Final Report Summary - INFIMAS (The study of Initial limiting steps for fimbrae assembly in E.coli) The control of infectious diseases caused by pathogens bearing adhesive organelles displayed on bacteria is a challenging task towards new alternatives to classical antibiotic treatments. In this regard, the OMS report of 2010 alerted of the raising of antibiotic resistance for the control of human infections. Many of these adhesive organelles, also known as pili or fimbriae, are assembled by specific secretory machinery called the chaperone-usher pathway (CU). In our project we investigated the initial steps required for the proper fimbriae assembly by the CU pathway and the sequences involved in it, using the E.coli Type 1 fimbriae prototype as the research model. Type I fimbriae of E.coli are present on uropathogenic bacteria and are known to mediate the attachment of these bacteria to urinary tract tissues, being responsible for urinary tract infections (UTIs). In humans these types of infections are nowadays one of the most common ones, causing serious health problem with important socio-economic consequences. With this work we have studied one of the initial steps that trigger the polymerization of Type 1 fimbriae and we have identified sequences in the key components involved in this step. Specifically, we have focused our research on the platform protein that catalyzes the polymerization of fimbriae subunits on the surface of the bacteria; this is the outer membrane (OM) protein (“usher”) FimD, and its activator subunit FimH. The starting point of our project was based on the discovery that FimH and the globular “plug” domain of FimD, which is occluding the channel in its non-active, close state, present a common sequence motif. Based on this finding, we hypothesized that the activator subunit provokes the displacement of the “plug” domain of FimD from a common binding site within the channel, which would trigger the molecular rearrangement of FimD usher to be fully active. Performing analysis of multiple alignments of sequences of the “plug” domain of FimD and FimH from Type 1 pili of different non related species we found that this homologous motif is conserved across the ushers and the activator subunits of Type 1 systems. Using both ELISA assays against the major pilus subunit FimA and against FimH, and negative-stain electron microscopy to quantify the levels of pili on the surface of the bacteria expressing single and double point mutations on FimH and FimD, we identified residues in the motif of both proteins that were important for proper pilus polymerization. Using the same FimH and FimD mutants in erythromycin sensitivity assays to detect changes in the permeability of the OM as a consequence of a defect of the activation of FimD usher, we confirmed that several residues in the common motif are required for usher activation. Our in vitro experiments based on in vitro polymerization reconstitution assay and FRET experiments, with purified FimD and FimH variants selected from the in vivo experiments also suggested that the same residues might be involved in the activation of the usher. Our results suggest that the common sequence motif shared by the FimD usher and its activator FimH is a key component for the activation of FimD, and could be the target for the design of inhibitory peptides repertoire that could potentially overcome bacterial resistance to common antibiotics and recurrence of UTIs in the future as a treatment of infectious diseases in EU and other countries.