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BfpU, a soluble protein essential for type IV pilus biogenesis in enteropathogenic Escherichia coli
Wiebke Schreiber1, Kelly D Stone1, Margaret A Strong1
1Molecular and Cellular Biology Program, Division of Infectious Diseases1 and Comparative Medicine Program, Department of Medicine,2 University of Maryland, Baltimore, School of Medicine, Baltimore, MD 21201, USA.
Abstract:
A cluster of 14 genes located on the large plasmid of enteropathogenic Escherichia coli (EPEC) strains is sufficient to direct the biogenesis of the type IV bundle-forming pilus (BFP) in a recombinant E. coli host. The fifth gene in the cluster, bfpU, encodes a protein that is predicted to be localized to the periplasmic space. To determine whether BfpU is necessary for pilus biogenesis, the authors constructed a non-polar bfpU mutant EPEC strain by allelic exchange. The mutant strain was unable to perform localized adherence and auto-aggregation, two phenotypes associated with BFP expression, and it failed to make BFP. These phenotypes were restored to the bfpU mutant by a plasmid containing bfpU. There was no difference between the wild-type and bfpU mutant strains in their expression or processing of the pre-pilin protein or in their localization of the pilin protein in the inner and outer membranes. Fractionation studies revealed that BfpU is completely soluble and is detected in both the periplasm and the cytoplasm. Thus, BfpU represents a novel protein required for type IV pilus assembly.
Insights
BfpU is essential for the assembly of type IV bundle-forming pili (BFP) in enteropathogenic Escherichia coli (EPEC). This protein is required for localized adherence and auto-aggregation, crucial for EPEC pathogenesis.
Area of Science:
- Microbiology
- Molecular Biology
- Bacterial Pathogenesis
Background:
- Enteropathogenic Escherichia coli (EPEC) utilizes type IV bundle-forming pili (BFP) for host cell adhesion.
- A 14-gene cluster on the EPEC large plasmid directs BFP biogenesis.
- The role of individual genes within this cluster, such as bfpU, remains to be fully elucidated.
Purpose of the Study:
- To investigate the necessity of the bfpU gene product for type IV bundle-forming pilus (BFP) biogenesis in EPEC.
- To characterize the function and localization of the BfpU protein.
Main Methods:
- Construction of a non-polar bfpU mutant EPEC strain using allelic exchange.
- Phenotypic analysis of the mutant strain, including localized adherence and auto-aggregation assays.
- Complementation studies to restore wild-type phenotypes.
- Fractionation studies to determine BfpU localization within the bacterial cell.
Main Results:
- The bfpU mutant strain failed to produce BFP and exhibited loss of localized adherence and auto-aggregation.
- Complementation with a bfpU-containing plasmid restored BFP production and associated phenotypes.
- BfpU is a soluble protein found in both the periplasm and cytoplasm.
- BfpU's absence did not affect pre-pilin or pilin protein expression, processing, or localization.
Conclusions:
- BfpU is a novel and essential protein required for type IV pilus assembly in EPEC.
- BfpU plays a critical role in the functional manifestation of BFP, impacting bacterial adherence and aggregation.
- The findings contribute to understanding the molecular mechanisms underlying EPEC virulence factor production.