Insight into Vibrio parahaemolyticus CHN25 response to artificial gastric fluid stress by transcriptomic analysis
ABSTRACT: Vibrio parahaemolyticus is the causative agent of food-borne gastroenteritis disease. Once consumed, human acid gastric fluid is perhaps one of the most important environmental stresses imposed on the bacterium. Herein, for the first time, we investigated Vibrio parahaemolyticus CHN25 response to artificial gastric fluid (AGF) stress by transcriptomic analysis. The bacterium at logarithmic growth phase (LGP) displayed lower survival rates than that at stationary growth phase (SGP) under a sub-lethal acid condition (pH 4.9). Transcriptome data revealed that 11.6% of the expressed genes in Vibrio parahaemolyticus CHN25 was up-regulated in LGP cells after exposed to AGF (pH 4.9) for 30 min, including those involved in sugar transport, nitrogen metabolism, energy production and protein biosynthesis, whereas 14.0% of the genes was down-regulated, such as ABC transporter and flagellar biosynthesis genes. In contrast, the AGF stress only elicited 3.4% of the genes from SGP cells, the majority of which were attenuated in expression. Moreover, the number of expressed regulator genes was also substantially reduced in SGP cells. Comparison of transcriptome profiles further revealed forty-one growth-phase independent genes in the AGF stress, however, half of which displayed distinct expression features between the two growth phases. Vibrio parahaemolyticus seemed to have evolved a number of molecular strategies for coping with the acid stress. The data here will facilitate future studies for environmental stresses and pathogenicity of the leading food-borne pathogen worldwide. When V.parahemolyticus CHN25 grown to log phase and stationary phase at 37°C in TSB-3% NaCl, different cultures were subsequently exposed to artificial gastric fluid at 37°C for 30 min. Two independent experiments were performed at each phase for microarray expreriments.
Project description:Naturally bacteria are commonly forced to remain in stationary phase. There is no increase in cell mass, however, cell division keep on. Vibrio (V.) parahaemolyticus is an aquatic bacterium capable of causing foodborne gastroenteritis outbreaks all over the world. So far, little is known about whole genomic expression of V. parahaemolyticus in the early stationary phase compared with the phase of exponential growth. Since under starvation cell sizes decrease and endogenous metabolism reduces, genes are considered to be highly repressed in the stationary phase. However, our data shows in total 172 induced genes, while 61 genes were repressed in the early stationary phase compared with exponential phase. In fatty acid and phospholipid metabolism functional category only induced genes were found, whereas in three other metabolic functional groups appeared no significant up-regulated genes (adjusted P-value<0.05). Genes in two metabolic functional categories remained stable in the early stationary phase. DAVID analyses were carried out exploring the gene regulation. In total, ten functional categories showed a total up-regulation in early stationary phase, while only three metabolic functional categories showed a down-regulation and four categories showed stably in early stationary phase. Early stationary phase gene expression was detected in total bacterial RNA of V. parahaemolyticus. Two phases (exponential phase and early stationary phase) were used in 8 biological replicates. Gene expression in exponential phase was used for normalization.
Project description:To investigate low-temperature tolerance of the bacterium, three in-frame gene deletion mutants of VpacspA and VpacspD were constructed using homologous recombination method. When compared to the wild type strain, the growth of ΔVpacspA mutant was strongly repressed at 10 0C, whereas the deletion of VpacspD gene greatly activated the bacterium growth at the low temperature. Transcriptome data revealed that 12.4% of the expressed genes in V. parahaemolyticus CHN25 was significantly changed in ΔVpacspA mutant grown at 10 0C, including those involved in amino acid degradation, ATP-binding cassette (ABC) transporters, secretion systems, sulfur and glycerophospholipid metabolisms, whereas the low temperature elicited 10.0% of the genes from ΔVpacspD mutant, such as phosphotransferase system, nitrogen and amino acid metabolisms. Moreover, the major changed metabolic pathways in dual-gene deletion mutant (ΔVpacspAD) differed radically from those in single-gene mutants. Comparison of transcriptome profiles further revealed a number of differentially expressed genes shared among the three mutants, as well as regulators specifically, coordinately and or antagonistically regulating in the adaptation of V. parahaemolyticus CHN25 to the low-temperature growth. mRNA profiles of mid-log phase WT(G), ΔVpacspA, ΔVpacspD and ΔVpacspAD at 10 °C were generated by deep sequencing using Illumina HiSeq 2500
Project description:Vibrio parahaemolyticus an emerging pathogen that is a causative agent of foodborne gastroenteritis when raw or undercooked seafood is consumed. Previous microarray data using a Vibrio parahaemolyticus RIMD2210633 chip has shown the master quorum-sensing regulator OpaR controls virulence, type III and type VI secretion systems, and flagellar and capsule production genes. In a parallel study, RNA-Seq was used to comparatively study the transcriptome changes of wild type Vibrio parahaemolyticus BB22 and a ΔopaR strain directly. Differences in mRNA expression were analyzed using next generation Illumina sequencing and bioinformatics techniques to align and count reads. A comparison with the previous microarray data showed good correlation between the shared genes. The RNA-Seq offered an insight into control of genes specific to the Vibrio parahaemolyticus BB22 strain as well as a new look at the sRNAs that are expressed. Eleven transcriptional regulators with greater than 4 fold regulation in the previous microarray study and 2 fold regulation in the RNA-Seq analysis, were chosen to validate the data using qRT-PCR and further characterized with electrophoretic mobility shift assays (EMSAs) to determine if they are direct targets of OpaR. The transcription factors chosen play key roles in virulence, surface motility, cell to cell interactions, and cell surface characteristics. One small RNA was identified in the RNA-Seq data to be quorum-sensing controlled and unidentified by other programs. The RNA-Seq data has aided in understanding and elucidating the hierarchy of quorum-sensing control of OpaR in Vibrio parahaemolyticus. The wild type Vibrio parahaemolyticus BB22 strain LM5312 and an opaR deletion strain LM5674 were analyzed for mRNA expression using RNA-Seq.
