Host defence related responses in bovine milk during an experimentally induced Streptococcus uberis infection.

BACKGROUND: Milk contains a range of proteins of moderate or low abundance that contribute to host defence. Characterisation of these proteins, the extent to which their abundance is regulated by pathogenic stimuli, and the variability of their response between and within individual animals would facilitate a better understanding of the molecular basis for this important function of milk. RESULTS: We have characterised the host defence proteins in bovine milk and their responses to intra-mammary infection by a common Gram positive mastitis pathogen, Streptococcus uberis, using a combination of 2D gel electrophoresis and GeLC mass spectrometry. In total, 68 host defence-associated proteins were identified, 18 of which have a direct antimicrobial function, 23 of which have a pathogen-recognition function, and 27 of which have a role in modulating inflammatory or immune signalling. The responsiveness of seven proteins was quantified by western blotting; validating the proteomic analyses, quantifying the within- and between animal variability of the responses, and demonstrating the complexity and specificity of the responses to this pathogen. CONCLUSIONS: These data provide a foundation for understanding the role of milk in host-microbe interaction. Furthermore they provide candidate biomarkers for mastitis diagnosis, and will inform efforts to develop dairy products with improved health-promoting properties.

The BPI-like/PLUNC family proteins in cattle.

Members of the protein family having similarity to BPI (bactericidal/permeability increasing protein) (the BPI-like proteins), also known as the PLUNC (palate, lung and nasal epithelium clone) family, have been found in a range of mammals; however, those in species other than human or mouse have been relatively little characterized. Analysis of the BPI-like proteins in cattle presents unique opportunities to investigate the function of these proteins, as well as address their evolution and contribution to the distinct physiology of ruminants. The present review summarizes the current understanding of the nature of the BPI-like locus in cattle, including the duplications giving rise to the multiple BSP30 (bovine salivary protein 30 kDa) genes from an ancestral gene in common with the single PSP (parotid secretory protein) gene found in monogastric species. Current knowledge of the expression of the BPI-like proteins in cattle is also presented, including their pattern of expression among tissues, which illustrate their independent regulation at sites of high pathogen exposure, and the abundance of the BSP30 proteins in saliva and salivary tissues. Finally, investigations of the function of the BSP30 proteins are presented, including their antimicrobial, lipopolysaccharide-binding and bacterial aggregation activities. These results are discussed in relation to hypotheses regarding the physiological role of the BPI-like proteins in cattle, including the role they may play in host defence and the unique aspects of digestion in ruminants.

The p100 EBNA-2 coactivator: a highly conserved protein found in a range of exocrine and endocrine cells and tissues in cattle.

The p100 transcriptional coactivator is an evolutionarily conserved protein that has been shown to be a coactivator of the Epstein-Barr virus-encoded transcription factor EBNA-2, as well as Stat5 and Stat6. However, the p100 genomic organisation, phylogeny and expression have not been analysed in detail and its physiological role is uncertain. The cDNA and amino acid sequence of bovine p100 was obtained, and the genomic organisation of the human p100 gene was determined. Homologues of p100 were found in the genomes of 21 diverse eukaryotes. Western blot and immunohistochemical analyses revealed that the bovine p100 protein is present in a range of exocrine and endocrine cells and tissues, including the lactating mammary gland, pancreas, adrenal, parotid, anterior pituitary, corpus luteum, ovarian follicular cells, placenta and small intestine. P100 was present in the nuclei of mammary epithelial cells and pancreatic acinar cells, but only in the extranuclear compartment of the other immunopositive tissues. These data indicate that the p100 protein plays a fundamental role in eukaryotic biology, and functions in secretory cells, at least in cattle.

Suppression of beta-casein gene expression by inhibition of protein synthesis in mouse mammary epithelial cells is associated with stimulation of NF-kappaB activity and blockage of prolactin-Stat5 signaling.

The protein synthesis inhibitor cycloheximide (Chx) suppresses prolactin-induced beta-casein gene expression in the mammary epithelial cell line COMMA-D. As the mechanism underlying this effect is unclear, the effects of protein synthesis inhibitors on interactions of transcription factors with the beta-casein promoter were examined. Suppression of prolactin-induced beta-casein gene expression occurred in both COMMA-D cells and primary mammary cell cultures with as little as 2 h protein synthesis inhibition. This was associated with changes in transcription factors interacting at a response element in the proximal region of the rat beta-casein promoter. Inhibition of protein synthesis was associated with NF-kappaB binding at a site immediately 3' to the Stat5-binding site at position 97-89 of the beta-casein promoter, suppression of Stat5 DNA-binding activity, and inhibition of Stat5 tyrosine phosphorylation. Treatment with the NF-kappaB inhibitor parthenolide failed to restore prolactin responsiveness. These results show that protein synthesis inhibition is associated with both blockage of prolactin-Stat5 signaling and NF-kappaB binding to the beta-casein promoter, but that the latter is not necessary for the suppression of beta-casein expression.