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Transcriptomic response of goat mammary epithelial cells to Mycoplasma agalactiae challenge – a preliminary study

ABSTRACT: Mycoplasma agalactiae (Ma) is one of the main aetiological agents of intramammary infections in small ruminants, causing contagious agalactia. To better understand the underlying disease patterns a primary goat mammary epithelial cell (pgMEC) culture was established from the mammary tissue and it was challenged with Ma. High-throughput mRNA sequencing was performed to reveal differentially expressed genes (DEG) at different time-points (3 h, 12 h, and 24 h) post infection (PI). The pathway enrichment analysis of the DEG showed that infection significantly affected pathways associated with immune response, steroid metabolism, fatty acid metabolism, apoptosis signalling, transcription regulation, and cell cycle regulation. Based on the results we suggest that mammary epithelial cells in vivo contribute to the immune system by the induced expression of cytokines and other chemotactic agents, activation of the complement system and apoptosis pathways, and expression of genes coding for antimicrobial molecules and peptides. In our study we attempted to interpret the detected transcriptomic changes in a biological context and infer mammary infection resistance candidate genes, interesting for further validation. Additionally, the results represent comprehensive goat mammary transcriptome information and demonstrate the applicability of the comparative genomics approach for annotation of goat data, using transcriptome information of a closely related species (Bos taurus) as a reference.

ORGANISM(S): Capra hircus

PROVIDER: GSE30379 | GEO | 2013/01/01

SECONDARY ACCESSION(S): PRJNA143425

REPOSITORIES: GEO

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Project description:Whole-transcriptome sequencing of luminal epithelial cells was performed in order to profile the transcriptomic response to infection and to elucidate the immune response signaling capability of milk producing epithelial cells. Primary goat mammary epithelial cell (pgMEC) culture model, mimicking the function of luminal cells in vivo, was established and infected with Mycoplasma agalactiae (Ma). After infection total RNA was extracted from the cells at different time-points post infection (PI) and sequenced using Illumina Gene Analyzer IIx. Four datasets were generated including controls and pgMECs challenged for 3 h, 12 h, and 24 h. The obtained 50 bp sequences were mapped against the bovine reference transcriptome. In addition to analysing individual genes for differential expression (DEG), we performed functional annotation of regulated genes to assess kinetics of the immune response on a more global scale. The DEGs were analysed for representative gene ontology (GO) enrichement terms, and biological processes as well as pathways regulated in response to Ma infection using different bioinformatic tools. The results show that in response to infection pgMECs are capable to induce genes involved in proinflammatory-cytokine signaling, activation of complement system, and can produce several bactericidal molecules of innate immune response. The pathway analyses revealed that in addition to immune response, the most regulated pathways were associated with lipid metabolism, apoptosis, transcription regulation, and cell cycle regulation. The obtained data is important for assessing the dynamics of the immune response signaling in MECs and opens new possibilities to idenitfy promising candidate genes, which could be beneficial for development of new therapeutic methods and introduction of a marker assisted selection towards enhanced mastitis resistance in breeding schemes. Transcriptome analysis of 12 samples; 3 replicated samples from control cells, and from cells 3 h, 12 h, and 24 h post infection.

Project description:Mastitis remains one of the most prevalent and costly diseases impacting the dairy industry worldwide. Escherichia coli is an environmental bacterium that frequently causes intramammary infections, the outcome of which depends on the capacity of the host to recognize and clear the bacterial pathogen. E. coli intramammary infection elicits localized and systemic responses, some of which have been characterized in mammary secretory tissue. However, nothing is known about early transcriptome-wide responses to infection that occur within regions of the teat and mammary parenchyma. Therefore, the objective of the current study was to use microarray analysis to characterize gene expression patterns and process networks that become activated in different regions of the mammary gland during the acute phase of experimentally induced intramammary infection with E. coli. Tissues evaluated were from Furstenberg’s rosette, teat cistern, gland cistern, and lobulo-alveolar regions of control and infected mammary glands, 12 and 24 h after bacterial (or control) infusions. For selected genes, quantitative RT-PCR was performed to confirm the microarray findings (Toll-like receptors; TLR1, TLR2, TLR4, TLR6) and evaluate the correspondence between mRNA level in tissues and protein level in milk (interleukin 8, tumor necrosis factor-alpha, haptoglobin, lipopolysaccharide-binding protein). The main networks activated by E. coli infection pertained to immune and inflammatory response, with marked induction of genes encoding proteins that function in chemotaxis, leukocyte activation and signaling. Genomic response was greatest in tissues of the teat and gland cisterns at 12 h post-infection, while tissue of the lobulo-alveolar region responded only, and most strongly of the regions, 24 h following the infection. Up regulation of TLR2, TLR4, IL8, TNF-alpha and bactericidal/permeability-increasing protein peaked at 12 h post infection in tissues of the teat and in the gland cistern, while the highest level of lipopolysaccharide-binding protein was reached at 24 h in the lobular alveoli region of infected quarters. Very few genes were down–regulated within the timeframe of this study. Similar genetic networks were impacted in all regions during early phases of E. coli intramammary infection, although regional differences throughout the gland were noted. Importantly, the tissues of the teat, which are first to encounter invading bacteria during a natural infection, responded rapidly and intensely, suggesting an important sentinel function for this region. Both resident mammary cells and infiltrating immune cells likely play an important role in recognition of pathogens and production of inflammatory mediators during mastitis.

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Transcriptomic response of goat mammary epithelial cells to Mycoplasma agalactiae challenge – a preliminary study

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