Project description:Serum is a valuable body fluid to diagnose cancer as it can be accessed with minimal invasive techniques. Studying the cancer serum proteome provides valuable insights into the pathophysiology of tumor progression. Gastric adenocarcinoma is an aggressive cancer resulting in poor prognosis, mainly due to the lack of specific early diagnostic biomarkers. To this end, we used an iTRAQ-based quantitative proteomic approach to identify differentially expressed proteins in the sera of patients diagnosed with gastric cancer. Our study resulted in the identification of 643 proteins in the serum, of which 48 proteins were found to be overexpressed and 11 proteins underexpressed in gastric cancer when compared with healthy controls. We used multiple reaction monitoring assays to validate the overexpression of potential biomarkers. This catalog of serum-based biomarkers will aid in diagnosis and prognosis of gastric cancer.
Project description:Vibrio parahaemolyticus is the leading bacterial cause for seafood-related gastroenteritis worldwide. As an intestinal pathogen, V. parahaemolyticus competes with other commensal bacteria for the same pool of nutrients. The major source of nutrition for intestinal bacteria is intestinal mucus. We wanted to determine the expression profile of wild-type V. parahaemolyticus in mouse intestinal mucus and then perform a differential expression analysis in a ∆luxO deletion mutant, in which the high cell density quorum sensing regulator OpaR is constitutively expressed and low cell density regulator AphA is repressed. Overall design: RNA-Seq analysis was performed on RNA isolated from two biological replicates of wild-type Vibrio parahaemolyticus strain RIMD2210633 and three biological replicates the ∆luxO mutant strain, grown in M9 minimal media supplemented with mouse intestinal mucus.
Project description:Vibrio (V.) parahaemolyticus is the leading cause of seafood borne gastro-intestinal infections in humans worldwide. It is widely found in marine environments and is isolated frequently from seawater, estuarine waters, sediments and raw or insufficiently cooked seafood. Within the food chain, V. parahaemolyticus encounters different temperature conditions that might alter the metabolism and pathogenicity of the bacterium. In this study, we performed gene expression profiling of V. parahaemolyticus RIMD 2210633 after exposure to 4°C, 15°C, 20°C, 37°C and 42°C to describe the cold and heat shock response. Analysis of transcriptomics data resulted in differential expression of 19 genes at 20°C, 193 genes at 4°C, 625 genes at 42°C and 639 genes at 15°C. Thus the highest portion of significantly expressed genes was observed at 15°C and 42°C with 13.3% and 13%, respectively. Genes of many functional categories were highly regulated even at lower temperatures. Our results showed that virulence associated genes (tdh1, tdh2, toxR, toxS, vopC, T6SS1, T6SS2) remained largely unaffected by heat or cold stresses. Along with folding and temperature shock depending systems, an overall temperature depended regulation of expression could be shown. Particularly the energy metabolism was affected most by changed temperatures. Whole-genome gene expression studies of food related pathogens such as V. parahaemolyticus reveal how these pathogens react to stress impacts for prediction of its behaviour under conditions like storage and transport. Temperature induced gene expression was detected in total bacterial RNA of V. parahaemolyticus. Five different temperatures (4°C, 15°C, 20°C, 37°C, 42°C) were used in at least 3 biological replicates (4 replicates for 37°C). Gene expression at 37°C was used for normalization.
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
Project description:Vibrio parahaemolyticus is a Gram-negative marine bacterium. A limited population of the organisms causes acute gastroenteritis in humans. Vibrio parahaemolyticus wild type strain RIMD 2210633 compared with the mutants of VtrA and VtrB have a winged helix-turn-helix DNA binding motif that genes encoded on pathogenicity island loci, at OD600=1.0 in Luria-Bertani containing medium 0.5 % NaCl at 37˚C. Our goal is to determine the VtrA or VtrB regulon. Overall design: Cohybridized wild type versus vtrA or vtrB mutants on a single array. Biological replicates: three of wild type, three of vtrA mutants and three of vtrB mutants were independently grown and harvested.
Project description:Vibrio parahaemolyticus is a Gram-negative marine bacterium. A limited population of the organisms causes acute gastroenteritis in humans. Vibrio parahaemolyticus wild type strain RIMD 2210633 compared with the mutants of VtrA and VtrB have a winged helix-turn-helix DNA binding motif that genes encoded on pathogenicity island loci, at OD600=1.0 in Luria-Bertani containing medium 0.5 % NaCl at 37˚C. Our goal is to determine the VtrA or VtrB regulon. Cohybridized wild type versus vtrA or vtrB mutants on a single array. Biological replicates: three of wild type, three of vtrA mutants and three of vtrB mutants were independently grown and harvested.
Project description:To determine Sigma 54 (SigL) reglons in Bacillus thuringiensis HD73 strain, A sigLmutant, HD(ΔsigL::kan), was constructed with insertion of kanamycin resistance gene cassete. We have employed whole genome microarray expression profiling as a discovery platform to identify the difference of gene expression between mutant and wild-type strains. 2 ml samples were separately harvested from B. thuringiensis HD73 and HD(ΔsigL::kan) strains grown in Schaeffer’s sporulation medium (SSM) at stages T7 of stationary phase (7 hours after the end of the exponential phase). Three independent repeats were performed for each stain.