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PapD chaperone function in pilus biogenesis depends on oxidant and chaperone-like activities of DsbA
F Jacob-Dubuisson1, J Pinkner, Z Xu
1Department of Molecular Microbiology, Washington University Medical School, St. Louis, MO 63110-1093.
Abstract:
Adhesive P pili of uropathogenic Escherichia coli were not assembled by a strain that lacks the periplasmic disulfide isomerase DsbA. This defect was mostly attributed to the immunoglobulin-like pilus chaperone PapD, which possesses an unusual intrasheet disulfide bond between the last two beta-strands of its CD4-like carboxyl-terminal domain. The DsbA-dependent formation of this disulfide bond was critical for PapD's proper folding in vivo. Interestingly, the absence of the disulfide bond did not prevent PapD from folding in vitro or from forming a complex with the pilus adhesin in vitro. We suggest that DsbA maintains nascently translocated PapD in a folding-competent conformation prior to catalyzing disulfide bond formation, acting both as an oxidant and in a chaperone-like fashion. Disulfide bond formation in pilus subunits was also mediated by DsbA even in the absence of PapD. However, the ability of pilus subunits to achieve native-like conformations in vivo depended on PapD. These results suggest that a productive folding pathway for subunits requires sequential interactions with DsbA and the PapD chaperone.
Insights
Uropathogenic Escherichia coli P pili assembly requires the periplasmic disulfide isomerase DsbA and the PapD chaperone. DsbA facilitates PapD folding and disulfide bond formation, crucial for P pili biogenesis in vivo.
Area of Science:
- Microbiology
- Molecular Biology
- Protein Folding
Background:
- Uropathogenic Escherichia coli (UPEC) utilize adhesive P pili for host colonization.
- Assembly of P pili involves complex protein-protein interactions and post-translational modifications.
- The periplasmic disulfide isomerase DsbA and the pilus chaperone PapD are implicated in P pili biogenesis.
Purpose of the Study:
- To investigate the specific roles of DsbA and PapD in P pili assembly.
- To elucidate the mechanism by which DsbA and PapD contribute to pilus subunit folding and disulfide bond formation.
- To understand the sequential requirements for DsbA and PapD in the productive folding pathway of P pili subunits.
Main Methods:
- Genetic analysis of UPEC strains lacking DsbA.
- In vitro folding and complex formation assays with PapD and pilus subunits.
- Assessment of P pili assembly and subunit conformation in vivo.
Main Results:
- A UPEC strain lacking DsbA exhibited defects in P pili assembly, primarily due to improper folding of the PapD chaperone.
- DsbA-dependent disulfide bond formation in PapD was essential for its correct in vivo folding, but not for in vitro folding or complex formation with adhesins.
- DsbA also mediated disulfide bond formation in pilus subunits independently of PapD, but PapD was necessary for achieving native-like conformations in vivo.
Conclusions:
- DsbA acts as both an oxidant and a chaperone, maintaining PapD in a folding-competent state before catalyzing disulfide bond formation.
- A productive folding pathway for P pili subunits necessitates sequential interactions with DsbA and the PapD chaperone.
- These findings highlight the intricate molecular mechanisms governing the assembly of bacterial adhesive pili.