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Gene Expression Profiles of Chicken Embryo Fibroblasts in Response to Salmonella Enteritidis Infection

ABSTRACT: The response of chicken to non-typhoidal Salmonella infection is becoming well characterised but the role of particular cell types in this response is still far from being understood. Therefore, in this study we characterised the response of chicken embryo fibroblasts (CEFs) to infection with two different S. Enteritidis strains by microarray analysis. The expression of chicken genes identified as significantly up- or down-regulated (≥3-fold) by microarray analysis was verified by real-time PCR followed by functional classification of the genes and prediction of interactions between the proteins using Gene Ontology and STRING Database. Finally the expression of the newly identified genes was tested in HD11 macrophages and in vivo in chickens. Altogether 19 genes were induced in CEFs after S. Enteritidis infection. Twelve of them were also induced in HD11 macrophages and thirteen in the caecum of orally infected chickens. The majority of these genes were assigned different functions in the immune response, however five of them (LOC101750351, K123, BU460569, MOBKL2C and G0S2) have not been associated with the response of chicken to Salmonella infection so far. K123 and G0S2 were the only 'non-immune' genes inducible by S. Enteritidis in fibroblasts, HD11 macrophages and in the caecum after oral infection. The function of K123 is unknown but G0S2 is involved in lipid metabolism and in β-oxidation of fatty acids in mitochondria. Increased levels of G0S2 might decrease the availability of fatty acids to mitochondria. In non-professional phagocytes such as CEFs, this may lead to the dysfunction of mitochondria, apoptosis of CEFs and release of intracellular Salmonella. In professional phagocytes, G0S2 might be involved in the control of mitochondrial respiration, resulting in a decrease of reactive oxygen species as respiration by-products and lower damage to tissue.

ORGANISM(S): Gallus gallus

PROVIDER: GSE67459 | GEO | 2015/06/12

SECONDARY ACCESSION(S): PRJNA279986

REPOSITORIES: GEO

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Project description:The response of chicken to non-typhoidal Salmonella infection is becoming well characterised but the role of particular cell types in this response is still far from being understood. Therefore, in this study we characterised the response of chicken embryo fibroblasts (CEFs) to infection with two different S. Enteritidis strains by microarray analysis. The expression of chicken genes identified as significantly up- or down-regulated (≥3-fold) by microarray analysis was verified by real-time PCR followed by functional classification of the genes and prediction of interactions between the proteins using Gene Ontology and STRING Database. Finally the expression of the newly identified genes was tested in HD11 macrophages and in vivo in chickens. Altogether 19 genes were induced in CEFs after S. Enteritidis infection. Twelve of them were also induced in HD11 macrophages and thirteen in the caecum of orally infected chickens. The majority of these genes were assigned different functions in the immune response, however five of them (LOC101750351, K123, BU460569, MOBKL2C and G0S2) have not been associated with the response of chicken to Salmonella infection so far. K123 and G0S2 were the only 'non-immune' genes inducible by S. Enteritidis in fibroblasts, HD11 macrophages and in the caecum after oral infection. The function of K123 is unknown but G0S2 is involved in lipid metabolism and in β-oxidation of fatty acids in mitochondria. Increased levels of G0S2 might decrease the availability of fatty acids to mitochondria. In non-professional phagocytes such as CEFs, this may lead to the dysfunction of mitochondria, apoptosis of CEFs and release of intracellular Salmonella. In professional phagocytes, G0S2 might be involved in the control of mitochondrial respiration, resulting in a decrease of reactive oxygen species as respiration by-products and lower damage to tissue. In this study we were interested whether chicken embryo fibroblast (CEFs) respond to S. Enteritidis infection and to what extent their response differs from that of other cells and caecal tissue. To address this, we characterised the gene expression of CEFs after infection with two different wild-type S. Enteritidis strains of poultry origin - SE 147 and SE 11 - using Agilen custom 8×15K microarrays. In total, 13,681 probes were designed to characterise the expression of ~9,000 transcripts of Gallus gallus.

Project description:Salmonella Enteritidis is the major food-borne pathogen primarily causing human infection through contaminated chicken meat and eggs. We recently demonstrated that S. Enteritidis strains from poultry differ in their ability to invade human intestinal cells and cause disease in orally challenged mice. Here we hypothesized that the differential pathogenicity of S. Enteritidis strains is due to the differential fitness in the adverse environments that may be encountered during infection in the host. The response of a panel of six S. Enteritidis strains to acid stress, oxidative stress, survival in egg albumen and the ability to cause infection in chickens were analyzed. This analysis allowed classification of strains into two categories: stress- sensitive and stress- resistant, with the former showing significantly (P<0.05) reduced survival in acidic (gastric phase of infection) and oxidative (intestinal and systemic phase of infection) stress. Stress-sensitive strains also showed impaired intestinal colonization and systemic dissemination in orally inoculated chickens and failed to survive/grow in egg albumen. Comparative genomic hybridization microarray analysis revealed no differences at the discriminatory level of the whole gene content between stress-sensitive and stress-resistant strains. However, sequencing of rpoS, a stress-regulatory gene, revealed that one of the three stress-sensitive strains carried an insertion mutation in the rpoS resulting in truncation of σS. Finding that one of the stress-sensitive strains carried an easily identifiable small polymorphism within a stress-response gene suggests that the other strains may also have small polymorphisms elsewhere in the genome, which likely impact regulation of stress or virulence associated genes in some manner. To determine if the differential stress response and virulence observed in this study could be due to the differences in the gene content between different strains, we compared the genomes of all the strains using CGH microarray designed based on the genomic information available for the sequenced strain. All strains (n=6) were hybridized in triplicate.

Project description:To determine the transcriptional response to Salmonella enterica serovar Enteritidis (SE) infection, a newly developed chicken 44K Agilent array was used to analyze total RNA of heterophils from SE-resistant (line A) and SEâsusceptible chickens (line B), treated with in vitro infection of SE (I) or non SE medium (N) for 1 hr. A dual-color balanced design was used to provide a direct comparison between SE-infected and non-infected groups (AI/AN, BI/BN) and between line A and line B (AN/BN, AI/BI). Data retrieved from 426 immunologically important genes were used for functional analysis. The results indicated that: In the comparisons of SE infection with non-infection, 39 genes were found differentially expressed (P < 0.05 with at least 2-fold) after Salmonella infection, while 21 genes were found differentially expressed between different lineages. Numerous of members in Toll-like receptor (TLR) signaling pathway were identified which include receptors: MD-2, TLR-4, -5, -15; adaptors: TLR adaptor molecule1 (TICAM1/TRIF), MAP kinase kinase 3 (MKK3) and NFkB-1, as well as cytokines, chemokines and costimulatory molecule: interleukin (IL)-1Î², IL-6, IL-8, IL-12, chemokine (C-X-C motif) ligand 1, chemokine (C-C motif) ligand 4, CCL 5, and CD80. Most of immune genes were up-regulated after SE infection, and the magnitude of up-regulation was higher in line A than line B in general. The results suggested that a similar TLR regulatory network exists in both lines with strong response in resistant line. This finding has laid the foundation for further study of molecular and cellular modulation of SE infection in chickens. Keywords: disease state analysis A dual color, balanced design was carried on for all of heterophils samples from sixteen chickens. Each sample type (AI, AN, BI and BN) includes four biological replication for labeling. A Dye swap was used in each pair of comparisons including AI/AN, BI/BN, and AI/BI, and AN/BN. Background subtracted signal intensity were collected from 16 arrays and normalized for data analysis.

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Gene Expression Profiles of Chicken Embryo Fibroblasts in Response to Salmonella Enteritidis Infection

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