DNA microarray-mediated transcriptional profiling of avian pathogenic Escherichia coli O2 strain E058 during its infection of chicken
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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.
Project description: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.
Project description:Avian Pathogenic Escherichia coli (APEC) are a group of extra-intestinal E. coli that infect poultry, and are able to cause a variety of diseases, systemic or localized, collectively designated as colibacillosis. Colibacillosis is the most common bacterial illness in poultry production, resulting in significant economic losses world-wide. Despite of its importance, pathogenicity mechanisms of APEC strains remain not completelly elucidated and available vaccines are not fully effectives. In order to better understand which genes could be related to pathogenicity in different APEC isolated, a microarray analyses of two APEC strains representing: Swollen Head Syndrome and Omphalitis was carried out. We used the microarray methodology to evaluate the expression profile of two different APEC strains
Project description:Avian pathogenic Escherichia coli (APEC) is a subset of extraintestinal pathogenic E. coli that causes detrimental losses to the poultry industry. Vaccines to reduce APEC in chickens have been partially successful, but many lack protection against multiple serotypes of APEC. Recombinant attenuated Salmonella vaccine (RASV) strains have been used to induce immunity against Salmonella in production chickens and can be modified to deliver foreign antigens as well. This study evaluated the transcriptome of chicken spleens and assessed prevention of APEC infection following vaccination with RASV strains, including a RASV carrying an E. coli antigen. Four-day-old White Leghorn chicks were orally vaccinated with RASV c8025(pYA3337) carrying an empty plasmid, c8025(pYA4428) carrying genes for E. coli common pilus (ECP), a combination of RASVs c8025(pYA3337) and c8025(pYA4428), or PBS (unvaccinated). To assess the host response to vaccination, antibody titers were measured by ELISA and spleen samples (n = 5) were collected from combination vaccinated and unvaccinated groups of four-week-old chickens for RNA sequencing. Five-week old chickens were challenged via air sac with APEC strains APEC-O2 and c7122 (O78). Blood was obtained 24 hours post-challenge, heart, liver, lung, and spleen were collected 48 hours post-challenge for enumeration of E. coli, and gross colibacillosis lesions were scored at necropsy. Chickens vaccinated with RASV strains elicited anti-E. coli EcpA, as well as cross reactive anti-E. coli IutA and IroN IgY antibodies. IgA results. In some organs, bacterial loads and lesions scores were numerically reduced, but no significant differences were detected for vaccinated compared to unvaccinated chickens. Transcriptome results. This data indicates that RASVs could be used to stimulate the immune system and is an initial step toward developing improved therapeutics to combat APEC infections in chickens.
Project description:Colisepticemia caused by avian pathogenic Escherichia coli (APEC) results in annual multimillion dollar losses to the poultry industry. Recent research suggests that APEC may have an important role in public health as well. Generally, colisepticemia follows a respiratory tract infection in which APEC penetrate the respiratory epithelium to enter the bloodstream. From the bloodstream, bacteria may spread to various internal organs resulting in perihepatitis, pericarditis, and other conditions. The aim of this study was to identify molecular mechanisms enabling APEC to survive and grow in the bloodstream. To do so, we compared the transcriptome of APEC O1 during growth in Luria-Bertani broth and chicken serum. Selected genes that were significantly up-regulated in chicken serum were then subjected to mutational analysis to confirm their role in APEC pathogenesis. Several categories of genes, predicted to contribute to adaptation and growth in the avian host, were identified. These included several known virulence genes and genes involved in adaptive metabolism, protein transport, biosynthesis pathways, stress resistance, and virulence regulation. Several genes with unknown function, which were localized to pathogenicity islands or APEC O1’s large virulence plasmid, were also identified, suggesting that they too contribute to survival in chicken serum. This genome-wide analysis provides novel insight into processes that are essential to APEC O1’s survival and growth in chicken serum. Two-condition experiment: LB vs. chicken serm; four biological replicates, independently grown and harvested.
Project description:RNA-Seq was performed on Avian pathogenic E. coli (APEC) WT and mutant to investigate the pathogenicity of ArcA (Aerobic Respiratory Control), a global regulator important for E. coli’s adaptation from anaerobic to aerobic conditions and control of that bacterium’s enzymatic defenses against ROS.
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 APEC chi7122 strain and its isogenic Pst mutant grown in rich phosphate medium using the Affymetrix GeneChip® E. coli Genome 2.0 Array. The Affymetrix GeneChip® 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%. Keywords: Escherichia coli, phosphate starvation response, Pho regulon, Pst system, Affymetrix, transcriptional analysis
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 GeneChip® 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 GeneChip® E. coli Genome 2.0 Array. The Affymetrix GeneChip® 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 37°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.
Project description:APEC cause avian colibacillosis in poultry, characterized by the systematic infection, such as septicemia, airsacculitis, and pericarditis. APEC mainly use two-component regulatory systems (TCSs) to deal with the stressing environments in host during their infection. Whereas most TCSs in E.coli are well characterized, the characterization of RstA/RstB in APEC has not been thoroughly investigated. To understand the whole picture of RstA/RstB regulation, especially its role in virulence regulation, transcriptional analysis of the effect of rstAB deletion was performed in vivo. We compared the transcriptome of rstAB mutant and its wild-type strain during their growth in bloodstream of challenged chickens. In total, the transcripts of 85 genes were down-regulated by rstAB deletion while 113 were up-regulated at least twofold (cutoff limitation for fold change >2 or <0.5 and Cuffdiff P-value <0.05 was used to select differential expression genes). Our data showed that the RstA/RstB was a by-function regulator system, acting as both an activator and a repressor. The RstAB mainly regulated systems involved in nitrogen metabolism, bacterial virulence, iron acquisition and acid resistance.