Pili are adhesive organelles of Gram-negative bacteria used to initiate an infection in the host. Uropathogenic Escherichia coli has two types of adhesive organelles: the type 1 (Fim) and the P (Pap) pili, which are assembled by the chaperone–usher (CU) pathway. Usher is an outer membrane protein that consists of a β-barrel domain in the membrane, flanked by an amino terminal domain (NTD) and two carboxyl terminal domains (CTD1 and CTD2) at the periplasmic side, and a plug domain that blocks the β-barrel in the resting apo state. Pilus biogenesis is a multistep process. Earlier studies have demonstrated that the usher NTD functions to recruit the chaperone–subunit complex, and the CTD1 and CTD2 facilitate subunit polymerization and protein secretion. However, how the chaperone–subunit complex is recruited by NTD and transferred from the NTD to the CTDs, and when the subunit is polymerized into the growing pilus, remain largely unanswered. By using protein engineering and single-particle cryo–electron microscopy, we captured a FimD–pilus tip assembly intermediate comprising FimDCFGH in a configuration in which the usher FimD is in the process of handing over FimC–FimF from its NTD to the CTDs. The pilus subunit polymerization occurs before subunit handover and reveals the mechanisms of subunit handover from FimD NTD to CTDs. We also found the cyclic conformational changes of the N-terminal tail which play crucial roles in the multi-step assembly process. In the second part of my thesis research, we captured three P pilus assembly intermediates in which the PapC usher is fully activated and is in the process ofrecruiting, polymerizing, and secreting P pilus tip subunits. Our cryo-EM structural analysis reveals new conformational dynamics of the usher and shows the movement of CTD2 to facilitate the NTD-to-CTD chaperone–subunit transfer process.
|Commitee:||Haab, Brian, Melcher, Karsten , Thanassi, David|
|School:||Van Andel Research Institute|
|School Location:||United States -- Michigan|
|Source:||DAI-B 82/7(E), Dissertation Abstracts International|
|Subjects:||Microbiology, Molecular biology, Biochemistry, Organic chemistry|
|Keywords:||Pili, Gram-negative bacteria, Host infection, Chaperone usher pathway, Polymerization , Protein secretion , Amino terminal domain (NTD), Biogenesis|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be