Burkholderia pseudomallei transcriptional adaptation in macrophage
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ABSTRACT:
ORGANISM(S): Burkholderia pseudomallei
SUBMITTER: Sheila Nathan
PROVIDER: E-GEOD-27558 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:This study reports the expression profile of intracellular B. pseudomallei following infection of human macrophage-like U937 cells. The transcriptome of intracellular B. pseudomallei harvested from macrophage cells over an infection period of 6 hr (1, 2, 4 and 6 hr post-infection) were compared to in vitro grown bacteria to identify genes whose expression is altered in response to intracellular growth.
2011-12-31 | GSE27558 | GEO
Project description:The potential for epigenetic changes in host cells following microbial infection has been widely suggested, but few examples have been reported. We assessed genome-wide patterns of DNA methylation in human macrophage-like U937 cells following infection with Burkholderia pseudomallei, an intracellular bacterial pathogen and the causative agent of human melioidosis. Our analyses revealed significant changes in host cell DNA methylation, at multiple CpG sites in the host cell genome, following infection. Infection induced differentially methylated probes (iDMPs) showing the greatest changes in DNA methylation were found to be in the vicinity of genes involved in inflammatory responses, intracellular signalling, apoptosis and pathogen-induced signalling. A comparison of our data with reported methylome changes in cells infected with M. tuberculosis revealed commonality of differentially methylated genes, including genes involved in T cell responses (BCL11B, FOXO1, KIF13B, PAWR, SOX4, SYK), actin cytoskeleton organisation (ACTR3, CDC42BPA, DTNBP1, FERMT2, PRKCZ, RAC1), and cytokine production (FOXP1, IRF8, MR1). Overall our findings show that pathogenic-specific and pathogen-common changes in the methylome occur following infection. The human leukemic monocyte lymphoma cell line (U937, ATCC CRL-1593.2) was maintained in RPMI 1640 supplemented with 10% foetal bovine serum (FBS) at 37°C. U937 cells were differentiated to macrophage-like cells following exposure to 20 ng/ml (final concentration) of phorbol 12-myristate 13-acetate (PMA) for 48 hours at 37°C and differentiation evidenced by increased adherence to tissue culture flasks. Overnight cultures of B. pseudomallei K96243 were diluted in L-15 medium and added to differentiated U937 cells at a multiplicity of infection (MOI) of 10. Uninfected controls were overlaid with L15 medium only. The cells were then incubated at 37°C to allow infection. The cells were washed 3 times with PBS and incubated with fresh L15 medium containing 1mg/ml kanamycin to kill extracellular bacteria. At appropriate time points the cells were washed 3 times in warm PBS and lysed with 0.1% (vol/vol) Triton X-100. DNA was isolated using an AllPrep kit (Qiagen) and stored at -80ºC until required. DNA yield was measured using a Nanodrop instrument with measurements between 22.8 â 50.6 ng/ul. Two experiments were carried out using the above procedure. In Experiment 1, DNA was collected from uninfected and infected cells at 2 hours (T2), and 4 hours (T4) post infection (2 biological replicates and 2 technical replicates from each group). In Experiment 2, DNA was collected from uninfected and infected cells at 1 hour (T1), 2 hours (T2), 3 hours (T3) and 4 hours (T4) post infection (1 sample from each group). The DNA methylation profile was determined using the Infinium HumanMethylation450 BeadChip (450K) (Illumina Inc.) following the manufacturerâs instructions. The data was extracted and the initial analysis was performed using GenomeStudio (2010.3) methylation module (1.8.5). Quality control checks and quantile normalisation were implemented using WateRmelon. Samples with more than 1% of sites with a detection p-value greater than 0.05 were removed as were probes with 1% of samples with a detection p-value greater than 0.05. Probes were removed if they had a bead count less than 3 in 1% of samples. Cross-hybridizing probes were removed, leaving 425496 probes for analysis. Here we report DNA methylation profile of 18 samples (10 infected and 8 control).
2016-06-16 | E-GEOD-83379 | biostudies-arrayexpress
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).
2009-04-04 | E-GEOD-7577 | biostudies-arrayexpress
Project description:New strategies are required to reduce the worldwide burden of tuberculosis. Intracellular survival and replication of Mycobacterium tuberculosis after macrophage phagocytosis is a fundamental step in the complex host-pathogen interactions that lead to granuloma formation and disease. Greater understanding of how the bacterium survives and thrives in these environments will inform novel drug and vaccine discovery programmes. Here, we use in-depth RNA sequencing of Mycobacterium bovis BCG from human THP-1 macrophages to describe the mycobacterial adaptations to the intracellular environment. We identify 329 significantly differentially regulated genes, highlighting cholesterol catabolism, methyl-citrate cycle and iron homeostasis as important for mycobacteria inside macrophages. Focused analysis of PE/PPE and cytochrome P450 gene families highlight additional pathways that are upregulated (35 and five respectively) 24h after infection. Comparison of the intracellular transcriptome to gene essentiality and immunogenicity studies identified 15 potential targets that are both required for intracellular survival and induced on infection, and eight genes upregulated that have been demonstrated to be immunogenic in TB patients. Further insight into these new and established targets will support drug and vaccine development efforts.
