Browsing publications of the research group Microbial Proteomics (MPRO) by Authors
Development and evaluation of a milk protein transcript depletion method for differential transcriptome analysis in mammary gland tissue.Brodhagen, Johanna; Weikard, Rosemarie; Thom, Ulrike; Heimes, Annika; Günther, Juliane; Hadlich, Frieder; Zerbe, Holm; Petzl, Wolfgang; Meyerholz, Marie M; Hoedemaker, Martina; et al. (BioMed Central (BMC), 2019-05-22)Background: In the mammary gland transcriptome of lactating dairy cows genes encoding milk proteins are highly abundant, which can impair the detection of lowly expressed transcripts and can bias the outcome in global transcriptome analyses. Therefore, the aim of this study was to develop and evaluate a method to deplete extremely highly expressed transcripts in mRNA from lactating mammary gland tissue. Results: Selective RNA depletion was performed by hybridization of antisense oligonucleotides targeting genes encoding the caseins (CSN1S1, CSN1S2, CSN2 and CSN3) and whey proteins (LALBA and PAEP) within total RNA followed by RNase H-mediated elimination of the respective transcripts. The effect of the RNA depletion procedure was monitored by RNA sequencing analysis comparing depleted and non-depleted RNA samples from Escherichia coli (E. coli) challenged and non-challenged udder tissue of lactating cows in a proof of principle experiment. Using RNase H-mediated RNA depletion, the ratio of highly abundant milk protein gene transcripts was reduced in all depleted samples by an average of more than 50% compared to the non-depleted samples. Furthermore, the sensitivity for discovering transcripts with marginal expression levels and transcripts not yet annotated was improved. Finally, the sensitivity to detect significantly differentially expressed transcripts between non-challenged and challenged udder tissue was increased without leading to an inadvertent bias in the pathogen challengeassociated biological signaling pathway patterns. Conclusions: The implementation of selective RNase H-mediated RNA depletion of milk protein gene transcripts from the mammary gland transcriptome of lactating cows will be highly beneficial to establish comprehensive transcript catalogues of the tissue that better reflects its transcriptome complexity.
Hepatic Transcriptome Analysis Identifies Divergent Pathogen-Specific Targeting-Strategies to Modulate the Innate Immune System in Response to Intramammary Infection.Heimes, Annika; Brodhagen, Johanna; Weikard, Rosemarie; Seyfert, Hans-Martin; Becker, Doreen; Meyerholz, Marie M; Petzl, Wolfram; Zerbe, Holm; Hoedemaker, Martina; Rohmeier, Laura; et al. (Frontiers, 2020-04-29)Mastitis is one of the major risks for public health and animal welfare in the dairy industry. Two of the most important pathogens to cause mastitis in dairy cattle are Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). While S. aureus generally induces a chronic and subclinical mastitis, E. coli is an important etiological pathogen resulting in an acute and clinical mastitis. The liver plays a central role in both, the metabolic and inflammatory physiology of the dairy cow, which is particularly challenged in the early lactation due to high metabolic and immunological demands. In the current study, we challenged the mammary glands of Holstein cows with S. aureus or E. coli, respectively, mimicking an early lactation infection. We compared the animals' liver transcriptomes with those of untreated controls to investigate the hepatic response of the individuals. Both, S. aureus and E. coli elicited systemic effects on the host after intramammary challenge and seemed to use pathogen-specific targeting strategies to bypass the innate immune system. The most striking result of our study is that we demonstrate for the first time that S. aureus intramammary challenge causes an immune response beyond the original local site of the mastitis. We found that in the peripheral liver tissue defined biological pathways are switched on in a coordinated manner to balance the immune response in the entire organism. TGFB1 signaling plays a crucial role in this context. Important pathways involving actin and integrin, key components of the cytoskeleton, were downregulated in the liver of S. aureus infected cows. In the hepatic transcriptome of E. coli infected cows, important components of the complement system were significantly lower expressed compared to the control cows. Notably, while S. aureus inhibits the cell signaling by Rho GTPases in the liver, E. coli switches the complement system off. Also, metabolic hepatic pathways (e.g., lipid metabolism) are affected after mammary gland challenge, demonstrating that the liver restricts metabolic tasks in favor of the predominant immune response after infection. Our results provide new insights for the infection-induced modifications of the dairy cow's hepatic transcriptome following mastitis.