The inner membrane subassembly of the enteropathogenic Escherichia coli bundle-forming pilus machine

Type IV pili (Tfps) are filamentous surface appendages expressed by Gram-negative microorganisms and play numerous roles in bacterial cell biology. Tfp biogenesis machineries are highly conserved and resemble protein secretion and DNA uptake systems. Although components of Tfp biogenesis systems have been identified, it is not known how they interact to form these machineries. Using the bundle-forming pilus (BFP) of enteropathogenic Escherichia coli as a model Tfp system, we provide evidence of a cytoplasmic membrane subassembly of the Tfp assembly machine composed of putative cytoplasmic nucleotide-binding and cytoplasmic membrane proteins. A combination of genetic, biochemical and biophysical approaches revealed interactions among putative cytoplasmic nucleotide-binding proteins BfpD and BfpF and cytoplasmic membrane proteins BfpC and BfpE of the BFP biogenesis machine. The polytopic membrane protein BfpE appears to be a central component of this subassembly as it interacts with BfpC, BfpD and BfpF. We report that BFP biogenesis probably requires interactions among BfpC, BfpD and BfpE, whereas BFP retraction requires interaction of the PilT-like putative ATPase BfpF with a conserved domain of BfpE. BfpE is the first protein that is not a member of the PilT family to be implicated in Tfp retraction. Furthermore, we found that the putative ATPases BfpD and BfpF play antagonistic roles in BFP biogenesis and retraction, respectively, by interacting with distinct domains of the BFP biogenesis machine.     

The type IV pilus assembly complex: biogenic interactions among the bundle-forming pilus proteins of enteropathogenic Escherichia coli

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.     

Structural and topographical studies of the type IV bundle-forming pilus assembly complex of enteropathogenic Escherichia coli

The type IV bundle-forming pili (BFP) of enteropathogenic Escherichia coli (EPEC) are required for virulence in orally challenged human volunteers and for the localized adherence and autoaggregation in vitro phenotypes. BFP filament biogenesis and function are encoded by the 14-gene bfp operon. The BFP assembly complex, containing a BfpB-His6 fusion protein, was chemically cross-linked in situ, and the complex was then purified from BFP-expressing EPEC by a combination of nickel- and BfpB antibody-based affinity chromatography. Characterization of the isolated complex by immunoblotting using BFP protein-specific antibodies showed that at least 10 of the 14 proteins specified by the bfp operon physically interact to form an oligomeric complex. Proteins localized to the outer membrane, inner membrane, and periplasm are within this complex, thus demonstrating that the complex spans the periplasmic space. A combination of immunofluorescence and immuno-gold thin-section transmission electron microscopy studies localized this complex to one pole of the cell.     

Role of bundle-forming pilus of enteropathogenic Escherichia coli in host cell adherence and in microcolony development

Enteropathogenic Escherichia coli (EPEC) adheres to epithelial cells and forms microcolonies in localized areas. Bundle-forming pili (BFP) are necessary for autoaggregation and the formation of microcolonies. In this study, we show that BFP, expressed by EPEC on epithelial cells, disappeared with the expansion of the microcolony. Bacterial dispersal and the release of BFP from the EPEC aggregates were induced by contact with host cellular membrane extract. In addition, BFP-expressing EPEC adhered directly to cell surfaces, in preference to attaching to pre-formed microcolonies on the cells. These results suggested that BFP mediate the initial attachment of EPEC through direct interaction with the host cell rather than through the recruitment of unattached bacteria to microcolonies on the cell.     

Bundle-forming pilus retraction enhances enteropathogenic Escherichia coli infectivity

Enteropathogenic Escherichia coli (EPEC) is an important human pathogen that causes acute infantile diarrhea. The type IV bundle-forming pili (BFP) of typical EPEC strains are dynamic fibrillar organelles that can extend out and retract into the bacterium. The bfpF gene encodes for BfpF, a protein that promotes pili retraction. The BFP are involved in bacterial autoaggregation and in mediating the initial adherence of the bacterium with its host cell. Importantly, BFP retraction is implicated in virulence in experimental human infection. How pili retraction contributes to EPEC pathogenesis at the cellular level remains largely obscure, however. In this study, an effort has been made to address this question using engineered EPEC strains with induced BFP retraction capacity. We show that the retraction is important for tight-junction disruption and, to a lesser extent, actin-rich pedestal formation by promoting efficient translocation of bacterial protein effectors into the host cells. A model is proposed whereby BFP retraction permits closer apposition between the bacterial and the host cell surfaces, thus enabling timely and effective introduction of bacterial effectors into the host cell via the type III secretion apparatus. Our studies hence suggest novel insights into the involvement of pili retraction in EPEC pathogenesis.     

