Characterization of the in vitro gene response of chicken cells to Salmonella Enteritidis

Salmonella Enteritidis (SE) is one of the most frequently reported causative agent of human gastroenteritis, originating mainly from poultry. Pathogenesis of SE infection in poultry is well-elucidated, but the complexity of the host cell response, and its relation to differring pathogenic potential of various strains is much less understood. Therefore we intended to provide a genome-wide comparative characterization of the gene expression profiles of chicken cells to wild type strains and virulence-related mutants of Salmonella Enteritidis. Freshly isolated chicken embryo fibroblast (CEF) cells co-incubated with Salmonella for 4 hrs were used to model gene response of young chickens to Salmonella infection and to measure the invasiveness of wild type strains SE147, SE11 and non-motile mutants of SE11 lacking the fliD gene and/or the virulence plasmid. Agilent custom 8×15K microarray was designed to profile the expression of 13741 chicken genes, with emphasis to those related to immune response. Significant gene expression changes with fold change ≥3 (in total of 31 genes) were verified by real-time PCR. Expression profile of infected CEF cells resulted in 314 genes significantly misregulated by the infection with the wild type strain SE147 (206 up-/108 down-regulations) while only 135 genes were significantly expressed as a result to SE11 infection (74 up-/61 down-regulations). There were 100 genes induced by both wild strains, among them CSF3 (colony-stimulating factor), IL-1β and IL-8 showing the highest upregulations. In contrast to this, infection with non-motile mutants lacking fliD gene and/or the virulence plasmid, did not cause any significant change in host gene expression. However real-time PCR results indicated that the cell cycle-related G0S2 switch-, and the enolase ENO2 genes were highly induced by the mutant strains, indicating that the reduced invasiveness of the mutants might have stimulated cell division and/or metabolism of the host cells. Results suggest that fliD gene plays a key role in the invasiveness of Salmonella strains, and could be considered as an important modulator of the chicken response to Salmonella infection. This work was supported by the EU FP6 NoE MedVetNet and OTKA 105635. Ama Szmolka is a holder of János Bolyai Research Scholarship of HAS

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

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

Impact of gene fliD and virulence plasmid pSEV on the host response by chicken embryo fibroblasts to Salmonella Enteritidis

While Salmonella Enteritidis is the most important serovar of poultry origin in human outbreaks, its complex interaction with avian cells remains less understood. Our previous observations on response of chicken embryo fibroblasts to the wild type strain S. Enteritidis 11 raised the question whether the virulence-defective mutants of this strain lacking flagellin gene fliD,only or both fliD and serovar-specific virulence plasmid pSEV, are inducing altered expression of fibroblast genes. As expected, deletion mutants of fliD and pSEV showed reduced invasiveness and moderate induction of immune genes, but induced a dramatically increased expression of the non-immune gene G0S2. We concluded that chicken fibroblasts prove to be a good model to differentiate host responses by wild type strains and virulence-defective mutants of S. Enteritidis. Our data indicate the importance of flagella and pSEV as key modulators of virulence and of fibroblast response including the modulation of survival genes such as G0S2

Gene expression in the chicken caecum in response to infections with non-typhoid Salmonella

International audienceChickens can be infected with Salmonella enterica at any time during their life. However, infections within the first hours and days of their life are epidemiologically the most important, as newly hatched chickens are highly sensitive to Salmonella infection. Salmonella is initially recognized in the chicken caecum by TLR receptors and this recognition is followed by induction of chemokines, cytokines and many effector genes. This results in infiltration of heterophils, macrophages, B- and T-lymphocytes and changes in total gene expression in the caecal lamina propria. The highest induction in expression is observed for matrix metalloproteinase 7 (MMP7). Expression of this gene is increased in the chicken caecum over 4000 fold during the first 10 days after the infection of newly hatched chickens. Additional highly inducible genes in the caecum following S. Enteritidis infection include immune responsive gene 1 (IRG1), serum amyloid A (SAA), extracellular fatty acid binding protein (ExFABP), serine protease inhibitor (SERPINB10), trappin 6-like (TRAP6), calprotectin (MRP126), mitochondrial ES1 protein homolog (ES1), interferon-induced protein with tetratricopeptide repeats 5 (IFIT5), avidin (AVD) and transglutaminase 4 (TGM4). The induction of expression of these proteins exceeds a factor of 50. Similar induction rates are also observed for chemokines and cytokines such as IL1β, IL6, IL8, IL17, IL18, IL22, IFNγ, AH221 or iNOS. Once the infection is under control, which happens approx. 2 weeks after infection, expression of IgY and IgA increases to facilitate Salmonella elimination from the gut lumen. This review outlines the function of individual proteins expressed in chickens after infection with non-typhoid Salmonella serovars

