MicroRNA profile of bovine monocyte-derived macrophages infected in vitro with Streptococcus agalactiae
ABSTRACT: In a subclinical infection such as bovine streptococcal mastitis, early recognition is a great challenge, and miRNAs profiling could potentially assist in the diagnosis and contribute to the understanding of pathogenicity and defense mechanisms. We have examined the miRNA repertoire during the early phase response of bovine macrophages to in vitro infection with live Streptococcus agalactiae. Next generation sequencing of 20 small RNA libraries from blood monocyte-derived macrophages exposed to two sequence types of S. agalactiae (ST103 and ST12) for 6 hours in vitro was performed. Analyzes of over 356 million of high quality sequence reads, revealed that 17 and 44 miRNAs were differentially expressed (P < 0.05) between the control unchallenged macrophages and the macrophages infected with ST103 and ST12, respectively. We also identified the expression of 31 potentially novel bovine miRNAs.
Project description:We determined dynamic behavior of S. agalactiae transcriptome during growth in THY medium and detected growth phase regulated genes Overall design: Three independent cultures (three biological replicates) of S. agalactiae NEM316 strain in rich laboratory medium were sampled at mid log, late log early and late stationary growth phase.
Project description:Analysis of S. agalactiae response to dltA mutation the role of DltR. This analysis is based on the comparison of the effect of dltA and dltR mutation on S. agalactiae transcription profile.
Project description:Macrophages from cattles with different infectious status of bovine tuberculosis have different responses to in vitro Mycobacterium bovis challenge. This is confirmed in our previous study exploring several immune-related genes using qPCR. Microarrays can help us better understand the differences by screening thousands of genes. Monocytes Derived Macrophages from 3 TB-infected cattles and 3 TB-free cattles were challenged with Mycobacterium bovis at a MOI of 10 at 6 hours, and the control group were the same unchallenged macrophages at 6 hours.
Project description:We determined dynamic behavior of S. agalactiae transcriptome during growth in THY medium and detected growth phase regulated genes Three independent cultures (three biological replicates) of S. agalactiae NEM316 strain in rich laboratory medium were sampled at mid log, late log early and late stationary growth phase.
Project description:During evolution, each bacterial strain shapes its metabolism in order to colonise a diversity of niches. Unraveling the biochemical reactions underlying bacteria metabolism is important for biotechnological purposes and for understanding relationships within a complex microbiome as well as the microbiome’s connection with its host. Here we propose a new approach to identifying active metabolic pathways, by integrating essentiality analysis and protein abundance. As an example, we used two bacterial species (Mycoplasma pneumoniae and Mycoplasma agalactiae) that share a high gene similarity yet show significant metabolic differences. After integrating all available metabolic knowledge about their enzymes, metabolites and reactions, we built detailed metabolic maps of their carbon metabolism. We determined the carbon sources that allow growth in M. agalactiae (as known for M. pneumoniae) and introduced glucose-dependent growth in M. agalactiae. By analyzing gene essentiality and performing quantitative proteomics, we could predict the active metabolic pathways and directionalities for the sugar, phospholipids, DNA/RNA precursors, glycoproteins, and glycolipids metabolism of these two bacteria. Comparison between predicted and experimentally determined active pathways shows an excellent agreement. Thus, protein essentiality profiling using transposon sequencing analysis combined with quantitative proteomics and metabolic maps could be used to determine and engineer metabolic fluxes.
Project description:In this study, we determined the miRNA expression profile of bovine alveolar macrophages, using next-generation sequencing strategy. On an Illumina HiSeq 2000 machine, we sequenced 8 miRNA libraries, prepared from small RNA fractions of alveolar macrophages isolated from 8 different healthy animals (Bos taurus). From the data, the potential novel miRNAs were predicted, and the expression levels of the known miRNAs were determined. We report that 80 known bovine miRNAs are expressed in bovine alveolar macrophages with >100 reads per million. The most highly expressed miRNA was bta-miR-21, followed by bta-miR-27a. Additionally, one putatively novel bovine miRNA was identified. To our knowledge, this is the first RNA-seq study to profile miRNA expression in bovine alveolar macrophages and provides an important reference dataset for investigating the regulatory roles miRNAs play in this important immune cell type. Examination of bovine alveolar macrophage miRNA profiles, using RNA-seq. Alveolar macrophages were isolated from lung lavages from 8 animals. Small RNA fractions were prepared from the cells using the Qiagen RNeasy Plus mini kit, and miRNA sequencing libraries were prepared using the Epicentre Scriptminer multiplex kit. The sequencing was performed on an Illumina HiSeq 2000 machine.