Global transcriptional response of porcine intestinal epithelial cell lines to Salmonella enterica serovar Typhimurium and Choleraesuis: IPI-2I infected with S. choleraesuis
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ABSTRACT: The GeneChip Porcine Genome Array was used to identify the transcriptional response upon either Salmonella typhimurium (ST) or Salmonella choleraesuis (SC) infection in two porcine epithelial cell lines (IPEC-J2, from jejunum and IPI-2I, from ileum) during 2 and 4 hours post infection. The objectives in this study were first, to identify the different response between the epithelial cell lines from different gut regions; second, to study how the Salmonella serotypes used could elicit a different host response; and third, to determine the effect of the time-points on the differentially gene expression. Epithelial cells were seeded into 6-well tissue culture plates and grown to confluence in 5% CO2 at 37ºC. Monolayers were infected for 1 h. with Salmonella typhimurium or Salmonella choleraesuis serotypes (MOI 1:10) or incubated with media alone (Control cells). Extracellular bacteria were removed, and cultures were further incubated during 2 and 4 h. in the presence of 50 µg/ml of the non-cell-permeant antibiotic gentamicin to kill remaining extracellular bacteria. The experiment was developed by triplicate in order to ensure a proper robustness in the microarray statistical analysis. An indirect ELISA was used to confirm cell activation by mean of IL8 detection in cell culture medium. After each time-point, cells were lysed and RNA extracted.
Project description:The GeneChip Porcine Genome Array was used to identify the transcriptional response upon either Salmonella typhimurium (ST) or Salmonella choleraesuis (SC) infection in two porcine epithelial cell lines (IPEC-J2, from jejunum and IPI-2I, from ileum) during 2 and 4 hours post infection. The objectives in this study were first, to identify the different response between the epithelial cell lines from different gut regions; second, to study how the Salmonella serotypes used could elicit a different host response; and third, to determine the effect of the time-points on the differentially gene expression. Epithelial cells were seeded into 6-well tissue culture plates and grown to confluence in 5% CO2 at 37ºC. Monolayers were infected for 1 h. with Salmonella typhimurium or Salmonella choleraesuis serotypes (MOI 1:10) or incubated with media alone (Control cells). Extracellular bacteria were removed, and cultures were further incubated during 2 and 4 h. in the presence of 50 µg/ml of the non-cell-permeant antibiotic gentamicin to kill remaining extracellular bacteria. The experiment was developed in triplicate in order to ensure a proper robustness in the microarray statistical analysis. An indirect ELISA was used to confirm cell activation by means of IL8 detection in cell culture medium. After each time-point, cells were lysed and RNA extracted.
Project description:The GeneChip Porcine Genome Array was used to identify the transcriptional response upon either Salmonella typhimurium (ST) or Salmonella choleraesuis (SC) infection in two porcine epithelial cell lines (IPEC-J2, from jejunum and IPI-2I, from ileum) during 2 and 4 hours post infection. The objectives in this study were first, to identify the different response between the epithelial cell lines from different gut regions; second, to study how the Salmonella serotypes used could elicit a different host response; and third, to determine the effect of the time-points on the differentially gene expression. Epithelial cells were seeded into 6-well tissue culture plates and grown to confluence in 5% CO2 at 37ºC. Monolayers were infected for 1 h. with Salmonella typhimurium or Salmonella choleraesuis serotypes (MOI 1:10) or incubated with media alone (Control cells). Extracellular bacteria were removed, and cultures were further incubated during 2 and 4 h. in the presence of 50 µg/ml of the non-cell-permeant antibiotic gentamicin to kill remaining extracellular bacteria. The experiment was developed by triplicate in order to ensure a proper robustness in the microarray statistical analysis. An indirect ELISA was used to confirm cell activation by mean of IL8 detection in cell culture medium. After each time-point, cells were lysed and RNA extracted.
Project description:The GeneChip Porcine Genome Array was used to identify the transcriptional response upon either Salmonella typhimurium (ST) or Salmonella choleraesuis (SC) infection in two porcine epithelial cell lines (IPEC-J2, from jejunum and IPI-2I, from ileum) during 2 and 4 hours post infection. The objectives in this study were first, to identify the different response between the epithelial cell lines from different gut regions; second, to study how the Salmonella serotypes used could elicit a different host response; and third, to determine the effect of the time-points on the differentially gene expression. Epithelial cells were seeded into 6-well tissue culture plates and grown to confluence in 5% CO2 at 37ºC. Monolayers were infected for 1 h. with Salmonella typhimurium or Salmonella choleraesuis serotypes (MOI 1:10) or incubated with media alone (Control cells). Extracellular bacteria were removed, and cultures were further incubated during 2 and 4 h. in the presence of 50 µg/ml of the non-cell-permeant antibiotic gentamicin to kill remaining extracellular bacteria. The experiment was developed by triplicate in order to ensure a proper robustness in the microarray statistical analysis. An indirect ELISA was used to confirm cell activation by mean of IL8 detection in cell culture medium. After each time-point, cells were lysed and RNA extracted.