The type IV bundle-forming pilus of enteropathogenic Escherichia coli undergoes dramatic alterations in structure associated with bacterial adherence, aggregation and dispersal

BFP, a plasmid-encoded type IV bundle-forming pilus produced by enteropathogenic Escherichia coli (EPEC), has recently been shown to be associated with the aggregation of bacteria and dispersal of bacteria from bacterial microcolonies. In standard 3 h HEp-2 cell assays, EPEC adhere in localized microcolonies; after 6 h, bacterial microcolonies are no longer present, indicating that bacterial aggregation and dispersal occurs in vitro during EPEC adhesion to cultured epithelial cells. To examine the role of BFP in EPEC aggregation and dispersal, we examined HEp-2 cell adhesion of strain E2348/69 and defined E2348/69 mutants by immunofluorescence and immunoelectron microscopy. BFP was expressed initially as approximately 40 nm diameter pilus bundles that promoted bacteria-bacteria interaction and microcolony formation. BFP subsequently underwent a striking alteration in structural organization with the formation of much longer and thicker ( approximately 100 nm diameter) pilus bundles, which frequently aggregated laterally to form even thicker bundles often arranged in a loose three-dimensional network; EPEC dispersal from bacterial microcolonies was associated with this transformation of BFP from thin to thick bundles. Bacterial dispersal and transformation of BFP from thin to thick bundles did not occur with a bfpF mutant of strain E2348/69. It is concluded that BFP promotes both the formation and the dispersal of EPEC microcolonies, that the dispersal phase requires BfpF and that dispersal is associated with dramatic alterations in the structure of BFP bundles.     

Species-specific cell adhesion of enteropathogenic Escherichia coli is mediated by type IV bundle-forming pili

Enteropathogenic Escherichia coli (EPEC) is a causative agent of diarrhoea in humans. Localized adherence of EPEC onto intestinal mucosa was reproduced in an in vitro adherence assay with cultured human epithelial cells. We found that the efficiency of EPEC adherence to a mouse-derived colonic epithelial cell line, CMT-93, was remarkably lower than its adherence to human-derived intestinal cell lines, such as Intestine-407 or Caco-2. Although EPEC did adhere to some cell lines derived from non-human species, fixing the cells with formalin to inactivate one or more formalin-sensitive factors allowed us to observe species-specific differences in EPEC adherence. In contrast to these results, an EPEC mutant that is defective in bundle-forming pili (BFP) production adhered as efficiently to CMT-93 cells as to Caco-2 cells. Furthermore, Citrobacter rodentium expressing BFP adhered to Caco-2 cells much more efficiently than to CMT-93 cells. Finally, a purified BfpA-His6 fusion protein showed higher affinity for Caco-2 cells than for CMT-93 cells, and inhibited EPEC adherence. Following BFP-mediated adherence, secretion of EspB from adherent bacteria and reorganization of F-actin in the host cells was observed. EPEC adhering to CMT-93 cells induced far less secretion of EspB, or reorganization of F-actin in the host CMT-93 cells, than did EPEC adhering to Caco-2 cells. These results indicated that BFP plays an important role in the cell-type-dependent adherence of EPEC and in the progression to the later steps in EPEC adherence.     

Enteropathogenic Escherichia coli: identification of a gene cluster coding for bundle-forming pilus morphogenesis.

Sequence flanking the bfpA locus on the enteroadherent factor plasmid of the enteropathogenic Escherichia coli (EPEC) strain B171-8 (O111:NM) was obtained to identify genes that might be required for bundle-forming pilus (BFP) biosynthesis. Deletion experiments led to the identification of a contiguous cluster of at least 12 open reading frames, including bfpA, that could direct the synthesis of a morphologically normal BFP filament. Within the bfp gene cluster, we identified open reading frames that share homology with other type IV pilus accessory genes and with genes required for transformation competence and protein secretion. Immediately upstream of the bfp gene cluster, we identified a potential replication origin including genes that are predicted to encode proteins homologous with replicase and resolvase. Restriction fragment length polymorphism analysis of DNA from six additional EPEC serotypes showed that the organization of the bfp gene cluster and its juxtaposition with a potential plasmid origin of replication are highly conserved features of the EPEC biotype.     

