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PapD, a periplasmic transport protein in P-pilus biogenesis
F Lindberg1, J M Tennent, S J Hultgren
1Department of Microbiology, University of Umeå, Sweden.
Insights
The papD gene product is essential for P pilus assembly in E. coli. PapD acts as a periplasmic transport protein, stabilizing pilus subunits for proper polymerization.
Area of Science:
- Microbiology
- Molecular Biology
- Bacterial Pathogenesis
Background:
- Uropathogenic Escherichia coli (UPEC) utilizes P pili for adhesion and infection.
- P pilus biogenesis involves complex protein interactions and assembly in the periplasm.
- The papD gene's role in P pilus formation was previously unclear.
Purpose of the Study:
- To elucidate the function of the papD gene product in P pilus biogenesis.
- To characterize the PapD protein and its interactions with other pilus components.
- To investigate the mechanism of PapD-mediated transport of pilus subunits.
Main Methods:
- Gene sequencing of papD.
- Purification and N-terminal sequencing of the PapD protein.
- Isolation and characterization of a PapD-PapE complex.
- Immunological assays using antibodies against PapD and the PapD-PapE complex.
Main Results:
- Mutations in papD lead to degradation of major pilus subunits (PapA, PapE, PapF) and partial breakdown of PapG.
- PapD was identified as a basic, hydrophilic, peripheral periplasmic protein.
- A stable complex between PapD and PapE was purified.
- Antibodies confirmed the association of PapD and PapE and their presence in wild-type P pili.
Conclusions:
- PapD is crucial for P pilus biogenesis, likely functioning as a periplasmic chaperone.
- PapD stabilizes pilus subunits, possibly at the inner membrane, and facilitates their transport for polymerization.
- This mechanism ensures the correct assembly of functional P pili in UPEC.
Abstract:
The product of the papD gene of uropathogenic Escherichia coli is required for the biogenesis of digalactoside-binding P pili. Mutations within papD result in complete degradation of the major pilus subunit, PapA, and of the pilinlike proteins PapE and PapF and also cause partial breakdown of the PapG adhesin. The papD gene was sequenced, and the gene product was purified from the periplasm. The deduced amino acid sequence and the N-terminal sequence obtained from the purified protein revealed that PapD is a basic and hydrophilic peripheral protein. A periplasmic complex between PapD and PapE was purified from cells that overproduced and accumulated these proteins in the periplasm. Antibodies raised against this complex reacted with purified wild-type P pili but not with pili purified from a papE mutant. In contrast, anti-PapD serum did not react with purified pili or with the culture fluid of piliated cells. However, this serum was able to specifically precipitate the PapE protein from periplasmic extracts, confirming that PapD and PapE were associated as a complex. It is suggested that PapD functions in P-pilus biogenesis as a periplasmic transport protein. Probably PapD forms complexes with pilus subunits at the outer surface of the inner membrane and transports them in a stable configuration across the periplasmic space before delivering them to the site(s) of pilus polymerization.