Browsing by Author "Fenselau, Catherine"

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    Extracellular vesicle proteomes reflect developmental phases of Bacillus subtilis
    (Springer Nature, 2016-03-09) Kim, Yeji; Edwards, Nathan; Fenselau, Catherine
    Extracellular vesicles (EV) are spherical membrane-bound vesicles with nano-scale diameters, which are shed to the extracellular region by most eukaryotic and prokaryotic cells. Bacterial EV are proposed to contribute to intercellular communication, bacterial survival and human pathogenesis as a novel secretion system. EV have been characterized from many Gram-negative species and, more recently, from several vegetative Gram-positive bacteria. Further characterization of EV and their molecular cargos will contribute to understanding bacterial physiology and to developing therapeutic approaches. Bacillus subtilis were observed to release EV to a similar extent during sporulation as during the vegetative growth phase. However, the two vesicular cargos show qualitatively and quantitatively different proteomes. Among 193 total proteins identified across both samples, 61 were shown to be significantly more abundant in EV shed by sporulating cells, with (log) ratio of spectral counts RSC > 1 and Fisher-exact test FDR < 5 %. Sixty-two proteins were found to be significantly more abundant in EV shed by vegetative cells. Membrane fusion was shown to take place between these EVs and Gram-positive cells. Biogenesis of EV is a continuous process over the entire life cycle of this sporulating bacterium. The formation of EV during sporulation is strongly supported by the delineation of protein content that differs from the proteome of EV formed by vegetative spores.
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    Extraction of Membrane Components from Neisseria gonorrhoeae Using Catanionic Surfactant Vesicles: A New Approach for the Study of Bacterial Surface Molecules
    (MDPI, 2020-08-20) Stein, Daniel C.; Stocker, Lenea H.; Powell, Abigail E.; Kebede, Salsawi; Watts, David; Williams, Emma; Soto, Nicholas; Dhabaria, Avantika; Fenselau, Catherine; Ganapati, Shweta; DeShong, Philip
    Identification of antigens is important for vaccine production. We tested extraction protocols using cetyltrimethylammonium tosylate (CTAT) and sodium dodecylbenzenesulfonate (SDBS) to formulate surfactant vesicles (SVs) containing components from Neisseria gonorrhoeae. Carbohydrate and protein assays demonstrated that protein and carbohydrates were incorporated into the vesicle leaflet. Depending on the extraction protocol utilized, 100–400 µg of protein/mL of SVs solution was obtained. Gel electrophoresis followed by silver staining demonstrated that SV extracts contained lipooligosaccharide and a subset of bacterial proteins and lipoproteins. Western blotting and mass spectral analysis indicated that the majority of the proteins were derived from the outer membrane. Mass spectrometric and bioinformatics analysis of SVs identified 29 membrane proteins, including porin and opacity-associated protein. Proteins embedded in the SVs leaflet could be degraded by the addition of trypsin or proteinase K. Our data showed that the incorporation of CTAT and SDBS into vesicles eliminated their toxicity as measured by a THP-1 killing assay. Incorporation of gonococcal cell surface components into SVs reduced toxicity as compared to the whole cell extracts, as measured by cytokine induction, while retaining the immunogenicity. This process constitutes a general method for extracting bacterial surface components and identification of antigens that might be included in vaccines.