Effect of the Mbov_0496 mutation on gene expression of Mycoplasma bovis under normal and high potassium conditions
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ABSTRACT: Cyclic di-AMP is an essential and ubiquitous second messenger that regulates bacterial potassium (K+) concentrations to maintain osmotic equilibrium. And its specific K+ regulation mechanism in Mycoplasma has rarely been investigated. We used the ruminant pathogen Mycoplasma bovis (M. bovis) to investigate this mechanism. We verified that MbovP496 is a c-di-AMP synthetase and its mutant T5.415 showed growth inhibition under high K+ condition. To deliver why the mutant strain T5.415 can regulate M. bovis growth, we held a RNA-seq analysis to reveal that the differentially expressed genes between WT and T5.415 under normal condition and high K+ condition. The K+ transport pathways and metabolism related pathways were enriched. In conclusion, we found that the mutation of c-di-AMP synthetase, MbovP496 can broadly influence not only the metal ions transport pathway, but also has an effect on metabolism pathways,which could significantly contribute to understanding the regulation of growth by c-di-AMP synthetase in mycoplasmas.
Project description:The immune response associated with mastitis caused by Mycoplasma bovis is a very complicated biological process in several type of cells, including immune cells, mammary epithelial cells and, endothelial cells. Thus, revealing of the microRNAs in the Mycoplasma bovis infected mammary gland tissues is particularly important for the immune response mechanism to Mycoplasma bovis. Firstly, mammary gland tissue samples were collected from Holstein cows and screened for Mycoplasma bovis. Then, total RNA was isolated from mycoplasma bovis infected tissues and RNA sequencing was performed. After bioinformatics analysis, GO and KEGG analysis of target genes of identified microRNAs were conducted. Our results revaled that 24 of the known microRNAs were expressed differently and 13 of the novel microRNAs were expressed differently in Mycoplasma bovis positive tissues. The target genes of these microRNAs were found to be associated with especially inflammation pathways. In conclusion, this study demonstrated that identified miRNAs may be involved in the signaling pathways during mastitis case caused by Mycoplasma bovis.
Project description:Streptococcus pneumoniae harbors two cyclic di-AMP (c-di-AMP) phosphodiesterases Pde1 and Pde2. Previously, we demonstrated that deletion of one or both of these proteins leads to growth retardation in culture media, defects in the bacterial stress response, and attenuation in mouse models of disease. All of these phenotypes are due to increased levels of c-di-AMP, since the nature and break down products of each protein are different, and mutations that lower c-di-AMP levels partially restore growth and stress tolerance in these mutants. However, how c-di-AMP mediates pneumococcal stress resistance and virulence is unknown. To establish how c-di-AMP affects the transcriptome, RNA-Seq analysis was employed to compare gene expression between wild-type and Δpde1Δpde2 (ST2734) pneumococci. Overall, the competence regulon was upregulated in the Δpde1Δpde2 mutant.
Project description:Mycoplasma species are highly contagious pathogens, and Mycoplasma infectious disease are a serious issue for the dairy industry. The bovine peripheral blood mononuclear cells play an important role for mycoplasma mastitis, however, the effects of M. bovis for immune response of peripheral blood mononuclear cells have not been fully clarified.We examined the transcription profiling of bovine peripheral blood mononuclear cells in intramammary infusion of M. bovis at day 7.
Project description:In this study, we showed for the first time that c-di-AMP is produced by C. difficile and controls the uptake of potassium, making it essential for growth. We found that c-di-AMP is involved in biofilm formation, cell wall homeostasis, osmotolerance as well as detergent and bile salt resistance in C. difficile. We identified BusR as a new regulator that binds c-di-AMP and represses the expression of the compatible solute transporter BusAA-AB. Interestingly, a busR mutant is highly resistant to a hyperosmotic or bile salt stress compared to the parental strain while a busAA mutant is more susceptible. A short exposure of C. difficile cells to bile salts resulted in a decrease of the c-di-AMP concentrations reinforcing the hypothesis that changes in membrane characteristics due to variations of the cellular turgor or membrane damages constitute a signal for the adjustment of the intracellular c-di-AMP concentration. In a colonization mouse model, a strain producing elevated c-di-AMP concentrations failed to persist in the gut in contrast to the parental strain. Thus, c-di-AMP is a signaling molecule with pleiotropic effects that controls osmolyte uptake to confer osmotolerance and bile salt resistance in C. difficile and that is important for colonization of the host.