2022-01-14 | E-MTAB-11107 | biostudies-arrayexpress
Project description:We present a detailed single cell time course of the macrophage response to Salmonella infection. By combining phenotypic fluorescent labels with single cell expression analysis we are able to identify gene modules associated with bacterial exposure and bacterial infection. We also identify other genetic clusters that are expressed heterogenously, ananlyzing both their regulation and their impact on infection Analysis of 192 single cells across 4 time points after Salmonella exposure (MOI 1:1) with one of three different fluorescent labels indicating whether a given cell contained no intracellular bacteria (non-fluorescent), contained dead intracellular bacteria (only pHrodo positive), or contained live intracellular bacteria (pHrodo and GFP positive)
2015-09-07 | E-GEOD-65528 | biostudies-arrayexpress
Project description:Microarrays were used to determine the transcriptional profile of Y. pestis that is growing inside macrophages. J774A.1 macrophage-like cells were infected with Y. pestis KIM5 and incubated in the presence of gentamicin in tissue culture media. RNA was isolated from intracellular bacteria at various time points post infection. Control bacteria were grown for 4 hours in tissue culture medium under the same conditions without macrophages or gentamicin. The transcriptional profiles of intracellular Y. pestis at different time points were compared to those of control Y. pestis using the 70-mer oligonucleotide microarrays obtained from Pathogen Functional Genomics Resource Center/J. Craig Venter Institute (Y. pestis microarray version 2). RNA from samples of Yersinia pestis KIM5 at 3 time points in were compared to 12 Yersinia pestis in DMEM for 4 hours.
2010-07-07 | E-GEOD-22168 | biostudies-arrayexpress
Project description:The potential for epigenetic changes in host cells following microbial infection has been widely suggested, but few examples have been reported. We assessed genome-wide patterns of DNA methylation in human macrophage-like U937 cells following infection with Burkholderia pseudomallei, an intracellular bacterial pathogen and the causative agent of human melioidosis. Our analyses revealed significant changes in host cell DNA methylation, at multiple CpG sites in the host cell genome, following infection. Infection induced differentially methylated probes (iDMPs) showing the greatest changes in DNA methylation were found to be in the vicinity of genes involved in inflammatory responses, intracellular signalling, apoptosis and pathogen-induced signalling. A comparison of our data with reported methylome changes in cells infected with M. tuberculosis revealed commonality of differentially methylated genes, including genes involved in T cell responses (BCL11B, FOXO1, KIF13B, PAWR, SOX4, SYK), actin cytoskeleton organisation (ACTR3, CDC42BPA, DTNBP1, FERMT2, PRKCZ, RAC1), and cytokine production (FOXP1, IRF8, MR1). Overall our findings show that pathogenic-specific and pathogen-common changes in the methylome occur following infection.
2016-06-16 | GSE83379 | GEO
Project description:Facultative intracellular Brucella infect and survive inside macrophages, and the outcome of macrophage-Brucella interaction is a basis for establishment of a chronic Brucella infection. The majority of Brucella are killed at the early infection stage. A subpopulation of virulent Brucella strains is instead trafficked to an intracellular replicative phagosome, and are resistant to further attack and begin to multiply dramatically. Virulent Brucella also inhibit macrophage apoptosis that in turn favors pathogen survival and replication. We used the Affymetrix mouse GeneChip 430 2.0 array to analyze mouse macrophage gene expression profiles during the time course of virulent B. melitensis strain 16M infection. Experiment Overall Design: Murine J774.A1 macrophage cells were infected with B. melitensis strain 16M at a MOI of 200:1. Brucella cultures derived from different Brucella colonies were used to infect different groups of macrophage cells to reflect independent infections. Following 4 h incubation, the cells were washed with PBS and treated with 50 ug/ml gentamicin to kill extracellular Brucella. At 0 h (no Brucella infection), 4 h, 24 h, and 48 h post-infection, cells were individually collected, and total RNA was isolated. The Affymetrix mouse GeneChip 430 2.0 array was used for microarray hybridization. Microarray intensity data were obtained by using Affymetrix GCOS software and further analyzed by GeneSpring and other software programs.
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Project description:Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a disease endemic to South-East Asia and Northern Australia. Clinical presentation is highly variable, ranging from asymptomatic to fatal septicaemia, and thus the outcome of infection can depend on the host immune responses. The aim of this study was to characterise the macrophage immune response to B. pseudomallei in the presence of novel inhibitors targeting the virulence factor, Macrophage Infectivity Potentiator (Mip) protein. To do this. murine macrophage J774A.1 cells were infected with B. pseudomallei K96243 in the presence and absence of two small-molecule inhibitors designed to target the Mip protein. Global transcriptional profiling of macrophages infected with B. pseudomallei was analysed by RNA-Seq four hours post-infection. In the presence of Mip inhibitors, we found a significant reduction in the expression of pro-inflammatory cytokines highlighting the potential to utilize Mip inhibitors to dampen potentially harmful pro-inflammatory responses resulting from B. pseudomallei infection in macrophages. We then performed gene expression profiling analysis using data obtained from RNA-seq of J774A.1 macrophages infected with Burkholderia pseudomallei in the presence of two Mip inhibitors or vehicle control 4 hours post-infection
2024-03-06 | GSE244127 | GEO