Project description:We performed comparative transcriptomic analysis of the outer membrane vesicles (OMVs) released from B. burgdorferi. We identified a total of ~1200 unique transcripts with at least one mapped read from the bacterial cell and its OMVs.
Project description:The antibiotic fosfomycin is widely recognized for treatment of lower urinary tract infections caused by Escherichia coli and lately gained importance as a therapeutic option to combat multidrug resistant bacteria. Still, resistance to fosfomycin frequently develops through mutations reducing its uptake. Whereas the inner membrane transport of fosfomycin has been extensively studied in E. coli, its outer membrane (OM) transport remains insufficiently understood. While evaluating minimal inhibitory concentrations in OM porin-deficient mutants, we observed that the E. coli ΔompCΔompF strain is five times more resistant to fosfomycin than the wild type and the respective single mutants. Continuous monitoring of cell lysis of porin-deficient strains in response to fosfomycin additionally indicated the relevance of LamB. Furthermore, the physiological relevance of OmpF, OmpC and LamB for fosfomycin uptake was confirmed by electrophysiological and transcriptional analysis. This study expands the knowledge of how fosfomycin crosses the OM of E. coli.
Project description:Large-scale production of bacterial extracellular vesicles is a hurdle for their development as novel cancer immunotherapeutic agents. Here, we developed manufacturing processes for mass production of Escherichia coli EVs known as outer membrane vesicles (OMVs). We present a comprehensive proteome of mass-produced E. coli OMVs.
Project description:Elexacaftor/tezacaftor/ivacaftor (ETI, Trikafta) is highly effective treatment for many cystic fibrosis patients, at least partly because it increases CFTR mediated Cl- and HCO3- secretion by airway epithelial cells leading to improved lung function and less frequent exacerbations and hospitalizations. However, little is known about how ETI affects airway epithelial cells in ways not related to CFTR mediated Cl- and HCO3- secretion, for example how ETI affects the expression of genes other than CFTR or how ETI might affect airway cells’ response to infection. It is established that CF airway cells bearing the delta F 508 mutation the CFTR gene respond characteristically differently from wild type CFTR cells, and we hypothesized that, as a highly effective CFTR modulator, ETI might make airway cells from CF donors respond to pathogen stimulation (Pseudomonas aeruginosa PA14 or outer membrane vesicles isolated from these bacteria) in more the same way that cells from wild type, healthy control cells do. We tested this hypothesis by measuring gene expression responses in polarized primary CF airway cells exposed to ETI alone or ETI in the presence of a pathogen challenge (PA14 or outer membrane vesicles). Responses of CFTR wild type primary CF airway cells to PA14 or outer membrane vesicles was also measured for comparison
Project description:Asthma is the most common chronic respiratory disease. Asthma that cannot be well controlled by steroid treatment is called steroid-resistant asthma. Steroid-resistant asthma accounts for only 5% of all asthma cases, but it accounts for 80% of asthma healthcare costs. Nontypeable Haemophilus influenzae (NTHi), as a Gram-negative bacterium, can release outer membrane vesicles (OMVs) and transfer biomolecules to host cells and the external environment by carrying lipopolysaccharides, proteins, peptidoglycans, outer membrane proteins, cell wall components, proteins, nucleic acids, ion metabolites, and signaling molecules. Thus, it plays a role in obtaining nutrition, stress, toxin delivery, adhesion, host immune surveillance evasion, and host immune response regulation. It becomes an essential way in bacterial pathogenesis. To further clarify whether NTHi OMVs could be inhaled to induce steroid-resistant asthma, we isolated and purified NTHi OMVs. In vivo experiments showed that NTHi OMVs could be inhaled and enter airway epithelial cells. Cosensitization with OVA induces steroid-resistant asthma in mice. Furthermore, through high-throughput sequencing, we found that the NTHi OMVs and OVA co-sensitized mice had significantly enriched inflammatory and immune-related signaling pathways, and the transcription and secretion of IL-1β were increased was the potential cause of SRA.
Project description:OMVs are nanoscale spherical bilayer-membrane vesicles naturally secreted during the growth of E. coli that retain outer membrane and periplasmic components similar to those of the source bacteria. The FimH-positive E. coli can enter macrophages through caveolin-mediated endocytosis, which enables them to bypass lysosome processing and to maintain a stable nanostructure. We performed proteomic analyses on the OMVs.
