Glenn T. Werneburg
B.S. Stony Brook University, 2010
5th Year MSTP
3rd Year Molecular Genetics & Microbiology Graduate Student
Advisor: David Thanassi, PhD
Department: Molecular Genetics & Microbiology
Graduate Program: Molecular Genetics & Microbiology
Title: Dissection of usher-chaperone-subunit interactions during pilus biogenesis in Escherichia coli
Abstract:
Gram-negative bacteria use the chaperone/usher (CU) pathway to construct virulence-associated surface structures known as pili. Type 1 pili, our model system, facilitate uropathogenic Escherichia coli bladder colonization. The CU pilus biogenesis pathway requires a periplasmic chaperone protein and an integral outer membrane protein, the usher. The chaperone facilitates folding of pilus subunits in the periplasm. The usher catalyzes the exchange of chaperone-subunit for subunit-subunit interactions, and promotes ordered polymerization and secretion of the pilus fiber. The usher (FimD), a putative multimer, has 5 domains: a periplasmic N-terminal domain (N), a transmembrane beta-barrel domain that is gated by an internal plug domain, and two periplasmic C-terminal domains (C1 and C2). Chaperone-subunit (FimC-FimH) complexes first bind to the N domain of the usher and then move to the C domains as the plug is expelled and the pilus subunits are inserted into the barrel channel. To understand the mechanism by which FimD catalyzes pilus assembly, we monitored interactions of FimC-FimH complexes with FimD domains using fluorescence-based approaches. Our data suggest that the C domains, FimD’s high-affinity binding site for FimC-FimH complex, are only accessible once FimC-FimH first binds FimD’s N domain, and that subsequently, the N to C domain handoff is driven by differential affinity. In the context of the full-length usher, our results indicate that the plug domain likely “masks” FimD’s C domains prior to usher activation. Further, in vivo we show that FimD is functional in trans, suggesting that the non-translocating FimD proteins actively recruit and incorporate chaperone-subunit complexes.
Publications:
(MSTP-supported publications indicated with an *)
Werneburg, GT, Chauhan, H, Henderson, NS, & Thanassi, DG (2016). MP24-04 THE UROPATHOGENIC ESCHERICHIA COLI PILUS USHER CONTROLS PROTEIN INTERACTIONS VIA DOMAIN MASKING AND IS FUNCTIONAL AS AN OLIGOMER. The Journal of Urology, 195(4), e271 (abstract).
Werneburg, G. T., Henderson, N. S., Portnoy, E. B., Sarowar, S., Hultgren, S. J., Li, H., & Thanassi, D. G. (2015). The pilus usher controls protein interactions via domain masking and is functional as an oligomer.
Nat Struct Mol Biol. 2015 Jul;22(7):540-6. doi: 10.1038/nsmb.3044. PMID: 26052892
Pham, T., Henderson, N. S., Werneburg, G. T., Thanassi, D. G., & Delcour, A. H. Electrostatic networks control plug stabilization in the PapC usher. Molecular membrane biology, 2015 Aug-Dec;32(5-8):198-207. doi: 10.3109/09687688.2016.1160450. Epub 2016 May 16. PMID: 27181766
Pham, T., Werneburg, G. T., Henderson, N. S., Thanassi, D. G., & Delcour, A. H. (2016). Effect of chaperone-adhesin complex on plug release by the PapC usher. Pham T, Werneburg GT, Henderson NS, Thanassi DG, Delcour AH. FEBS Lett. 2016 Jul;590(14):2172-9. doi: 10.1002/1873-3468.12257. PMID: 27313078
Sarowar S, Hu J, Werneburg G.T., Thanassi DG, Li H. (2016). The Escherichia coli P and type 1 pilus assembly chaperones PapD and FimC are monomeric in solution. J Bacteriol. 2016 Jun 27. Pii: JB.00366-16 [Epub ahead of print] PMID: 27353649
Rubin ET, Werneburg G.T., Pales Espinosa E, Thanassi DG, Allam, B. Identification and characterization of peptidases secreted by quahog parasite unknown (QPX), the protistan parasite of hard clams. 2016. Submitted.
*Werneburg G.T., Thanassi, D.G. (2014). Pilus Biogenesis in Escherichia coli by the Chaperone/Usher Pathway. EcoSal Plus. In preparation.
