treatment groups were analyzed using a common reference approach.
Project description:This SuperSeries is composed of the following subset Series: GSE28481: Transcriptome analysis of the zebrafish embryonic host response to Edwardsiella tarda infection using a static immersion systems [experiment A] GSE28485: Transcriptome analysis of the zebrafish embryonic host response to Edwardsiella tarda infection [experiment B] Refer to individual Series
Project description:We use the zebrafish embryo model to study the similarities and differences in the innate immune response against three different bacterial pathogens. Therefore, we injected E. tarda, Salmonella, or M. marinum into the caudal vein of 28 hours post fertilization (hpf) zebrafish embryos and analysed their gene expression profile at 8 hours or 4 days after infection by microarrays. The results show that infections with the gram-negative bacteria E. tarda and S. typhimurium, which are lethal within 1-2 days, induce a strong early immune response at 8 hours after infection. In contrast, infection with M. marinum leads to a chronic infection that only induces a strong response at 4 days post infection. This microarray study was designed to determine the gene expression profile during infection with Salmonella typhimurium, Mycobacterium marinum, and Edwardsiella tarda. RNA was isolated from single embryos and each treatment group consisted of three embryos: (1) Wildtypes injected with PBS 8 hours post infection (hpi), (2) S. typhimurium-infected wildtypes 8hpi, (3) wildtypes injected with PBS/2%PVP 8hpi , (4) M. marinum-infected wildtypes 8hpi, (5) wildtypes injected with PBS/2%PVP 4 days post infection (dpi), (6) M. marinum-infected wildtypes 4dpi, (7) wildtypes injected with PBS 8hpi (E. tarda control), (8) E. tarda-infected wildtypes 8hpi. Embryos were grown at 28.5–30°C in egg water and manually dechorionated at 24 hours post fertilization (hpf). Subsequently, embryos were infected at 28 hpf by micro-injecting 200 colony forming units (CFU) of S. typhimurium SL1027, E. tarda or Mycobacteria marinum M20 bacteria into the caudal vein, or were mock-injected with buffer as a control. After injections embryos were transferred into fresh egg water and incubated for 8 h or 4 days at 28°C. After the incubation period, single embryos were snap-frozen in liquid nitrogen and RNA was isolated for microarray analysis. All treatment groups were analyzed using a common reference approach.
Project description:MyD88 is an adaptor protein in Toll-like receptor and interleukin 1 receptor mediated signaling pathways that plays an essential role in activation of immune responses following pathogen recognition. We investigate that role in the zebrafish embryo model by using a zebrafish mutant line that contains a premature stop condon in the gene encoding MyD88, leading to a truncated protein that lacks domains important for its normal function. We infected these MyD88 mutants and wildtype individuals with Salmonella typhimurium and Edwardsiella tarda to compare the resulting immune response by transcriptome profiling on total RNA isolated from single embryos. The data derived from these microarray experiments confirms the vital role of MyD88 in pathogen recognition and provides many leads for further research. This microarray study was designed to determine the effect of a truncation of the MyD88 protein on the innate immune response of zebrafish embryos during infection with Salmonella typhimurium and Edwardsiella tarda. Embryos used in this study are derived from an incross between parents heterozygous for the mutation. Both homozygous mutants and their wildtype siblings were selected by genotyping after being injected with the bacteria or PBS as control. RNA was isolated from single embryos and each treatment group consisted of three embryos: (1) Homozygous mutants injected with PBS 8 hours post infection (hpi), (2) wildtype siblings injected with PBS 8hpi, (3) S. typhimurium-infected homozygous mutants 8hpi, (4) S. typhimurium-infected wildtype siblings 8hpi, (5) Homozygous mutants injected with PBS 8 hours post infection (hpi) (E.tarda control), (6) wildtype siblings injected with PBS 8hpi (E. tarda control), (7) E. tarda-infected homozygous mutants 8hpi, (8) E. tarda-infected wildtype siblings 8hpi. Embryos were grown at 28.5–30°C in egg water and manually dechorionated at 24 hours post fertilization (hpf). Subsequently, embryos were infected at 28 hpf by micro-injecting 200 colony forming units (CFU) of S. typhimurium SL1027 or E. Tarda FL-F60, or were mock-injected with buffer as a control. After injections embryos were transferred into fresh egg water and incubated for 8 h or 4 days at 28°C. After the incubation period, single embryos were snap-frozen in liquid nitrogen and RNA was isolated for microarray analysis. All treatment groups were analyzed using a common reference approach.
