Transcriptome Analysis of T3SS1 in Vibrio parahaemolyticus
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ABSTRACT:
ORGANISM(S): Vibrio parahaemolyticus
PROVIDER: GSE51423 | GEO | 2014/01/21
SECONDARY ACCESSION(S): PRJNA223069
REPOSITORIES: GEO
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Project description:Comparative transcriptional mRNA profiles were generated of bacterial pathogen Vibrio parahaemolyticus under conditions that induce activity of virulence factor type III secretion system 1 (T3SS1). Induction conditions included growth of the bacteria in Dulbecco's Modified Eagle Medium (DMEM), overexpression of transcriptional activator exsA and contact with HeLa cells in Hank's Balanced Salt Solution (HBSS), while non-inducing conditions included growth in Luria-Bertani medium supplemented with 2.5% (w/v) NaCl (LB-S) and overexpression of transcriptional anti-activator exsD. Transcriptome profiles of induction conditions were cross-compared against background non-inducing conditions and time points during HeLa cell infection (2 hr, 3 hr, 4 hr, 6 hr, 8 hr) were compared against pre-infection (0 hr) to identify genes important in T3SS1 activity and pathogenesis. Duplicate mRNA transcriptome profiles of V. parahaemolyticus grown in T3SS1 inducing (DMEM, exsA expression) and non-inducing (LB-S, exsD expression) conditions as well as during HeLa cell infection (0 hr, 2 hr, 3 hr, 4 hr, 6 hr, 8 hr) were generated by deep sequencing using an Illumina MiSeq
2014-01-21 | E-GEOD-51423 | biostudies-arrayexpress
Project description:Here we probe the global response to calcium in the marine bacterium and pathogen Vibrio parahaemolyticus by using transcriptome, reporter and phenotypic analyses. Swarming gene expression and motility were enhanced by calcium. Calcium also stimulated expression and function of one of the organismM-bM-^@M-^Ys two type three secretion systems (T3SS1 but not the T3SS2). Although low calcium is an inducing signal for the T3SS of many organisms, calcium stimulation of T3SS has not been reported before. EGTA was also a stimulus for T3SS1 expression and activity; however this appeared to be the consequence of iron rather than calcium chelation. LafK, a key regulator of swarming genes, was found necessary for calcium induction of T3SS1 gene transcription and cytotoxicity. Regulation of swarming and T3SS1 were additionally linked by a negative feedback loop propagated by ExsA. Overexpression of exsA was used to probe the extent of the T3SS1 regulon and verify its coincident induction by calcium and EGTA. The calcium transcriptome analysis revealed a calcium-repressed LysR-type transcriptional regulator; CalR was shown to repress swarming and T3SS1 gene expression. Thus in V. parahaemolyticus, calcium influences gene expression and behavior and seems a signal pertinent for surface colonization and virulence. The gene expression profiles of Vibrio parahaemolyticus cells grown on rich medium or medium supplemented with CaCl2 or EGTA were compared using Affymetrix custom microarrays.
2010-11-05 | E-GEOD-22190 | biostudies-arrayexpress
Project description:Here we probe the global response to calcium in the marine bacterium and pathogen Vibrio parahaemolyticus by using transcriptome, reporter and phenotypic analyses. Swarming gene expression and motility were enhanced by calcium. Calcium also stimulated expression and function of one of the organism’s two type three secretion systems (T3SS1 but not the T3SS2). Although low calcium is an inducing signal for the T3SS of many organisms, calcium stimulation of T3SS has not been reported before. EGTA was also a stimulus for T3SS1 expression and activity; however this appeared to be the consequence of iron rather than calcium chelation. LafK, a key regulator of swarming genes, was found necessary for calcium induction of T3SS1 gene transcription and cytotoxicity. Regulation of swarming and T3SS1 were additionally linked by a negative feedback loop propagated by ExsA. Overexpression of exsA was used to probe the extent of the T3SS1 regulon and verify its coincident induction by calcium and EGTA. The calcium transcriptome analysis revealed a calcium-repressed LysR-type transcriptional regulator; CalR was shown to repress swarming and T3SS1 gene expression. Thus in V. parahaemolyticus, calcium influences gene expression and behavior and seems a signal pertinent for surface colonization and virulence.
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Project description:Analysis of platelet-derived growth factor (PDGF)-stimulated fibroblasts. Results provide insight into novel pro-tumorigenic factors induced by PDGF-activation of fibroblasts. 1.2 x 10e6 BJ-hTERT fibroblasts were cultured in 10cm dish with Dulbecco's Modified Eagle Medium (DMEM; Gibco Life Technologies, Gergy-Pontoise, France) containing 1% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, penicillin (100 units/ml) and streptomycin (100 ng/ml) at 37ºC in a 5% CO2 humidified atmosphere during 24h. Cultures were stimulated with or without 20ng/mL PDGF-BB (Peprotech, New Jersey, USA) for another 24h prior to harvest. Unstimulated fibroblasts were used as control samples. Cultures of both unstimulated and stimulated fibroblasts were done in triplicate.
2012-09-10 | E-GEOD-40720 | biostudies-arrayexpress
Project description:SGH10 bacteria were grown under DMEM (Dulbecco's Modified Eagle Medium) and LB(Lysogeny broth). Bacteria were harvested by centrifugation, the pellet was resuspended in TE buffer, mixed with lysosyme (SigmaAldrich) in a final concentration of 0.9 mg/ml, and immediately frozen in liquid nitrogen. The frozen pellet was subjected to two cycles of thaw and freeze at 37°C and in liquid nitrogen, respectively, and RNA was extracted using Tri-Reagent (Sigma-Aldrich). RNA-seq libraries were constructed by the RNAtag-seq method. RNA-seq libraries were sequenced by single end sequencing using Nextseq 500 Sequencer (Illumina). sequence fragments were mapped to SGH10 which contains a chromosome NZ_CP025080.1 and a plasmid NZ_CP025081.1. For each growth condition, three biological repeats were done.
2023-12-01 | E-MTAB-13341 | biostudies-arrayexpress
Project description:Abstract: The type three secretion system 1 (T3SS1) of the marine bacterium, Vibrio parahaemolyticus, orchestrates a host cell death comprised of distinct stages defined by the action of specific effectors. To understand how the host responds to the T3SS1, we analyzed gene expression changes over time in infected primary fibroblasts by RNA-seq. The host cell’s transcriptional response was rapid, robust and temporally distinct. Despite the lethal action of the T3SS1 effectors, network analysis indicated that the integrated host transcriptional response to T3SS1 effectors activated cell survival and repressed cell death networks, suggesting that T3SS1 masks its cytotoxicity to the host cell. Pathway analysis revealed that signaling through several pathways, including MAPK pathways, was specifically altered by T3SS1. Overall, this temporal analysis of T3SS1-mediated subversion of host cell signaling revealed cytoprotective pathways that are likely hijacked by many bacterial pathogens to ensure survival. Methods: We employed genome-wide transcriptional profiling methods (RNA-seq) to measure changes in gene expression over time in response to infection with POR4:T3SS1- or POR3:T3SS1+. Time points were collected 45, 60, 75 and 90 minutes post-infection. In total, libraries from 27 samples (triplicates of uninfected, POR3/4 at t=45, 60, 75, and 90 minutes) were sequenced and mapped to the human genome. EdgeR was used for differential expression analysis using statistical cutoffs of of false discovery rate (FDR) ≤0.01, log2counts-per-million (log2CPM) ≥0 and fold change (FC) cutoffs of -1.5≥ FC ≥1.5
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