The Type IV Pilus Assembly Complex: Biogenic Interactions among the Bundle-Forming Pilus Proteins of Enteropathogenic Escherichia coli

Abstract

Production of type IV bundle-forming pili (BFP) by enteropathogenic Escherichia coli (EPEC) requires the protein products of 12 genes of the 14-gene bfp operon. Antisera against each of these proteins were used to demonstrate that in-frame deletion of individual genes within the operon reduces the abundance of other bfp operon-encoded proteins. This result was demonstrated not to be due to downstream polar effects of the mutations but rather was taken as evidence for protein-protein interactions and their role in the stabilization of the BFP assembly complex. These data, combined with the results of cell compartment localization studies, suggest that pilus formation requires the presence of a topographically discrete assembly complex that is composed of BFP proteins in stoichiometric amounts. The assembly complex appears to consist of an inner membrane component containing three processed, pilin-like proteins, BfpI, -J, and -K, that localize with BfpE, -L, and -A (the major pilin subunit); an outer membrane, secretin-like component, BfpB and -G; and a periplasmic component composed of BfpU. Of these, only BfpL consistently localizes with both the inner and outer membranes and thus, together with BfpU, may articulate between the Bfp proteins in the inner membrane and outer membrane compartments. The virulence of enteropathogenic Escherichia coli (EPEC)for orally challenged volunteers (3) requires genes encoded on the 69-kb EPEC adherence factor (EAF) plasmid (31) and within the chromosomal locus of enterocyte effacement (7). The EAF plasmid carries a 14-gene operon that encodes the bundle-forming pilus (BFP), a member of the widely distributed type IV family of pilin proteins (28,29). This operon is required for the production of BFP filaments and virulence; in addition, functional studies of bfp operon mutants show that expression of the operon confers two readily assayable in vitro phenotypes. The localized adherence (LA) phenotype is characterized by circumscribed clusters of bacteria attached to the surface of cell culture monolayers (6, 16). The autoaggregation phenotype (AA) is evident when an overnight culture of dispersed EPEC is inoculated into tissue culture medium; 45 to 60 min later, the bacteria coalesce into dynamic, spherical assemblies which disaggregate after 3 to 4 h, again yielding a suspension of single cells (3).The bfp operon, together with its transcriptional activator BfpTVW/PerABC, which is located elsewhere on the EAF plasmid, (9, 32), is sufficient for expression of BFP filaments and the LA and autoaggregation phenotypes when it is harbored in E. coli strains that normally do not express type IV pili. Thus, the operon's 14 genes appear to encode the minimal set of functions, exclusive of transcription factors and the periplasmic protein, DsbA (34), that are specifically required for BFP biogenesis and function.Details of the environmentally responsive transcriptional regulation of the bfp operon are emerging (21); however, the interactions among the proteins expressed by the operon rema...

The Platform Protein Is Essential for Type IV Pilus Biogenesis

“…Similar studies of platform proteins in the T4bP and T2S systems have also supported a critical role in assembly (23,29). However, the equivalent platform protein PilG in the T4aP system of Neisseria meningitidis was reported to be dispensable for pilus assembly (26,30), as mutation of pilG in a retractiondeficient background resulted in wild-type surface piliation and adhesive properties.…”

Structural and Topographical Studies of the Type IV Bundle-Forming Pilus Assembly Complex of Enteropathogenic Escherichia coli

“…The topographical features of this complex have been explored by localizing individual proteins to compartments of the cell through the use of protein-specific antibodies and immunoblot assays to detect their presence in compartment-specific cell fractions. In this manner, BfpB and BfpG have been shown to localize exclusively to the outer membrane (12,17); BfpU and BfpH mainly localize to the periplasmic space (see references 13 and 18 and unpublished data), and BfpA (as a pool of unassembled pilins), BfpC and BfpE, and BfpI, BfpJ, and BfpK (the last three are stoichiometrically minor pilin-like proteins of the assembly complex) localize to the inner membrane (3,13). By contrast, BfpL (while predominantly an inner membrane protein) can be consistently detected in small amounts in outer-membrane fractions prepared from French pressure cell-disrupted bacteria and sucrose gradient centrifugation (13).…”

“…In this manner, BfpB and BfpG have been shown to localize exclusively to the outer membrane (12,17); BfpU and BfpH mainly localize to the periplasmic space (see references 13 and 18 and unpublished data), and BfpA (as a pool of unassembled pilins), BfpC and BfpE, and BfpI, BfpJ, and BfpK (the last three are stoichiometrically minor pilin-like proteins of the assembly complex) localize to the inner membrane (3,13). By contrast, BfpL (while predominantly an inner membrane protein) can be consistently detected in small amounts in outer-membrane fractions prepared from French pressure cell-disrupted bacteria and sucrose gradient centrifugation (13). BfpP, which encodes the prepilin peptidase that processes BfpA (the major repeating subunit of the pilus filament) (30) and BfpI, BfpJ, and BfpK (13), is presumed (on the basis of its functional role) to localize to the cytoplasmic face of the inner membrane, but biochemical evidence for this prediction has not been reported (21,30).…”

“…In-frame disruption of each of the 14 bfp operon genes (designated bfpA to bfpL) ( Table 1) and analysis of the resulting mutants show that with the exception of bfpH, each of the remaining 13 genes is required for normal BFP filament production and phenotype expression (1,13). These findings for the BFP system and similar results from the study of other type IV pili have led to the idea that bfp operon-encoded proteins comprise an oligomeric structural and functional complex (4,10,14,15,22,24).…”

“…BfpP, which encodes the prepilin peptidase that processes BfpA (the major repeating subunit of the pilus filament) (30) and BfpI, BfpJ, and BfpK (13), is presumed (on the basis of its functional role) to localize to the cytoplasmic face of the inner membrane, but biochemical evidence for this prediction has not been reported (21,30). BfpD and BfpF, which contain Walker box motifs that are presumed to hydrolyze ATP and thus energize the extrusion and retraction of the pilus filament, respectively, have not been localized (13,21).…”