Project description:The GeneChip Porcine Genome Array was used to identify the transcriptional response upon either Salmonella typhimurium (ST) or Salmonella choleraesuis (SC) infection in two porcine epithelial cell lines (IPEC-J2, from jejunum and IPI-2I, from ileum) during 2 and 4 hours post infection. The objectives in this study were first, to identify the different response between the epithelial cell lines from different gut regions; second, to study how the Salmonella serotypes used could elicit a different host response; and third, to determine the effect of the time-points on the differentially gene expression.
Project description:The GeneChip Porcine Genome Array was used to identify the transcriptional response upon either Salmonella typhimurium (ST) or Salmonella choleraesuis (SC) infection in two porcine epithelial cell lines (IPEC-J2, from jejunum and IPI-2I, from ileum) during 2 and 4 hours post infection. The objectives in this study were first, to identify the different response between the epithelial cell lines from different gut regions; second, to study how the Salmonella serotypes used could elicit a different host response; and third, to determine the effect of the time-points on the differentially gene expression.
Project description:The GeneChip Porcine Genome Array was used to identify the transcriptional response upon either Salmonella typhimurium (ST) or Salmonella choleraesuis (SC) infection in two porcine epithelial cell lines (IPEC-J2, from jejunum and IPI-2I, from ileum) during 2 and 4 hours post infection. The objectives in this study were first, to identify the different response between the epithelial cell lines from different gut regions; second, to study how the Salmonella serotypes used could elicit a different host response; and third, to determine the effect of the time-points on the differentially gene expression.
Project description:The GeneChip Porcine Genome Array was used to identify the transcriptional response upon either Salmonella typhimurium (ST) or Salmonella choleraesuis (SC) infection in two porcine epithelial cell lines (IPEC-J2, from jejunum and IPI-2I, from ileum) during 2 and 4 hours post infection. The objectives in this study were first, to identify the different response between the epithelial cell lines from different gut regions; second, to study how the Salmonella serotypes used could elicit a different host response; and third, to determine the effect of the time-points on the differentially gene expression.
Project description:To understand the host transcriptional response to S. enterica serovar Choleraesuis (S. Choleraesuis), the first generation Affymetrix porcine GeneChip® was used to identify differentially expressed genes in the mesenteric lymph nodes responding to infection at acute (8 hours (h), 24h, 48h post-inoculation (pi)) and chronic stages (21 days (d) pi); The objectives of this study were to identify and examine the stereotypical gene expression response within the host mesenteric lymph nodes to S. Choleraesuis infection, and to characterize the global host responses by revealing the specific features of the hostâs innate immunity. Experiment Overall Design: Fifteen piglets from Salmonella spp.-free sows were weaned at 10 days (d) of age, shipped to the National Animal Disease Center, Ames, IA and raised in isolation facilities. To confirm that all piglets were free of Salmonella spp. prior to challenge, bacteriological cultures were performed on rectal swabs twice. Seven week old pigs were randomly divided into 2 groups, 3 non-infected pigs and 12 infected pigs. Three non-infected control pigs were necropsied 3 days prior to experimental infection. On day 0, pigs in the infected groups were intranasally challenged with 1 billion CFU of Salmonella enterica serotype Choleraesuis x3246. Three infected pigs were necropsied at 8 hours post-inoculation (hpi), 24 hpi, 48 hpi and 21 day post-inoculation (dpi). Tissue samples from the mesenteric lymph nodes (MLN) were collected and immediately frozen in liquid nitrogen for RNA isolation.
Project description:To understand the host transcriptional response to S. enterica serovar Choleraesuis (S. Choleraesuis), the first generation Affymetrix porcine GeneChip® was used to identify differentially expressed genes in the mesenteric lymph nodes responding to infection at acute (8 hours (h), 24h, 48h post-inoculation (pi)) and chronic stages (21 days (d) pi) The objectives of this study were to identify and examine the stereotypical gene expression response within the host mesenteric lymph nodes to S. Choleraesuis infection, and to characterize the global host responses by revealing the specific features of the host’s innate immunity. Keywords: time course
Project description:Aggregatibacter actinomycetemcomitans (Aa) is a Gram-negative bacterial pathogen associated with periodontitis and non-oral diseases like rheumatoid arthritis and Alzheimer´s disease. Aa isolates with the serotypes a, b and c are globally most prevalent. Importantly, isolates displaying these serotypes have different clinical presentations. While serotype b isolates are predominant in severe periodontitis, serotypes a and c are generally encountered in mild periodontitis or healthy individuals. It was so far not known how these differences are reflected in the overall secretion of virulence factors. Therefore, this study was aimed at a comparative analysis of exoproteomes from different clinical Aa isolates with serotypes a, b or c by mass spectrometry, and a subsequent correlation of the recorded exoproteome profiles with virulence. Overall, we identified 425 extracellular proteins. Significant differences in the exoproteome composition of isolates with different serotypes were observed in terms of protein identification and abundance. In particular, serotype a isolates presented more extracellular proteins than serotype b or c isolates. These differences are mirrored in their virulence in infection models based on human salivary gland epithelial cells and neutrophils. Remarkably, serotype a isolates displayed stronger adhesive capabilities and induced more lysis of epithelial cells and neutrophils than serotype b or c isolates. Conversely, serotype c isolates showed relatively low leukotoxicity, while provoking NETosis to similar extents as serotype a and b isolates. Altogether, we conclude that the differential virulence presentation by Aa isolates with the dominant serotypes a, b or c can be explained by their exoproteome heterogeneity.