Novel Roles for Two Proteins Required for Type IV Pilus Expression in Enteropathogenic Escherichia coli

Abstract

Enteropathogenic Escherichia coli (EPEC) causes diarrhea among infants in developing countries. The Bundle Forming Pilus (BFP) is a type IV pilus found on the surface of EPEC and essential for pathogenesis. The machinery for BFP polymerization is encoded by an operon of 14 genes. To examine the role of BfpI, a putative pilin-like protein, and BfpL, a protein of unknown function, in expression of BFP, deletions of bfpI and bfpL were created in wild type EPEC and a bfpF mutant deficient for an ATPase required for pilus retraction. I found that both bfpI and bfpL mutants were deficient for BFP expression and BFP associated phenotypes in a wild type EPEC background. However a bfpF bfpI double mutant still expressed BFP while a bfpF bfpL double mutant did not. Western blots of sheared pilus preparations did not suggest that BfpL is a component of BFP. Topology studies using C-terminal truncations and a dual-reporter revealed that the majority of BfpL lies in the periplasm. In addition I demonstrated that BfpL interacts with the periplasmic face of BfpC, a bitopic inner membrane protein, by yeast 2 hybrid assays and confirmed this finding by fluorescence anisotropy. I examined the rate of pilus extension and retraction over time using flow cytometry and found that the bfpF/I double mutant expresses more surface bundlin than the bfpF mutant, but when these strains were complemented with a vector containing bfpF and placed in conditions inducing retraction, the double mutant had less surface expression over time than the bfpF mutant. These data indicate that BfpL is absolutely required for bundle-forming pilus biogenesis and associates with the inner membrane subassembly complex, while BfpI is not absolutely required for biogenesis and impedes pilus retraction.