Project description:The bacterial cell envelope is the frontier line of the adaptational defense that protects cells against environmental and intrinsic stressors to maintain homeostasis. Deciphering the molecular pathways involved in envelope biology is of high importance for both fundamental sciences and translational applications. Peptidoglycan (PG) is one of the main structural components of bacterial cell envelopes. It protects bacteria against mechanical and osmotic stress, confers cell shape and physical integrity. In a previous study, we identified that the Escherichia coli ElyC protein containing a highly conserved domain of an unknown function is required for envelope integrity at low temperature. ElyC was shown to be essential for PG assembly at room temperature (21°C) and to be involved in the metabolism of the lipid carrier necessary for the membrane step of the biosynthesis pathways for PG and other bacterial envelope polysaccharides. PG assembly was completely blocked in ElyC-defective cells grown at 21°C, leading to a terminal phenotype of cell lysis. The physiological importance of ElyC in bacterial cells grown at the physiological temperature of 37°C had not yet been investigated. To study this, we performed phenotypic characterization and transcriptomic profiling of ElyC-defective E. coli cells were grown at 37°C and 21°C, compared to wild-type cells. Even if ∆elyC mutant cells grow as well as wild-type cells at 37°C, microscopic analysis revealed that cellular morphology is altered by the lack of ElyC at this temperature. PG quantification and analysis confirmed that PG biosynthesis is significantly inhibited in the absence of ElyC at 37°C. We further have shown that ∆elyC mutant cells are more sensitive to PG-targeting β-lactam antibiotics than wild-type cells at 37°C. The RNA-Seq transcriptomic study showed that elimination of the ElyC evokes a highly defective cellular envelope when elyC mutant grows at 21°C, whereas at 37°C, the absence of ElyC leads to a moderate cellular response. While the PG defect of cells inactivated for ElyC is lethal at 21°C, ElyC-defective cells maintain enough PG integrity to grow and survive at 37°C. However, those cells are morphologically defective, are coping with envelope stress and are more sensitive to PG-targeting antibiotics. Overall, our data support a crucial role for ElyC in the metabolic flux for the PG biosynthesis pathway at optimal and suboptimal temperatures.

Project description:The main constituents of electronic cigarette liquids are nicotine, propylene glycol (PG), and vegetable glycerin (VG), together with distilled water and flavors. To assess the toxicity of PG/VG mixtures with and without nicotine as basic components of liquids used in e-cigarettes, a 90-day rat inhalation study according to the Organization for Economic Co-operation and Development test guideline 413 was conducted. Sprague-Dawley (SD) rats were nose-only exposed, 6 h/day, 5 days/week to filtered air, or nebulized vehicle (saline), or three concentrations of PG/VG (0.174 mg PG/l + 0.210 mg VG/l; 0.520 mg PG/l + 0.630 mg VG/l; 1.520 mg PG/l + 1.890 mg VG/l) – with (test item) and without (reference item) 23 µg nicotine/L. Standard toxicological endpoints were complemented by molecular analyses using transcriptomics, proteomics, and lipidomics. Compared to vehicle exposure, the tested PG/VG aerosols showed only very limited biological effects with no signs of toxicity, both for the standard toxicological endpoints (e.g., histopathology, clinical chemistry) and the systems toxicological analyses (transcriptomics, proteomics, and lipidomics). The addition of nicotine to the PG/VG aerosols (23 µg/l) resulted in effects in line with nicotine effects in previous studies. These included up-regulation of xenobiotic enzymes (Cyp1a1 and Fmo3) in the lung and metabolic effects, e.g., reduction in serum lipid concentrations and changes in the expression of metabolic enzymes in the liver. Signs of a generalized stress response to nicotine exposure such as decreased thymus weights were observed; and likely, a subset of the observed metabolic alterations was interlinked with this generalized stress response. Under the conditions of this 90-day SD rat inhalation study, no toxicologically relevant effects of PG/VG aerosols (up to 1.520 mg PG/l + 1.890 mg VG/l) were observed, and the no observed adverse effect level (NOAEL) for PG/VG/nicotine was determined to be 438/544/6.7 mg/kg/day. Further the study demonstrated how complementary systems toxicology analyses can reveal, also in the absence of observable adverse effects, subtoxic and adaptive responses to pharmacologically active compounds such as nicotine.

