Unknown,Transcriptomics,Genomics,Proteomics

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DNA microarray-mediated transcriptional profiling of avian pathogenic Escherichia coli O2 strain E058 during its infection of chicken

ABSTRACT: APEC most often infect chickens, turkeys, ducks, and other avian species, and therefore pose a significant economic burden on the poultry industry worldwide. Few studies have analyzed the genome-wide transcriptional profile of APEC during infection in vivo. In this study, we examined the genome-wide transcriptional response of APEC O2 strain E058 in an in vivo chicken infection model to better understand the factors necessary for APEC colonization, growth, and survival in vivo. An Affymetrix multigenome DNA microarray, which contains most of the genomic open reading frames of E. coli K-12 strain MG1655, uropathogenic E. coli strain CFT073, and E. coli O157:H7 strain EDL 933, was used to profile the gene expression in APEC E058.The genes highly expressed during infection were involved in metabolism, iron acquisition or transport, virulence, response to stress, and biological regulation. Many genes encoding putative or hypothetical proteins were also strongly upregulated, implying that some undiscovered mechanism may underlie APEC pathogenesis. We identified the in vivo transcriptional response of APEC E058 bacteria collected directly from the blood of infected chickens. Significant differences in expression levels were detected between the in vivo expression profile and the in vitro expression profile in LB medium.

ORGANISM(S): Escherichia coli APEC O2

SUBMITTER: Qingqing Gao

PROVIDER: E-GEOD-81974 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Avian pathogenic Escherichia coli (APEC) is considered one of the most common infectious bacterial diseases resulting in significant economic losses in poultry industry worldwide. In order to investigate the association between host immune resistance and miRNA expression in the pathogenic process induced by APEC, miRNA expression profiles in broilers spleen were performed by Solexa deep sequencing from three different treatment groups including non-challenged (NC), challenged-mild pathology (MD), and challenged-severe pathology (SV).In total, 3 462 706, 3 586 689, and 3 591 027 clean reads were obtained for NC, MD, and SV, respectively. After comparing the miRNA expression patterns, 27 differentially expressed miRNAs were identified among the three response groups, which included 13 miRNAs between NC and MD, 17 between NC and SV, and 14 between MD and SV. For these miRNAs, different expression in MD and SV suggested they may have resistance activity in APEC infection. Through integrated analysis of miRNA and mRNA expression patterns, 43 negative pairs between miRNA and mRNA (r < -0.80) were obtained. 4 miRNAs were validated to be significant negatively correlated to targets by quantitative real time PCR: gga-miR-21 (CLEC3B and GGTLA1), gga-miR-429 (TMEFF2, CDC20, SHISA2 and NOX4), gga-miR-146b (LAT2 and WNK1), and gga-miR-215 (C7 and ASL2). Additionally, the expression of gga-miR-21 and gga-miR-146b was significantly up-regulated by LPS induced in HD11 macrophage cell. In contrast, gga-miR-429 has no significant change. In summary, we present the first report that characterized the miRNA profiles of chicken spleen in response to APEC infection, and identified several candidate miRNAs which might accelerate host immune response through down-regulating their specific target genes. Through the intra-air sac route into the left thoracic air sac, 240 non-vaccinated males at 4 weeks of age were challenged with 0.1 ml APEC O1 (10^8 colony forming units) and another 120 non-vaccinated males were non-challenged but treated with 0.1 ml PBS. Detailed information on the APEC O1 strain and challenge process was described by previously described study. Necropsy was performed at 1 day post challenge, and a summarized lesion ranging from 0 to 7 was determined for each APEC-challenged bird. Birds with lesions scoring 0-2 were regarded as mild infection, and those scoring 4-7 were designated as severe infection. The mild and severe pathology meant that birds were resistant and susceptible to APEC infection, respectively. Then, spleens from three groups, consisting of non-challenged, challenged-mild pathology and challenged-severe pathology were subjected to Solexa deep sequencing to investigate the dynamics of chicken miRNA expression.

Project description:Many reports show an association between the Pst system, the Pho regulon related genes and bacterial virulence. Our previous results showed that a functional Pst system is required for full virulence, resistance to serum, polymyxin B and acid shock However, the interplay between the Pst system and virulence has an unknown molecular basis. To understand global APEC virulent strain responses to Pho regulon activation, we conducted transcriptome profiling experiments comparing the isogenic Pst mutant with its wild type strain chi7122 using the Affymetrix GeneChipÂ® E. coli Genome 2.0 Array. To understand the global responses to Pho regulon activation of APEC, we use the Affymetrix technology. We conducted transcriptome profiling experiments comparing the APEC chi7122 strain and its isogenic Pst mutant grown in rich phosphate medium using the Affymetrix GeneChipÂ® E. coli Genome 2.0 Array. The Affymetrix GeneChipÂ® E. coli Genome 2.0 Array contains the genome of the E. coli MG1655 and three pathogenic E. coli strain (EDL933, Sakai and CFT073) representing 20,366 genes. While comparing genes expression between Pst mutant and the wild type chi7122 strain, 471 genes are either up- (254) or down-regulated (217) of at least 1.5-fold, with a p-value inferior or equal to 0.05 and a false discovery rate of 2.71%. Experiment Overall Design: RNA extraction was performed onto the chi7122 and K3 strains on four biological replicates. An overnight culture grown at 37Â°C was diluted 100-fold into 5 ml of LB broth and was allowed to grow to mid-log phase (OD600 0,6). Cultures were centrifuged and RNAs were isolated by using the RiboPureâ¢-Bacteria Kit (Ambion, Austin, TX), according to the manufacturerâs recommendations, with the exception that the DNAse 1 treatment was performed twice. RNA extractions were performed on four different days (the chi7122 and K3 RNA were extracted at the same time) and a total of eight hybridizations were performed. Hybridization was carried out at McGill University & Genome Quebec Innovation Centre according to the Affymetrix recommendation (Affymetrix Expression Manual Section 3 701029 rev. 4). GeneChip scan was carried out at McGill University & Genome Quebec Innovation Centre according to the Affymetrix recommendation (Affymetrix Expression Manual Section 3 701029 rev. 4), and data were processed using the FlexArray software (Michal Blazejczyk, Mathieu Miron, Robert Nadon (2007). FlexArray: A statistical data analysis software for gene expression microarrays. Genome Quebec, Montreal, Canada). Raw data were normalized using the RMA algorithm and log2 were generated. The expression value was generated by subtracting the mean value of each replicate of the mutant strain by wild-type strain.

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DNA microarray-mediated transcriptional profiling of avian pathogenic Escherichia coli O2 strain E058 during its infection of chicken

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