Project description:Transcriptional profiling to investigate the effect of drug treatment on the E. faecalis cells. For microarray analysis, E. faecalis OG1RF was grown in FMC medium supplemented with 10 mM glucose to an optical density at 600 nm (OD600) of 0.3 and the cultures were divided in 7 aliquots. One aliquot was collected by centrifugation and immediately frozen (untreated control cells). The other aliquots were treated for 30 or 60 min with 1.25 X the minimum inhibitory concentration (MIC) of one of the following CW inhibitors: ampicillin (20 μg ml-1), bacitracin (80 μg ml-1), or cephalothin (80 μg ml-1). After an exposure time of 30 or 60 minutes, each of these cultures was also centrifuged and the pellets frozen. RNA was then isolated from each pellet for microarray analysis. This process was repeated 3 additional times, for a total of four replicates of each condition.
Project description:Transcriptional profiling to investigate the effect of drug treatment on the E. faecalis cells. For microarray analysis, E. faecalis OG1RF was grown in FMC medium supplemented with 10 mM glucose to an optical density at 600 nm (OD600) of 0.3 and the cultures were divided in 7 aliquots. One aliquot was collected by centrifugation and immediately frozen (untreated control cells). The other aliquots were treated for 30 or 60 min with 1.25 X the minimum inhibitory concentration (MIC) of one of the following CW inhibitors: ampicillin (20 μg ml-1), bacitracin (80 μg ml-1), or cephalothin (80 μg ml-1). After an exposure time of 30 or 60 minutes, each of these cultures was also centrifuged and the pellets frozen. RNA was then isolated from each pellet for microarray analysis. This process was repeated 3 additional times, for a total of four replicates of each condition. RNA was extracted from four replicate samples of each condition of interest (control cells grown to OD600 = 0.3 in FMC medium supplemented with 10mM glucose, then treated with ampicillin, bacitracin, or cephalothin for 30 or 60 minutes) and labeled with Cy3. For each replicate, labeled RNA was hybridized to slides along with Cy5-labeled reference RNA, extracted from E. faecalis OG1RF cells grown in BHI medium to mid-log.
Project description:Transcriptional profiling to investigate the effect of drug treatment on the E. faecalis cells. For microarray analysis, E. faecalis OG1RF was grown in FMC medium supplemented with 10 mM glucose to an optical density at 600 nm (OD600) of 0.3 and the cultures were divided in 3 aliquots. One aliquot was collected by centrifugation and immediately frozen (untreated control cells). The other aliquots were treated for 30 or 60 min with 1.25 X the minimum inhibitory concentration (MIC) of vancomycin (10 μg ml-1). After an exposure time of 30 or 60 minutes, each of these cultures was also centrifuged and the pellets frozen. RNA was then isolated from each pellet for microarray analysis. This process was repeated 3 additional times, for a total of four replicates of each condition.
Project description:Transcriptional profiling to investigate the effect of drug treatment on the E. faecalis cells. For microarray analysis, E. faecalis OG1RF was grown in FMC medium supplemented with 10 mM glucose to an optical density at 600 nm (OD600) of 0.3 and the cultures were divided in 3 aliquots. One aliquot was collected by centrifugation and immediately frozen (untreated control cells). The other aliquots were treated for 30 or 60 min with 1.25 X the minimum inhibitory concentration (MIC) of vancomycin (10 M-NM-<g ml-1). After an exposure time of 30 or 60 minutes, each of these cultures was also centrifuged and the pellets frozen. RNA was then isolated from each pellet for microarray analysis. This process was repeated 3 additional times, for a total of four replicates of each condition. RNA was extracted from four replicate samples of each condition of interest (control cells grown to OD600 = 0.3 in FMC medium supplemented with 10mM glucose, then treated with vancomycin for 30 or 60 minutes) and labeled with Cy3. For each replicate, labeled RNA was hybridized to slides along with Cy5-labeled reference RNA, extracted from E. faecalis OG1RF cells grown in BHI medium to mid-log.
Project description:BackgroundEnterococcus faecalis is a dominant pathogen in the root canals of teeth with persistent apical periodontitis (PAP), and osteoblast apoptosis contributes to imbalanced bone remodelling in PAP. Here, we investigated the effect of E. faecalis OG1RF on apoptosis in primary human calvarial osteoblasts. Specifically, the expression of apoptosis-related genes and the role of anti-apoptotic and pro-apoptotic members of the BCL-2 family were examined.MethodsPrimary human calvarial osteoblasts were incubated with E. faecalis OG1RF at multiplicities of infection corresponding to infection time points. Flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, caspase-3/-8/-9 activity assay, polymerase chain reaction (PCR) array, and quantitative real-time PCR were used to assess osteoblast apoptosis.ResultsE. faecalis infection increased the number of early- and late-phase apoptotic cells and TUNEL-positive cells, decreased the mitochondrial membrane potential (ΔΨm), and activated the caspase-3/-8/-9 pathway. Moreover, of all 84 apoptosis-related genes in the PCR array, the expression of 16 genes was upregulated and that of four genes was downregulated in the infected osteoblasts. Notably, the mRNA expression of anti-apoptotic BCL2 was downregulated, whereas that of the pro-apoptotic BCL2L11, HRK, BIK, BMF, NOXA, and BECN1 and anti-apoptotic BCL2A1 was upregulated.ConclusionsE. faecalis OG1RF infection triggered apoptosis in human calvarial osteoblasts, and BCL-2 family members acted as regulators of osteoblast apoptosis. Therefore, BCL-2 family members may act as potential therapeutic targets for persistent apical periodontitis.
Project description:Transcriptional profiling to define the stringent response regulon and significance of basal (p)ppGpp levels in E. faecalis OG1RF. RNA was extracted from four replicate samples of each strain of interest and labeled with Cy3. For each replicate, labeled RNA was hybridized to slides along with Cy5-labeled reference RNA, extracted from E. faecalis OG1RF cells grown to mid-log.
Project description:Liquid cultures of Enterococcus faecalis OG1RF and OG1RF Δbph were grown in tryptic soy broth without added dextrose (TSB-D) for 2 and 4 hr. At each time point, the transcriptomes were compared to identify differentially expressed genes in the Δbph mutant.
Project description:Vancomycin resistance is conferred upon vancomycin-resistant enterococci (VRE) through the replacement of peptidoglycan (PG) stem terminal d-Ala-d-Ala with d-Ala-d-Lac. The d-Ala-d-Lac incorporation can affect both the fitness and virulence of VRE. Here we comprehensively investigate the changes to PG composition in vancomycin-resistant Enterococcus faecalis following the growth in presence of vancomycin using liquid chromatography-mass spectrometry. Using high-resolution mass spectrometry, 104 unique muropeptides fragments were identified and the relative abundance of each fragment was accurately quantified by integrating the ion current of a selected ion using extracted-ion chromatogram. The analysis indicates reduced PG cross-linking, increased carboxypeptidase activities, increased N-deacetylation, and increased O-acetylation in VRE when grown in the presence of vancomycin. We found that O-acetylation preferentially occurred on muropeptides fragments with reduced cross-linking with a pentapeptide stem that terminated in d-Ala-d-Lac. These findings show that O-acetylation preferentially occurred in regions of the cell wall with reduced PG cross-linking on PG units that have stems terminating in d-Ala-d-Lac, serving as markers to prevent both the PG-stem modification by carboxypeptidases and the cell wall degradation by autolysins. Accurate quantitative PG composition analysis provided compositional insights into altered cell wall biosynthesis and modification processes in VRE that contribute to lysozyme resistance and enhanced virulence for VRE grown in the presence of vancomycin.