Project description:The main constituents of electronic cigarette liquids are nicotine, propylene glycol (PG), and vegetable glycerin (VG), together with distilled water and flavors. To assess the toxicity of PG/VG mixtures with and without nicotine as basic components of liquids used in e-cigarettes, a 90-day rat inhalation study according to the Organization for Economic Co-operation and Development test guideline 413 was conducted. Sprague-Dawley (SD) rats were nose-only exposed, 6â€‰h/day, 5 days/week to filtered air, or nebulized vehicle (saline), or three concentrations of PG/VG (0.174 mg PG/l + 0.210 mg VG/l; 0.520 mg PG/l + 0.630 mg VG/l; 1.520 mg PG/l + 1.890 mg VG/l) with (test item) and without (reference item) 23 µg nicotine/L. Standard toxicological endpoints were complemented by molecular analyses using transcriptomics, proteomics, and lipidomics. Compared to vehicle exposure, the tested PG/VG aerosols showed only very limited biological effects with no signs of toxicity, both for the standard toxicological endpoints (e.g., histopathology, clinical chemistry) and the systems toxicological analyses (transcriptomics, proteomics, and lipidomics). The addition of nicotine to the PG/VG aerosols (23 Âµg/l) resulted in effects in line with nicotine effects in previous studies. These included up-regulation of xenobiotic enzymes (Cyp1a1 and Fmo3) in the lung and metabolic effects, e.g., reduction in serum lipid concentrations and changes in the expression of metabolic enzymes in the liver. Signs of a generalized stress response to nicotine exposure such as decreased thymus weights were observed; and likely, a subset of the observed metabolic alterations was interlinked with this generalized stress response. Under the conditions of this 90-day SD rat inhalation study, no toxicologically relevant effects of PG/VG aerosols (up to 1.520 mg PG/l + 1.890 mg VG/l) were observed, and the no observed adverse effect level (NOAEL) for PG/VG/nicotine was determined to be 438/544/6.7 mg/kg/day. Further the study demonstrated how complementary systems toxicology analyses can reveal, also in the absence of observable adverse effects, subtoxic and adaptive responses to pharmacologically active compounds such as nicotine.

Project description:Veillonella parvula is a biofilm-forming commensal found in the lungs, vagina, mouth, and gastro-intestinal tract of humans, yet it may also develop into an opportunistic pathogen. Furthermore the presence of Veillonella has been associated with the development of a healthy immune system in infants. Veillonella belongs to the Negativicutes, a diverse clade of bacteria that represent an evolutionary enigma: they are phylogenetically placed within Gram-positive (monoderm) Firmicutes yet maintain an outer membrane (OM) with lipopolysaccharide similar to classic Gram-negative (diderm) bacteria. The OMs of Negativicutes have unique characteristics, such as the replacement of Braun’s lipoprotein by OmpM for anchoring the outer membrane to the peptidoglycan. Through phylogenomic analysis, we have recently provided the first bioinformatic annotation of the Negativicute diderm cell envelope. We showed that it is a unique type of envelope that was present in the ancestor of present-day Firmicutes and lost multiple times independently in this phylum, giving rise to the monoderm architecture. However, little experimental data is presently available for any Negativicute cell envelope. Here, we have performed the first experimental proteomic characterization of the cell envelope of these atypical diderm Firmicutes, producing an OM proteome of Veillonella parvula. We initially conducted a thorough bioinformatics analysis of all 1844 predicted proteins from Veillonella parvula DSM 2008’s genome using seven different localization prediction programs. These results were then complemented by protein extraction with surface exposed protein tags and subcellular fractionation, which were then sequenced by liquid chromatography tandem mass spectrometry. The merging of proteomics and bioinformatics results allowed identification of 76 OM proteins. Their annotation markedly extends previous inferences on the nature of the cell envelope of Negativicutes, and provides important information on the role of OM systems in the lifestyle of Veillonella.