Gene expression of chicken cells influenced by plasmidless non-motile mutants of Salmonella Enteritidis

Salmonella Enteritidis is still a significant pathogen of human gastroenteritis, originating mainly from poultry (ECDC/EFSA supporting publication 2014). Recently we have shown that chicken embryo fibroblast cells (CEFs) proved to be useful for in vitro assessment of the innate immune response of chicks to S. Enteritidis infection. Therefore we intended to use CEFs to estimate and the significance of certain virulence determinants of S. Enteritidis in chicken. Our objective was to characterize the gene expression profiles of CEFs as a response to non-motile mutants of S. Enteritidis 11 (SE 11) lacking the serovar-specific virulence plasmid and/or the fliD gene in comparison to the wild type parental strain. Freshly isolated CEFs incubated with Salmonella for 4 hrs were used to measure the invasiveness of parental strain SE 11 and its virulence-defective mutants (Imre et al., 2011). We used Agilent custom 8×15K microarray to profile the expression of CEFs, with emphasis to genes related to the immune response. Expression of chicken genes identified as significantly up- or downregulated (≥3-fold) was verified by real-time PCR. Invasiveness of both mutants of SE11 proved to be significantly lower than that of parental strain. Infection with SE 11 induced 26 genes and led to the suppression of 39 genes. Out of them the colony-stimulating factor gene CSF3 and cytokine genes for IL-1β and IL-8 showed the highest upregulations. In contrast, infection with non-motile mutants lacking the virulence plasmid and/or fliD did not cause any significant change in host gene expression. However real-time PCR results indicated that the cell cycle G0S2 switch gene (cell division), and the enolase ENO2 gene (metabolism) were highly induced by the mutant strains, indicating that the reduced invasiveness of the mutants was concomitant to stimulated cell division and/or metabolism of the host cells. Based on these results fliD seems to be more important for the invasiveness of Salmonella Enteritidis than the serovar-specific virulence plasmid. This is in harmony with our earlier in vivo studies about minor significance of the virulence plasmid in the pathogenicity of S. Enteritidis 11. In contrast, fliD could be considered as a modulator of the chicken response to Salmonella infection. Interestingly stimulation of non-immune genes such as G0S2 and ENO2 was much stronger by the virulence-defective mutants demonstrating that plasmid- and/or flagellin of Salmonella may influence host cell metabolism and regeneration. This work was supported by the EU FP6 NoE MedVetNet and FP7 PROMISE. Ama Szmolka is a holder of János Bolyai Research Scholarship of Hungarian Academy of Sciences. A. Imre’s present address is: CEVA—Phylaxia, Szállás u.5, H-1107 Budapest, Hungary

Colony morphology of the wild-type <i>S</i>. Enteritidis, SPI1- <i>lon</i>::Cm-<i>fliC</i> mutant and SPI1-<i>lon</i>::Cm-<i>fliC</i>-<i>rcsB</i>::Kan mutant.

<p>Inactivation of <i>lon</i> resulted in a mucoid colony phenotype which was observed in all the mutants with the <i>lon</i> mutation except for the mutant in which the <i>rcsB</i> mutation has been introduced. The overproduction of capsular polysaccharides in the vaccine strain enables simple differentiation of the vaccine strain from those circulating in the environment.</p

List of primers used in this study for the construction of <i>fliC</i>, <i>rcsB</i> and <i>lon</i> mutants.

*<p>For primers used for the amplification of pKD3 or pKD4, only the gene specific overhangs are shown. „CTR“ primers, either Forward (F) or Reverse (R) were used for the verification of the final contructs.</p

Electron microscopy of flagella in <i>S</i>. Enteritidis.

<p>Flagella could be visualised in all the strains and mutants with intact <i>fliC</i> after negative staining with ammonium molybdate.</p