Project description:The rise of antimicrobial resistant pathogens calls for new antibacterial treatments, but potent new compounds are scarce. Development of new antibiotics is difficult, especially against Gram-negative bacteria, as here uptake is strongly hindered by the additional outer membrane. Most antimicrobial agents against Gram-negatives use the porin mediated pathway to cross the outer membrane, which limits the choice of an antibiotic, as it has to fit by size, charge and hydrophilicity. In E. coli, the major porins OmpF and OmpC are associated with antibiotic translocation and therefore also with unspecific antibiotic cross-resistance. In this regard, alternative uptake routes are of interest. We were interested in the uptake opportunities of the small, natural product antibiotic negamycin and thereby found new uptake pathways across the outer membrane of E. coli. Besides OmpF and OmpC, we investigated the role of the minor porins OmpN and ChiP in negamycin translocation. We detected an effect of OmpN and ChiP on negamycin susceptibility and confirmed passage by electrophysiological assays. The structure of OmpN was resolved in order to analyze the negamycin translocation mechanism by computational simulations. As abundancy of these minor porins was low in E. coli, their transcript levels were analyzed by RNA-Seq. Increased transcripts levels of ompN and chiP were observed upon negamycin treatment, hinting at a role in antibiotic uptake. These new, additional uptake pathways across the outer membrane of E. coli highlight the antibiotic potential of negamycin, especially as resistance development is low due to availability of multiple uptake routes at both the outer and inner membranes
Project description:This project compares the outer membrane proteome to the proteome of outer membrane vesicles of the bacterium Shewanella onedensis. S. oneidensis outer membrane was purified via the sarkosyl method described in (Brown, 2010). A 50 mL overnight culture of cells were harvested by centrifugation at 10,000× g for 10 min. cell pellet suspended in 20 mL of 20 mM ice-cold sodium phosphate (pH 7.5) and passed four times through a French Press (12000 lb/in2). The lysate was centrifuged at 5,000× g for 30 min to remove unbroken cells. The remaining supernatant was centrifuged at 45,000 × g for 1 h to pellet membranes. Crude membranes were suspended in 20 mL 0.5% Sarkosyl in 20 mM sodium phosphate and shaken horizontally at 200 rpm for 30 min at room temperature. The crude membrane sample was centrifuged at 45,000 × g for 1 h to pellet the OM. The pellet of OM was washed in ice-cold sodium phosphate and recentrifuged. The cells were pelleted by centrifugation at 5000 x g for 20 min at 4°C, and the supernatant was filtered through 0.45 μm pore size filters to remove remaining bacterial cells. Vesicles were obtained by centrifugation at 38,400 x g for 1 h at 4°C in an Avanti J-20XP centrifuge (Beckman Coulter, Inc). Pelleted vesicles were resuspended in 25 ml of 50 mM HEPES (pH 6.8) and filtered through 0.45 μm pore size filters. Vesicles were again pelleted as described above and finally resuspended in 50 mM HEPES, pH 6.8. Extracellular DNA, flagella, and pili can all be co-purified. Protocol was adapted from (Perez-Cruz, 2013).
Project description:Helicobacter pylori, a bacterium that colonizes the human stomach, like all Gram-negative bacteria spontaneously shed outer membrane vesicles (OMVs). OMVs, which act as a delivery system for bacterial components, are involved in bacterial-host interactions and thus contribute to pathogenesis. In this study, to understand the gene expression changes that human gastric epithelial cells might undergo when exposed to H. pylori-OMVs, we profiled the transcriptomic changes of the MKN74 gastric cell line induced by OMVs compared to control cells and H. pylori-infected cells, using the Ion AmpliSeq™ Transcriptome Human Gene Expression Panel. The top enriched pathways in the OMVs challenge condition included amino acid-related metabolic pathways, mitogen-activated protein kinase signaling, autophagy, and ferroptosis. The cell cycle, DNA replication, and repair pathways were the top diminished pathways. The transcriptomic changes induced by OMVs were largely consistent with those of the bacteria, although often at low expression levels, suggesting that their effects will mostly reinforce those of the bacterium itself. Our data provide a valuable portrayal of the transcriptomic remodeling of gastric cells by H. pylori-OMVs, which can be further dissected regarding the underlying molecular mediators and explored to understand the pathobiology of the full-spectrum of H. pylori-mediated diseases.