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dc.contributor.advisorVazquez-Boland, Jose
dc.contributor.advisorScortti, M.
dc.contributor.authorBielecka, Magdalena Kamila
dc.date.accessioned2015-06-03T14:07:16Z
dc.date.available2015-06-03T14:07:16Z
dc.date.issued2011-06-27
dc.identifier.urihttp://hdl.handle.net/1842/10431
dc.description.abstractPathogenic Listeria are the causative agents of listeriosis, a severe food-borne infection. They are able to invade various non-phagocytic cell types including epithelial cells. The life cycle of these intracellular parasites involves penetrating into host cells, rupturing of the phagocytic vacuole, rapidly proliferating in the cytosol, and directly spreading cell to cell. Each step of the listerial intracellular infection involves activation of virulence factors dependent on PrfA, the master regulator of Listeria virulence. PrfAmediated virulence gene activation occurs within host cells by mechanisms that remain unknown. This thesis explores several aspects of PrfA regulation and its impact in the host-pathogen interaction. Methods for assessing PrfA-dependent gene expression were first developed and standardized, including a highly sensitive and accurate quantitative reverse-transcription real-time PCR (RT-QPCR), as well as procedures to investigate the correlation with virulence using cell culture-based assays. These techniques were applied in an investigation into the structure-function of PrfA. We studied the role of a solvent-accessible pocket identified in the N-terminal domain of PrfA, homologous to the cyclic nucleotide-binding (CNB) domain of Crp and other cAMP-regulated proteins, in intracellular virulence gene activation. Site-directed PrfA mutants were constructed. Our data support the notion that PrfA activity is allosterically regulated and are consistent with a role for the pocket as putative binding site for the PrfA-activating allosteric effector. The characterization of spontaneously occurring PrfA mutations that identified in our laboratory as PrfA*- suppressor or attenuator mutations, A129T, E173G and C229Y, allowed us to gain additional insight into PrfA structure-function. The role of the C229Y in sugar-mediated repression was investigated and found to explain the anomalous phenotype of strain NCTC 7973, a prfA* (G145S) mutant that carries this second mutation and is repressed by cellobiose but not glucose. We also carried out experiments to address the intriguing activation of PrfAdependent virulence genes upon addition of an adsorbent to the culture medium, the socalled "charcoal effect". Using a chemically defined culture medium and resin, Amberlite™ XAD-4, we provided evidence that the virulence gene activation may involve the sequestration of a medium component rather than a bacteria-derived autorepressor, as initially thought. We also explored the role of PrfA and the sigma factor σB in L. monocytogenes entry into host cells. ΔsigB mutants in different prfA regulation backgrounds were constructed. We showed that σB has no major effect on host cell invasion, and that L. monocytogenes invasiveness is a strictly PrfA-dependent trait. Our results also demonstrate a differential role of σB in L. monocytogenes serotypes. σB apparently plays no role in stress tolerance in serotype 4b, whereas it is important in serotype 1/2a for maintenance of bacterial fitness in stress conditions. Finally, we investigated the occurrence of apoptosis in Listeria-infected cells and developed normalized methods to accurately determine and quantify this cellular response in infected cell monolayers.en_US
dc.language.isoenen_US
dc.publisherThe University of Edinburghen_US
dc.relation.hasversionGuillet, C., O. Join-Lambert, A. L. Monnier, A. Leclercq, F. Mechai, M. F. Mamzer- Bruneel, M. K. Bielecka, M. Scortti, O. Disson, P. Berche, J. Vazquez- Boland, O. Lortholary & M. Lecuit, (2010) Human listeriosis caused by Listeria ivanovii. Emerging infectious diseases 16: 136-138.en_US
dc.subjectlisteriosisen_US
dc.subjectintracellular parasitesen_US
dc.subjectPrfA regulationen_US
dc.subjecthost-pathogen interactionen_US
dc.titleExploration into the virulence mechanisms of Listeriaen_US
dc.typeThesis or Dissertationen_US
dc.type.qualificationlevelDoctoralen_US
dc.type.qualificationnamePhD Doctor of Philosophyen_US


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