Project description:C-di-AMP is primarily associated with the regulation of carbon utilization as well as other central traits, central metabolism, and bacterial stringent response to environmental changes. Elevated c-di-AMP levels result in aberrant physiology for most c-di-AMP synthesizing organisms, drawing particular attention to the importance of the c-di-AMP homeostasis and the molecular mechanisms pertaining to nucleotide metabolism and signal transduction. Here we show that c-di-AMP binds the GntR-family regulator DasR, uncovering a direct link between c-di-AMP and GlcNAc signaling. Further, we show c-di-AMP functions as an allosteric activator of DasR activity. GlcNAc is necessary for cell-surface structure from bacteria to humans, as well as a signal for bacterial development and antibiotic production. DasR is a global repressor that oversees GlcNAc metabolism and antibiotic production, which enables Actinobacteria to cope with stress and starvation. Our in vivo studies reveal the important biological role of allosteric regulation by c-di-AMP in metabolic imbalance and the transduction of a series of signals. Notably, DasR also controls intracellular c-di-AMP level through direct repression on disA. Overall, we identify a function of allosteric regulation between c-di-AMP and DasR in global signal integration and c-di-AMP homeostasis in bacteria, which is likely widespread in Actinobacteria.
Project description:Mycoplasma bovis is one of the major causative pathogens of the bovine respiratory complex disease that is characterized by enzootic pneumonia, mastitis, pleuritis and polyarthritis. M. bovis enters and colonizes the bovine respiratory epithelia through inhalation of aerosol from contaminated air. The nature of the interaction between M. bovis and bovine innate immune system is not well understood. We hypothesized that M. bovis invades blood monocytes and regulates cellular function to support its persistence and systemic dissemination. We used bovine-specific peptide kinome arrays to identify cellular signaling pathways that could be relevant to M. bovis-monocyte interaction in vitro. We validated these pathways using functional, protein and gene expression arrays. Here we show that infection of blood monocytes with M. bovis delays spontaneous or TNF-α/staurosporine-driven apoptosis, activates NF-κβ p65 subunit and inhibits caspase-9 activity. We also report that M. bovis infected bovine monocytes do not produce IFN-γ and TNF-α, although production of IL-10 is elevated. Our findings suggest that M. bovis takes over the cellular machinery of bovine monocytes to prolong bacterial survival and to possibly facilitate subsequent systemic distribution.
Project description:Mycoplasma species are highly contagious pathogens, and Mycoplasma infectious disease are a serious issue for the dairy industry. The bovine neutrophils play an important role for the eradication of pathogens which cause mycoplasmal infection, however the effects of M. bovis for immune response of neutrophils have not been fully clarified. We examined the transcription profiling of bovine neutrophils on the stimulation with M. bovis for 3h (3 stimuli, 3 control).
Project description:Nucleotide signaling pathways are found in all kingdoms of life and are utilized to coordinate a rapid response to changes in the environment. One more recently discovered signaling nucleotide is the secondary messenger cyclic diadenosine monophosphate (c-di-AMP), which is widely distributed among bacteria and is also found in several archaea. This cyclic nucleotide has been shown to involve in several important cellular processes, including maintenance of DNA integrity, cell wall metabolism, stress tolerance, transcription regulation and virulence. However, the mechanisms by which c-di-AMP modulates these physiological changes have remained largely unknown.In the present study, we identified and characterized a c-di-AMP synthase (CdaA) in S. mutans UA159. Furthermore, we investigated the role of CdaA in S. mutans cell physiology and global gene expression by utilizing cdaA gene in-frame deletion mutant. Our findings suggest that CdaA is an important global modulator of optimal growth and environmental adaption in this pathogen.
Project description:Mycoplasma species are highly contagious pathogens, and intramammary Mycoplasma infection is a serious issue for the dairy industry. The bovine mammary epithelial cells (bMEC) play an important role for the eradication of pathogens which cause intramammary infection, however the effects of M. bovis for immune response of bMEC have not been fully clarified. We examined the transcription profiling of bMEC on the stimulation with M. bovis for 6h (3 stimuli, 3 control).
Project description:Mycoplasma species are highly contagious pathogens, and Mycoplasma infectious disease are a serious issue for the dairy industry. The bovine peripheral blood mononuclear cells (PBMCs) play an important role for the eradication of pathogens which cause mycoplasmal infection, however the effects of M. bovis for immune response of PBMCs in vitro have not been fully clarified.We examined the transcription profiling of bovine PBMCs on the stimulation with M. bovis for 6h (3 stimuli, 3 control).