Structure of the cell-adhesion fragment of intimin from enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) induce gross cytoskeletal rearrangement within epithelial cells, immediately beneath the attached bacterium. The C-terminal 280 amino acid residues of intimin (Int280; 30.1 kDa), a bacterial cell-adhesion molecule, mediate the intimate bacterial host-cell interaction. Recently, interest in this process has been stimulated by the discovery that the bacterial intimin receptor protein (Tir) is translocated into the host cell membrane, phosphorylated, and after binding intimin triggers the intimate attachment. Using multidimensional nuclear magnetic resonance (NMR) and combining perdeuteration with site-specific protonation of methyl groups, we have determined the global fold of Int280. This represents one of the largest, non-oligomeric protein structures to be determined by NMR that has not been previously resolved by X-ray crystallography. Int280 comprises three domains; two immunoglobulin-like domains and a C-type lectin-like module, which define a new family of bacterial adhesion molecules. These findings also imply that carbohydrate recognition may be important in intimin-mediated cell adhesion.     

The Cpx envelope stress response affects expression of the type IV bundle-forming pili of enteropathogenic Escherichia coli

The Cpx envelope stress response mediates adaptation to potentially lethal envelope stresses in Escherichia coli. The two-component regulatory system consisting of the sensor kinase CpxA and the response regulator CpxR senses and mediates adaptation to envelope insults believed to result in protein misfolding in this compartment. Recently, a role was demonstrated for the Cpx response in the biogenesis of P pili, attachment organelles expressed by uropathogenic E. coli. CpxA senses misfolded P pilus assembly intermediates and initiates increased expression of both assembly and regulatory factors required for P pilus elaboration. In this report, we demonstrate that the Cpx response is also involved in the expression of the type IV bundle-forming pili of enteropathogenic E. coli (EPEC). Bundle-forming pili were not elaborated from an exogenous promoter in E. coli laboratory strain MC4100 unless the Cpx pathway was constitutively activated. Further, an EPEC cpxR mutant synthesized diminished levels of bundle-forming pili and was significantly affected in adherence to epithelial cells. Since type IV bundle-forming pili are very different from chaperone-usher-type P pili in both form and biogenesis, our results suggest that the Cpx envelope stress response plays a general role in the expression of envelope-localized organelles with diverse structures and assembly pathways.     

Cloning and characterization of the bundle-forming pilin gene of enteropathogenic Escherichia coli and its distribution in Salmonella serotypes

bfp, the structural gene of the major repeating bundle-forming pilus (BFP) subunit, was cloned from the enteroadherent factor (EAF) plasmid of enteropathogenic Escherichia coli (EPEC) strain B171 (0111:NM). The bfp open reading frame encoded a 193-amlno-acid protein; comparison of this sequence with the biochemically determined N-terminal amino acid sequence showed that the mature pilin protein is comprised of 180 amino acids, that this sequence is similar to other members of the type IV pilin family, and that it is preceded by a 13-amino-acid signal peptide. Expression of the cloned bfp structural gene in an EPEC strain that had been cured of the EAF plasmid yielded a 21000 dalton protein that co-migrated with the BFP precursor protein. Thus, other genes, probably carried by the EAF plasmid, are required for the maturation of the bfp product and for the production of extracellular pilus filaments. Use of bfp as a hybridization probe showed that homologous sequences are present in all tested EPEC strains and in 13 of 16 tested Salmonelia serotypes. Fifty per cent of these bfp probe-sensitive salmonellae exhibited the localized-adherence (LA) phenotype when incubated with tissue culture cell monolayers, a trait previously associated with EAF plasmid-containing EPEC strains. Scanning electron micrographs of a bfp probe-positive, LA-positive Salmonella dublin strain showed that it grows as adherent colonies on infected monolayers and that within these colonies, BFP-like fibres form inter-bacterial linkages. For EAF plasmid-containing EPEC strains and for severai Salmonella serotypes, BFP expression may lead to the development of adherent colonies on epithelial surfaces early in the infective process.     

BfpB, an outer membrane lipoprotein required for the biogenesis of bundle-forming pili in enteropathogenic Escherichia coli.

