The main causative agent of human urinary tract infections is the uropathogenic Escherichia coli (UPEC) pathotype. It may cause disease due to its ability to express a number of bacterial virulence factors. Fimbrial adhesins are particularly important for the initial establishment of infection in the urinary tract. The fimbriae are hair-like structures protruding from the bacterial cell and by attaching to specific receptors in the urinary tract they mediate adherence to different cell types, allowing the bacteria to resist the shear forces from urine flow. The UPEC strains generally carry multiple determinants for fimbrial adhesins. Previous studies have indicated that there is a co-regulation between different fimbrial genes and one factor that has been implicated in this is the PapB protein, acting as a transcriptional regulator of P-fimbrial expression. The PapB protein can be regarded as the prototype of a family of fimbrial regulators that show high homology between different fimbrial operons. One homolog is FocB, regulator of F1C fimbriae. In this study, the role of the FocB protein in the regulation of F1C fimbriae as well as in the co-regulation with other fimbrial genes was investigated. It was observed that FocB binds to DNA, similarly to PapB, in an oligomeric fashion and that PapB and FocB can form hetero-oligomeric complexes, which appear to have a repressive role in the regulation of the F1C fimbriae. In addition, the FocB protein also had a repressive effect on transcription of the fim operon, which encodes theType 1 fimbriae. For further analysis of FocB in vitro, we developed efficient procedures for purification of the protein and established conditions for its crystal formation with the aim to conduct X-ray diffraction studies. By the hanging-drop vapour-diffusion method, we obtained crystals that in the X-ray analysis diffracted sufficiently well to allow modelling of a high resolution structure of FocB. The structural model was considered in relation to the DNA binding properties of the protein. The FocB analysis represents the first structural model of this family of transcriptional factors. This model should aid in further understanding of the roles and functions of these proteins in the regulation of the UPEC fimbrial operons. The complexity of the system, with multiple factors involved in the regulation of fimbrial operons, was revealed in earlier studies of the PapI protein showing that PapI activates transcription of the pap operon as a part of a complex with the global regulator Lrp. However, PapI itself did not appear to bind to DNA and its mode of action has remained unclear. By genetic analyses and in vitro studies we show that PapI may interact also with the α subunit of the RNA polymerase. This finding indicates that PapI might directly interact with the transcriptional apparatus and thus aid in the activation of pap expression. Bacteria are frequently releasing outer membrane vesicles (OMVs) from their surface. We studied the release of the haemolysin toxin from E. coli in connection with formation of OMVs and found that the toxin was tightly associated with the vesicles in an active form. By overproduction of the PapB or PapI regulators in order to maximise the population of bacteria expressing fimbriae, we could detect P fimbriae proteins associated with OMVs that displayed specific adhesion to receptor-coated beads. This suggests a possible scenario in which the vesicles canfunction as directed vehicles of bacterial virulence factors.

Human Papilloma Viruses (HPVs) are a large family of viruses with a capsid constituted by the L1 and L2 proteins, which bind to receptors of the basal epithelial cells, thus promoting virus entry. The majority of sexually active people become exposed to HPV, which is the most common cause of cervical cancer affecting more than 600.000 women every year. Moreover, every year more than 13.000 new cases of HPV-related cancers, including anal, penile and head and neck cancers, are diagnosed in men. Three vaccines are available based on the L1 capsid protein, which self-assembles and forms virus-like particles (VLPs) when expressed in yeast and insect cells. Although very effective, these vaccines are HPV type-restricted, and their costs limit broad vaccination campaigns, especially in low- and middle- income countries. Recently, vaccine candidates based on the conserved L2 epitope from serotypes 16, 18, 31, 33, 35, 6, 51 and 59 were shown to elicit broadly neutralizing anti-HPV antibodies, reaching a protection around 90% against all the HPV serotypes. During my research activity, we have tested whether E. coli Outer Membrane Vesicles (OMVs) could be successfully decorated with L2 polytopes and whether the engineered OMVs could induce neutralizing antibodies. OMVs represent an attractive vaccine platform for their intrinsic adjuvanticity and their low production costs. We show that strings of L2 epitopes could be efficiently expressed on the surface of the OMVs and a polypeptide constituted by the L2 epitopes from serotypes 18, 33, 35 and 59 provided broad cross-protective activity against a large panel of HPV serotypes as judged by the in vitro pseudovirus neutralization assay. In order to better characterize the vesicle and in perspective of future clinical studies of our HPV candidate vaccine, we also worked on the setting-up of a simple and reproducible production process at laboratory scale ready to be transferred at industrial level.Moreover, we focused our attention on the strategy used for the engineering of the OMVs with the L2 epitopes and in particular on the carrier used for the delivery of the fusion construct in the surface of the vesicle. More in detail, since part of the carrier is a human cancer epitope, we tested whether a similar scaffold, with less homologies to the human gene could maintain the same properties in terms of: i) expression level of the fused epitopes in the OMVs, ii) localization on the surface of the vesicle and iii) 9 immunogenicity and efficiency to stimulate the immune system in order to produce anti L2 antibodies. Considering all the results described in this work combined with the points of strength of the OMV-based vaccine platform, as the simplicity of the production process, the yields of vaccine doses and the low cost/dose, our data provide a very promising prototype of universal anti-HPV vaccine.

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