Project description:The obligate intracellular bacterium Chlamydia alternates between two functional forms during its developmental cycle: elementary body (EB) and reticulate body (RB). However, the molecular mechanisms governing the transitions between these forms are unknown. Here, we present evidence cyclic di-AMP (c-di-AMP) is a key factor in triggering the transition from RB to EB (i.e., secondary differentiation) in the chlamydial developmental cycle. We made strains producing different levels of c-di-AMP, which we linked to changes in secondary differentiation status. Increases in c-di-AMP resulted in an earlier increase in transcription of EB-associated genes, and this was further manifested in earlier production of EBs. In contrast, when c-di-AMP levels were decreased, secondary differentiation was delayed. Based on these data, we conclude there is a threshold level of c-di-AMP needed to trigger secondary differentiation in Chlamydia. This is the first study to define a mechanism of secondary differentiation in Chlamydia.
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:Background: Recessive Dystrophic Epidermolysis Bullosa (RDEB) is characterized by wounds chronically colonized with Staphylococcus aureus associated with local and systemic inflammation. This study aimed to clarify how the immune response is shaped by S. aureus in RDEB patients with focus on Mucosal-Associated invariant T (MAIT) cells. Methods: Fifteen RDEB children (moderate phenotype n=5; severe phenotype n=10) and controls (n=18) were enrolled. A novel proteomic pipeline was used to evaluate over 800 proteins in plasma. Patient-specific cytokine, immune T cell and S. aureus signatures were evaluated. The effect of the patient's S. aureus strain on T-cell function was analyzed in vitro and correlated with the bacterial secretome. Findings: We detected a unique plasma proteomic signature that enabled the differentiation of RDEB children and controls. Profiling of immune cells (n= 30 subsets and cytokine-producing cells) and cytokines (n=38) identified a severe inflammatory response and activation of CD4+ T and MAIT cells in severe RDEB patients with increased frequency of IL-17A-producing CD4+ T and MAIT cells. Positive S. aureus cultures from the skin of 12 of the 15 RDEB patients allowed whole-genome sequencing of patient strains, assessment of the primary keratinocyte response to bacterial challenge, and identification of potential immunomodulatory bacterial determinants. Conditioned media from keratinocytes challenged with S. aureus strains of severe RDEB patients induced a strong Th17 response. Interpretation: This study uncovers a huge systemic inflammatory response associated with a high level of circulating IL-17A-producing CD4+ T and MAIT cells in RDEB patients and supports the involvement of patient S. aureus strains in biasing the host immune response.
Project description:Deep-sequencing of the engineered production genes in five E coli production chassis strains (BL21(DE3), MG1655, TOP10, W and W3110) producing two case metabolic products, 2,3-butanediol and mevalonic acid
Project description:The increasing resistence and/or bacterial tolerance to bactericides, such as chlorhexidine, causes worrisome public health problems. Using transcriptomical and microbiological studies, we analysed the molecular mechanisms associated with the adaptation to chlorhexidine in two carbapenemase-producing strains of Klebsiella pneumoniae belonging ST258-KPC3 and ST846-OXA48.
Project description:High throughput sequencing was used to investigate the production of small RNAs from in Chlamydomonas in different strains and different stages of the life cycle. The association between these and methylation was assessed using genomic sequencing, comparing a sequenced genome with one enriched for methylated DNA sequence by immuno-precipitation. Examination of small RNA production in several strains and of the methylation of regions producing these small RNAs.
Project description:Recessive Dystrophic Epidermolysis bullosa (RDEB) is characterized by wounds colonized with Staphylococcus aureus associated with local and systemic inflammation. However, it remains unclear how the immune response and S. aureus interact. A novel proteomic pipeline was used to evaluate over 800 proteins in plasma, resulting in a unique proteomic signature that enabled the differentiation of RDEB patients (n=15); mild phenotype n=5; severe phenotype n=10; median 7 years) and controls (n=18; median 5 years). Immune cell (n= 30 subsets and cytokine-producing cells) and cytokine (n=38) profiling identified a severe inflammatory response and activation of conventional CD4+ T and innate-like T cells as Mucosal-Associated invariant T (MAIT) and gamma delta T cells in RDEB severe patients with increased frequency of IL-17A-producing CD4+ T and MAIT cells. Positive S. aureus cultures from skin of 12 out of 15 RDEB patients allowed whole-genome sequencing of patient's strains and assessment of primary keratinocyte immune response upon bacterial challenge. Conditioned media from keratinocytes challenged with S. aureus strains of severe RDEB patients induced a strong Th17 response. In conclusion, this study uncovers a huge systemic inflammatory response associated with a high level of circulating IL-17A-producing CD4+ T and MAIT cells in RDEB patients and supports the involvement of patient S. aureus strains in biasing the host immune response.
Project description:To investigate gene expression differences of different tylosin high-producing strains, transcriptomes of three tylosin high-producing engineered strains (TLPH08-2, TLPH11 and TLPH17) and the vector control strain TLSET152 were analyzed by RNA-Seq. Different strains (TLSET152, TLPH08-2, TLPH11 and TLPH17) were harvested at 96 h of fermentationat and then RNA isolation, transcriptome sequencing and data analysis were conducted.
Project description:Responses of Escherichia coli DH5alpha as they overexpress pUC at different ODs in LB + Amp Escherichia coli DH5alpha expressing pUC sampled at different ODs (0.2, 0.5, 0.9) in LB + Amp vs cells not producing pUC