The bundle-forming pili (BFP) of enteropathogenic Escherichia coli are believed to play a role in pathogenesis by causing the formation of bacterial microcolonies that bind epithelial surfaces of the small intestine. This in vivo process is mimicked in vitro by the autoaggregation and localized adherence phenotypes. Expression of BFP, a member of the type IV pilus family, requires the enteroadherence factor (EAF) plasmid, which contains bfpA, the gene that encodes the principal structural subunit of BFP. Immediately downstream of bfpA are 13 open reading frames transcribed in the same direction as bfpA; together with bfpA, these compose the bfp gene cluster. Disruption of bfpB, the second open reading frame downstream of bfpA, was performed by allelic exchange. The resulting mutant, B171-8deltaB, did not exhibit the autoaggregation or localized adherence phenotype or produce BFP filaments. Thus, BfpB is required for pilus biogenesis. However, BfpA was produced at wild-type levels and processed normally by B171-8deltaB, indicating that BfpB acts at a step in the BFP biogenic pathway after production and processing of the structural subunit. Biochemical and cell fractionation studies showed that BfpB is a 58-kDa lipoprotein that is located primarily in the outer membrane. Assays of bfpA and bfpB mRNAs and protein expression showed that both genes are cotranscribed as part of an environmentally responsive operon that is regulated by growth phase and ammonium.     

A cluster of fourteen genes from enteropathogenic Escherichia coli is sufficient for the biogenesis of a type IV pilus

Enteropathogenic Escherichia coli (EPEC) adhere to epithelial cells in microcolonies, a pattern termed localized adherence (LA). LA is dependent upon the presence of 50–70MDa plasmids, termed EPEC adherence factor (EAF) plasmids. Expression of an EAF plasm id-encoded type IV fimbria, the bundle-forming pilus (BFP), is associated with the LA phenotype. TnphoA insertions in bfpA, the gene encoding the major structural subunit of the BFP, abolish LA. While bfpA::TnphoA mutants cannot be complemented for LA by plasmids carrying the bfpA gene alone in trans, this work shows that they can be complemented by plasmids carrying the bfpA gene, as well as approximately 10kb of downstream sequence, suggesting that such mutations have polar effects on downstream genes. The identification and characterization of a cluster of 13 genes immediately downstream of bfpA are described. The introduction into a laboratory Escherichia coli strain of a plasmid containing these 14 bfp gene cluster genes, along with pJPN14, a plasmid containing another fragment derived from the EAF plasmid, confers LA ability and BFP biogenesis. However, when a mutation is introduced into the last gene of the bfp cluster, neither LA nor BFP biogenesis is conferred. This work also provides evidence to show that the fragment cloned in pJPN14 encodes a factor(s) which results in increased levels of the pilin protein. Finally, it is shown that expression of the 14 genes in the bfp cluster from an IPTG-inducible promoter, in the absence of pJPN14, is sufficient to reconstitute BFP biogenesis in a laboratory E. cob strain, but is insufficient for LA. This is the first report demonstrating the reconstitution of a type IV pilus in a laboratory E. coli strain with a defined set of genes. The 8FP system should prove to be a useful model for studying the molecular mechanisms of type IV pilus biogenesis.     

Characterisation of maltase from enteropathogenic Escherichia coli

Abstract Enteropathogenic strains of faecal Escherichia coli produced significantly ( P < 0.01) more maltase than the non-pathogenic strains of the organism. The enzyme was induced by maltose but repressed by glucose and fructose. The maltase was partially purified by ammonium sulphate precipitation, followed by dialysis and gel permeation chromatography. The partially purified maltase had an M _r of 144500 and an apparent K _m of approx. 7.6 mM for maltose. The enzyme was stimulated by Ca²⁺, inhibited by Cu²⁺, Hg²⁺, Uo²⁺, IAA and EDTA, and exhibited optimum activity at pH 6.5 at 30°C.     

3D structure of EspA filaments from enteropathogenic Escherichia coli

The type III secretion system (TTSS) is a modular apparatus assembled by many pathogenic Gram-negative bacteria and is designed to translocate proteins through the bacterial cell wall into the eukaryotic host cell. The conserved components of the TTSS comprise stacks of rings spanning the inner and outer bacterial membrane and a narrow, needle-like structure projecting outwards. The TTSS of enteropathogenic E. coli is unique in that one of the translocator proteins, EspA, polymerizes to form an extension to the needle complex which interacts with the host cell. In this study we present the 3D structure of EspA filaments to c. 26 A resolution determined from electron micrographs of negatively stained preparations by image processing. The structure comprises a helical tube with a diameter of 120 A enclosing a central channel of 25 A diameter through which effector proteins may be transported. The subunit arrangement corresponds to a one-start helix with 28 subunits present in five turns of the helix and an axial rise of 4.6 A per subunit. This is the first report of a 3D structure of a filamentous extension to the TTSS.