Project description:Two-component systems (TCS) are often used by bacteria to rapidly assess and respond to environmental changes. ChvG/ChvI (ChvGI) is a TCS conserved in γ-proteobacteria and is known for regulating expression of genes related to exopolysaccharide production, virulence and growth. The sensor kinase ChvG autophosphorylates upon yet unknown signals and phosphorylates the response regulator ChvI to activate transcription. Recent studies in Caulobacter crescentus showed that chv mutants are sensitive to vancomycin treatment and fail to grow in synthetic minimal media. In this work, we identified the osmotic imbalance as the main cause of growth impairment in synthetic minimal media. We also determined the ChvI regulon and confirmed that ChvI regulates cell envelope architecture at different levels by controlling outer membrane, peptidoglycan assembly/recycling and inner membrane proteins. Furthermore, we identified genes with osmoregulatory properties and confirmed that osmotic upshift is a signal triggering ChvG-dependent phosphorylation of ChvI. In addition, we challenged chv mutants with other cell envelope related stress and found that targeting with antibiotics the transpeptidation of peptidoglycan during cell elongation impairs growth of the mutant. Moreover, these antibiotics activate expression of the chvIG-hprK operon in ChvI-dependent and independent ways. ChvI phosphorylation is also shown to be activated upon antibiotic treatment with vancomycin. Finally, we observed that the sensor kinase ChvG fused to a fluorescent protein relocates from a patchy-spotty distribution to distinctive foci after transition from complex to synthetic minimal media. Interestingly, this pattern of (re)location has been described for proteins involved in cell growth control and peptidoglycan synthesis upon osmotic shock. Overall, our data support that the ChvGI TCS is mainly used to monitor and respond to osmotic imbalances and damages in the peptidoglycan layer.

Project description:Periodontitis affects 47.1% of adult population in the U.S. Porphyromonas gingivalis is an opportunistic oral pathogen that colonizes the oral mucosa, invades myeloid dendritic cells and accesses the bloodstream, brain, placenta and other organs in human with periodontitis. Periodontitis also sustains a chronic long-term pro-inflammatory immune disorder, potentially contributing to other systemic conditions such as cardiovascular disease, type 2 diabetes mellitus, adverse pregnancy outcomes, and osteoporosis. However, the role of P. gingivalis minor and major fimbriae in DC-SIGN-TLR2 crosstalk during traverses from oral mucosa to these distant sites and its influence on survival of P. gingivalis within DCs and its immune-mechanism involve at molecular/transcriptome level has not been examined. In this study to address the role of fimbriae we utilized defined bacterial mutants that solely express minor fimbriae (Mfa1+Pg), major fimbriae (FimA+Pg) or are deficient in both fimbriae (MFB) and compared with un-infected control. P. gingivalis strains were maintained anaerobically (10% H2, 10% CO2, and 80% N2) in a Forma Scientific anaerobic system glove box model 1025/1029 at 37Â°C in Difco anaerobe broth MIC. Mutant strains were maintained using erythromycin (5 Î¼g/ml) for mutant Mfa1+Pg, tetracycline (2 Î¼g/ml) for mutant FimA+Pg and both erythromycin and tetracycline for double fimbriae mutant MFB. Bacterial suspensions were washed five times in PBS and re-suspended for spectrophotometer reading at OD 660 nm of 0.11, which previously determined to be equal to 5 x 107 CFU. For bacterial CFSE staining, the suspension were washed (3 times) and re-suspended in 5 Î¼M of CFSE in PBS. The bacteria were incubated for 30 min at 37Â°C in the dark. MoDCs were pulsed with Pg381, Mfa1+Pg, FimA+Pg and MFB at 10 MOI and incubated with the MoDCs for 12 hours and each experimental condition was performed in triplicate. To facilitate our understanding on host immunity and defense mechanism of this pathogen, here we used the Illumina High-throughput RNA-seq transcriptome profiling to investigate the myeloid dendritic cells response to oral Amphibiont (1. Pg381, 2. Mfa1+Pg, 3. FimA+Pg, 4. MFB and 5. Un-infected control group).

			Action	DRS
	11315.mgf	Mgf
	11315.pride.mgf.gz	Mgf
	11708.mgf	Mgf
	11708.pride.mgf.gz	Mgf
	12294.mgf	Mgf

Dataset Information

Study of Brucella abortus / Agrobacterium tumefaciens outer membrane proteins covalently bound to peptidoglycan

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