Itraq-Proteomics Identification Of Mammary Tissue From Cows With Clinical Mastitis Infected By Staphylococci Aureus And Expression Regulation Analysis Of Its Candidate Genes

Mastitis is the most costly disease affecting dairy cattle production and has a negative impact on animal welfare and product quality. Mastitis mainly involves a complex set of interactions between an invading pathogen and immune systems of the host. Staphylococci aureus is a major pathogenic bacteria. Proteomics and the associated bioinformatics are considered as complimentary tools for the study the dynamic interactions between the immune system and pathogens. The use of proteomic methodologies to obtain a more complete and unbiased characterization of host responses during clinical mastitis could lead to the identification of a biomarker, indicative of the disease and decrease the economic losses. Here we present an exploration of the mastitis-induced changes in the proteome of mammary gland tissue using the iTRAQ system to find differentially expressed proteins between mastitis mammary gland and mammary gland. Differentially expressed proteins were used for bioinformatics analyses to find some ones associated with inflammation, immune and tissue damage. Then we further investigate the regulation of intresting candidate gens expression in different ways. The thesis was described as follows:1 Proteomic expression profiles of mammary tissue from cows with clinical mastitis due to natural infection with Staphylococci aureusThe aim of the present study was to use isobaric tags for relative and absolute quantification (iTRAQ) to screen potential proteins associated with mastitis at late infectious stage. Healthy and mastitic cows’ mammary gland tissues were analyzed using iTRAQ combined with two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS). Bioinformatics analysis of differentially expressed proteins was by means of Gene Ontology, transcriptional regulation networks using ProteinPilot software and the Dynamic Impact Approach. Western blotting and Immunohistochemical staining methods were used to analyze a subset of affected proteins. At a false discovery rate of 5%, a total of 768 proteins were identified from 6,499 peptides, which were matched with 15,879 spectra. Compared with healthy mammary gland tissue,36 proteins were significantly up-regulated (>1.5-fold) while 19 were significantly down-regulated (<0.67-fold) in response to mastitis due to natural infections with Staphylococci aureus. Up-regulation of collagen, type I, alpha 1 (COL1A1) and inter-alpha (Globulin) inhibitor H4 (ITIH4) in the mastitis-infected tissue was confirmed by Western blotting and Immunohistochemistry. This paper is investigated to show the late response to a mastitic pathogen, thus, revealing mechanisms associated with host tissue damage. The bioinformatics analyses highlighted the effects of mastitis on proteins such as collagen, fibrinogen, fibronectin, casein alpha and heparan sulfate proteoglycan 2. The up-regulated expression of COLl Al and ITIH4 in the mastitic mammary gland may be associated with tissue damage and repair during late stages of infection.2 The methylation pattern of COL1A2 gene promoter is associated with its expression and mastitis in dairy cowThis study was to investigate the methylation pattern of COL1A2 gene promoter and its effect on gene expression between the healthy cow’s mammary tissues (n=3) and mastitiic cow’s tissues (n=3) to provide clues for disease-resistant breeding and prevention of mastitis in dairy cows. The promoter CpG islands and its transcription factors were analyzed by bioinformatics.The methylation status of a CpG island in the COL1A2 gene core promoter region and the expression of COL1A2 mRNA were detected by the bisulfite sequencing PCR and RT-PCR in two groups, the methylation of the CpG island of two groups were not significantly different(P>0.05) and were hypomethylated (<50% of CpG sites on a given methylated strand), while the significant difference methylation status were found at the 4th and 5th CpG sites within a SP1 binding site, between the healthy mammary tissues (30% and 60%) and the mastitis-infected tissues (0% and 10%), which was consist with the result of differential expression of COL1A2 mRNA in two groups (P<0.05).Conclusion:the different methylation patterns at the 4th and 5th CpG sites may contribute to the higher expression of the COL1A2 gene in mastitis-infected tissues than the healthy ones. COL1A2 gene can play an important role in the processes against mastitis by up-regulating COL1A2 expresion via promoter methylation mechanism.