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Characterization of Outer Membrane Vesicles and Nanotubes in Francisella

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dc.contributor.advisor Thanassi, David G en_US
dc.contributor.advisor van der Velden, Adrianus en_US
dc.contributor.author McCaig, William Daniel en_US
dc.contributor.other Department of Genetics en_US
dc.date.accessioned 2017-09-20T16:53:05Z
dc.date.available 2017-09-20T16:53:05Z
dc.date.issued 2012-12-01 en_US
dc.identifier.uri http://hdl.handle.net/11401/77630 en_US
dc.description 185 pgs en_US
dc.description.abstract <italic>Francisella</italic> spp. are highly infectious and virulent bacteria that cause the zoonotic disease tularemia. The identification of virulence factors and mechanisms of virulence factor secretion by <italic>Francisella</italic> spp. are not well understood. Gram-negative bacteria constitutively release vesicles from their cell surface, and these outer membrane vesicles (OMV) may function in the delivery of virulence factors to host cells. In addition, prokaryotic and eukaryotic cells have been shown to produce membrane-enclosed projections, termed nanotubes (NT), which appear to function in cell-cell communication and exchange of molecules. Examination of <italic>Francisella</italic> bacteria revealed the presence of NT extending out from the bacterial surface, and purification of OMV resulted in a heterogeneous mixture of OMV and NT. Proteomic analysis of gradient-purified OMV and NT identified 292 protein constituents, including known <italic>Francisella</italic> secreted proteins and virulence factors. <italic>Francisella</italic> produced the OMV and NT in a regulated manner. In contrast to previously characterized NT, the <italic>F. novicida</italic> NT were produced by bacteria grown in liquid as well as on solid medium, and were derived from the outer membrane rather than the cytoplasmic membrane. An increase in the number of OMV and NT was observed when bacteria are grown in brain heart infusion (BHI) medium, a growth condition which has been shown to more closely resemble infection of host cells. In addition, infection of host cells stimulated the production of NT by <italic>F. novicida</italic>. The OMV/NT are effective at shielding cargo proteins from extracellular proteases and the NT structure is resistant to numerous forms of chemical disruption. NT appear to be sensitive to treatment with high levels of heat, as evidenced by disruption of these structures when so treated. The effects of purified OMV/NT on host cells were examined and their use as a potential subunit vaccine explored. Purified OMV/NT incubated with primary murine macrophages show a minor cytotoxic effect at high doses over long periods of time. Interestingly, at earlier time points and lower doses, proinflammatory cytokines are released when purified OMV/NT are incubated with macrophages. The OMV/NT must be intact for the majority of this cytokine response, as OMV/NT disrupted by heat treatment showed a marked reduction in levels of cytokines released by host cells. Mice vaccinated intranasally with purified OMV/NT and subsequently challenged with high doses of wild-type <italic>F. novicida</italic> were delayed in time to death or survived the challenge entirely. This work shows that <italic>Francisella</italic> produces OMV and NT in a regulated manner and reveals a novel class of bacterial NT. The presence of known virulence factors and effects of the vesicles on host cells suggests roles for the OMV and NT in the pathogenesis of tularemia and opens up the possibility for generation of an effective component-based vaccine. en_US
dc.description.sponsorship This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree. en_US
dc.format Monograph en_US
dc.format.medium Electronic Resource en_US
dc.language.iso en_US en_US
dc.publisher The Graduate School, Stony Brook University: Stony Brook, NY. en_US
dc.subject.lcsh Microbiology en_US
dc.subject.other Francisella, nanotubes, OMV, outer membrane vesicles en_US
dc.title Characterization of Outer Membrane Vesicles and Nanotubes in Francisella en_US
dc.type Dissertation en_US
dc.mimetype Application/PDF en_US
dc.contributor.committeemember Furie, Martha en_US
dc.contributor.committeemember London, Erwin en_US
dc.contributor.committeemember Ratner, Adam en_US

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