Project description:Transcriptional profiling of the zebrafish embryonic host response to infection by injection of 200 CFUs of Edwardsiella tarda (strain FL6-60) All infection experiments were performed using mixed egg clutches of Albino strain zebrafish. Embryos were staged at 28 hours post fertilization (hpf) by morphological criteria and approximately 200 cfu of mCherry expressing E. tarda bacteria were injected into the caudal vein close to the urogenital opening. As a control an equal volume of PBS was likewise injected. Single embryos of the infected and control group were collected 8 hours post infection (hpi).
Project description:Xanthomonas oryzae pv. oryzae strain PXO99A, so called Xoo, is disable to infect in rice cultivar carrying Xa21 gene. A set of experiments indicated that Ax21 is quorum sensing factor in PXO99 strain. To observe fine-tuned details how Ax21 controls expression of PXO99 in response to change of cell population density, transcriptional profiling analysis was perform by using published two channel oligo Xo microarray platform (Seo et al.,2008 BMC microbiology). Keywords: Comparative transcription profiling between low cell density and high cell density with same genetic background Three biological and dye-swap replicates, total six samples for each dataset. Two datasets contained; (i) PXO99 at 106 CFU/ml vs 108 CFU/ml, and (ii) PXO99∆ax21at 106 CFU/ml vs PXO99∆ax21 108 CFU/ml. 106 CFU/ml and 108 CFU/ml are cell density used in this study as representation of low and high cell density. All bacteria cultures were grown in PS (Peptone sucrose) broth media.
Project description:To establish the effect of DADS on the P. aeruginosa proteome, 50-mL LB medium samples were inoculated at 108 CFU/mL with exponential growth phase P. aeruginosa PAO1. DADS was then added at a concentration of 0 (control) or 0.64 mg/mL, in triplicate. The six experimental groups were incubated for 5 h in a water bath shaker at 37 ºC with a shaking rate of 180 rpm. Cells from the three control groups and three DADS treatment groups were then sampled and centrifuged. The cell precipitate from each experiment group was snap-frozen at -80 ºC.
Project description:Host response to systemic bacterial challenge (sepsis) with E. coli strains CFT073 and F11 at 6 and 12 hours post inoculum 48 hpf embryos were manually dechorianated, briefly anesthetized with 0.77 mM ethyl 3-aminobenzoate methanesulfonate salt (tricaine) (Sigma-Aldrich), and embedded in 0.8% low melt agarose (MO BIO Laboratories) without tricaine. After the agarose solidified, embryos were immersed in E3 media lacking methylene blue. Prior to injection, 1 ml of bacterial culture was pelleted, washed with 1 ml sterile PBS, and re-suspended in PBS to obtain ~1X109 CFU/ml. PBS. One nl of this bacterial suspension containing ~1000 CFU was microinjected into the bloodstream via the circulation valley using an Olympus SZ61 or SZX10 stereomicroscope together with a YOU-1 micromanipulator (Narishige), a Narishige IM-200 microinjector, and a JUN-AIR model 3-compressor. For each experiment, the average CFU per injection was determined by adding 10 1-nl drops to 1 ml of 0.7% NaCl, which was then serial diluted and plated on Luria-Bertani (LB) agar plates. Mock-infected controls were inoculated with 1 nl sterile PBS. Following injection, embryos were removed from agar and placed individually into wells of a 48-well plate (Nunc) containing E3 medium and incubated at 28.5°C. Experiments were performed in biological quadruplicate.
