ABSTRACT: Antimicrobial resistance (AMR) represents a global public health crisis, with an estimated 10 million deaths per year expected by 2050 without significant public health intervention and the development of new classes of antibiotics. Towards combating this crisis, the parent grant funding of this supplement proposal aims to better understand bacterial virulence factors in order to develop new strategies to prevent, diagnose, and treat antibiotic-resistant infections. In project 2 of the parent U19 grant, we have further elucidated the mechanism by which FimD, the outer membrane (OM) usher of the type I chaperone usher pathway (CUP) pilus system, critical for both mouse and human cystitis, accommodates the tip adhesin during pilus biogenesis, and developed monoclonal antibodies that inhibit this process by binding to and inhibiting usher function. While we have made tremendous progress towards understanding usher function during pilus biogenesis, the process by which ushers are folded and inserted into the OM has remained obscure. Each usher is -800 amino acids and consists of five domains; i) a periplasmic N-terminal domain (NTD); ii) two periplasmic C-terminal domains (CTD1 and CTD2); iii) a 24-stranded β-barrel translocation domain (TD); and iv) a plug domain (PD) that sits in the TD when the usher is inactive and is moved into the periplasm to interact with the NTD when the usher is active. We hypothesize that like other Gram-negative OM β-barrel proteins tested to date, all CUP ushers are folded and inserted into the OM by the β-barrel Assembly Machinery (BAM). However, the presence of soluble periplasmic terminal domains (NTD, CTD1, CTD2) and a luminal plug domain between strands 136 and 137 of the translocation domain raises yet untested questions on how the β-barrel Assembly Machinery (BAM) accommodates these domains and whether they play a role during usher folding by BAM. In this proposal, we will establish that ushers are folded through the canonical BAM-mediated pathway by conducting photocrosslinking experiments between BamA and FimD and investigate the role of the PD on FimD folding by comparing the crosslinking efficiency between wild type and a plugless FimD (which is genetically deleted for the PD). We will also determine which components of the BAM complex are required to accommodate the different soluble domains of the FimD usher. These studies will not only enhance our understanding of the mechanism by which ushers are folded and inserted into the OM by BAM, but also how BAM handles large multidomain OM proteins; revealing folding intermediates that can be targeted for future therapeutic development.