Project description:Goal was to identify yeast genes whose expression changed as a function of the shift from growth in bulk culture to growth in an air-liquid interfacial biofilm. Experiment Overall Design: Cells were grown 24 h at 30 C in YEPD to a density of about 500,000,000 cells/ml. At harvest, sugar was found to be depleted as measured by an enzymatic dip stick (Diastic, Bayer). Cells were pelleted and washed twice in sterile distilled water by centrifugation and then diluted 10-fold into 100 ml of Flor medium (YNB + 4% ethanol + leucine + histidine + uracil) in triplicate 250 ml beakers. Cultures were then grown statically in Flor medium at 27 C. Within a few hours following inoculation, the bulk liquid appeared to be clear, no biofilm was evident by visual observation, and a layer of settled cells was evident at the bottom of the beakers. After 48 h, a visible air-liquid interfacial biofilm covered the entire surface while the thickness of the layer of settled cells appeared unchanged. After 48 h, biofilm cells (floaters) were collected by aseptic aspiration. Once the biofilm cells were removed, cells at the bottom of the beaker (sinkers) were collected similarly. Cells from both populations were washed once in sterile distilled water by centrifugation prior to RNA isolation. Significant cell clumping was evident in both populations of cells by microscopic observation. While cell viability was estimated by plating on YEPD, the resultant cfu/ml values could not be directly correlated with cell counts in a hemacytometer because clumps of cells containing at least one viable cell presumably produced only a single colony. Further, counting individual cells accurately in the numerous clumps containing large numbers of cells was not possible. Nonetheless, when cells were counted (clumps were counted as single cells) and compared to cfu/ml for the same suspension, the cfu/ml values were about 2-fold higher than the corresponding values for cell counts using the hemacytometer for both biofilm and bottom layer cells.
Project description:Characterization of the zebrafish embryonic host response to systemic bacterial infection with Salmonella typhimurium wild type strain (SL1027) and its isogenic LPS O-antigen mutant Ra (SF1592) by means of a time-resolved global expression analysis. All infection experiments were performed using mixed egg clutches from three tanks of AB strain zebrafish. Embryos were staged at 27 hours post fertilization (hpf) by morphological criteria (Kimmel et al., 1995) and approximately 250 cfu of DsRed expressing S. typhimurium wt and Ra mutant bacteria were injected into the caudal vein close to the urogenital opening. As a control an equal volume of PBS was likewise injected. Injections were controlled using a Leica MZ Fluo 3 stereomicroscope with epifluorescence attachment together with a Femtojet microinjector (Eppendorf) and a micromanipulator with pulled microcapillary pipettes. Pools of 20-40 embryos were collected at 2, 5, 8 and 24 hours post infection (hpi). For the microarray analysis, the whole infection procedure was preformed in triplicate on separate days. The triplicates are marked A,B and C. The order of injecting wt bacteria, Ra bacteria and PBS control was randomized in the different experiments. The general reference sample is a mixture of all RNA samples from this infection study and it is named Common Reference (CR).
Project description:Gene expression profiles of human cell (THP-1) lines exposed to a novel Daboiatoxin (DbTx) isolated from Daboia russelli russelli, and specific cytokines and inflammatory pathways involved in acute infection caused by Burkholderia pseudomallei. Experiment Overall Design: 1. Group I:- Human monocytic macrophage (THP-1) cell lines grown in the culture medium without any bacterial infection served as untreated control group (Three Biological Replicates). Experiment Overall Design: 2. Group II:- THP-1 cells were infected with Burkholderia pseudomallei (A600 nm = OD 0.6, ~5 x 107 cfu/ml) for 24h served as a disease control group (Three Biological Replicates). Experiment Overall Design: 3. Group III:- THP-1 cells were infected with B. pseudomallei and treated with Daboiatoxin (0.5 mM) isolated from Daboia russelli russelli venom served as a treatment group (Three Biological Replicates). Experiment Overall Design: 4. Group IV:- THP-1 cells were infected with B. pseudomallei (A600 nm = OD 0.6, ~5 x 107 cfu/ml) treated with standard antimicrobial drug ceftazidime (10mg/ml) served as a drug control (Three Biological Replicates). Experiment Overall Design: 5. Group V:- THP-1 cells were exposed to Daboiatoxin (0.5 mM) without bacterial infection (Three Biological Replicates).