Structure of the bundle-forming pilus from enteropathogenic Escherichia coli

Bundle-forming pili (BFP) are essential for the full virulence of enteropathogenic Escherichia coli (EPEC) because they are required for localized adherence to epithelial cells and auto-aggregation. We report the high resolution structure of bundlin, the monomer of BFP, solved by NMR. The structure reveals a new variation in the topology of type IVb pilins with significant differences in the composition and relative orientation of elements of secondary structure. In addition, the structural parameters of native BFP filaments were determined by electron microscopy after negative staining. The solution structure of bundlin was assembled according to these helical parameters to provide a plausible atomic resolution model for the BFP filament. We show that EPEC and Vibriocholerae type IVb pili display distinct differences in their monomer subunits consistent with data showing that bundlin and TcpA cannot complement each other, but assemble into filaments with similar helical organization.     

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.     

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.     

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.     

Genetic diversity of the gene cluster encoding longus, a type IV pilus of enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) strains produce a type IV pilus named Longus. We identified a 16-gene cluster involved in the biosynthesis of Longus that has 57 to 95% identity at the protein level to CFA/III, another type IV pilus of ETEC. Alleles of the Longus structural subunit gene lngA demonstrate a diversity of 12 to 19% at the protein level with strong positive selection for point replacements and horizontal transfer.     

Enteropathogenic Escherichia coli Tir translocation and pedestal formation requires membrane cholesterol in the absence of bundle-forming pili

Enteropathogenic Escherichia coli (EPEC) is a significant cause of paediatric diarrhoea worldwide. Virulence requires adherence to intestinal epithelial cells, mediated in part through type IV bundle-forming pili (BFP), and the EPEC protein Tir. Tir is inserted into the enterocyte plasma membrane (PM), resulting in the formation of actin-rich pedestals. Tir is translocated by the type III secretion system (TTSS), through a pore comprised of EPEC proteins inserted into the PM. Here, we demonstrate that in the absence of BFP, EPEC adherence, effector translocation and pedestal formation are dependent on lipid rafts. Lipid raft disruption using methyl-beta-cyclodextrin (MbetaCD) decreased adherence by an EPEC BFP-deficient strain from 85% to 1%. Translocation of the effectors Tir and EspF was blocked by MbetaCD treatment, although the TTSS pore still formed. MbetaCD treatment after Tir delivery decreased pedestal formation by EPEC from 40% to 5%, but not by the related pathogen E. coli O157:H7 which uses a different Tir-based mechanism. In contrast, EPEC expressing the BFP can circumvent the requirement for membrane cholesterol. This suggests that lipid rafts play a role in virulence of this medically important pathogen.     

Enteropathogenic Escherichia coli: cellular harassment

The mechanisms by which enteropathogenic Escherichia coli (EPEC) mediates diarrhea remain a mystery. Recently a number of interesting and at times surprising results have come from studying EPEC interactions with host cells. Identification and characterization of bacterial factors, including Tir, EspA, EspB and EspD, and host responses have expanded our grasp of the diverse effects of EPEC on host cells.     

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.     

Cloning and characterization of the Escherichia coli gene coding for alkaline phosphatase.

The Escherichia coli structural gene for alkaline phosphatase, phoA, and a promoter-like mutant of phoA, called pho-1003(Bin) phoA+, were cloned by using plasmid vectors. Initially, these genes were cloned on deoxyribonucleic acid fragments of 28.9 kilobases (kb). Subsequently, they were subcloned on fragments and 4.8 and then 2.7 kilobases. A restriction map was developed, and phoA was localized to a 1.7-kb region. The promoter end of the gene was inferred by its proximity to another gene cloned on the same deoxyribonucleic acid fragment, proC. The stability of the largest plasmid (33.3 kb) was found to be recA dependent, although the subcloned plasmids were stable in a recA+ strain. Synthesis of alkaline phosphatase directed by the phoA+ and pho-1003(Bin) phoA+ plasmids in a phoA deletion strain was assayed under repressing and derepressing levels of phosphate. These data were compared with the copy numbers of the plasmids. It was found that synthesis of alkaline phosphatase was tightly regulated, even under derepressing conditions: a copy number of 17 enabled cells to synthesize only about twofold more enzyme than did cells with 1 chromosomal copy of phoA+. Enzyme levels were also compared for cells containing pho-1003(Bin) phoA+ and phoA+.     

Expression in Escherichia coli of a synthetic gene coding for horse heart myoglobin

A gene for expression of horse heart myoglobin in Escherichia coli has been constructed in one step from long synthetic oligonucleotides. The synthetic gene contains an efficient translation initiation signal and used codons that are commonly found in E. coli. Unique restriction sites are placed throughout the gene. It has been inserted in a phagemid vector and is expressed from the lac promoter in E. coli at high efficiency, the soluble heme protein representing approximately 10% of soluble protein. Two versions of horse heart myoglobin were produced with aspartic acid or asparagine at residue 122. Comparison of chromatographic mobilities of these two proteins with authentic horse heart myoglobin identified aspartic acid as the correct residue 122. The availability of this gene, which is designed to facilitate oligonucleotide mutagenesis or cassette mutagenesis, will allow systematic structure-function analysis of horse heart myoglobin.     

Molecular cloning of the Escherichia coli B L-fucose-D-arabinose gene cluster.

To metabolize the uncommon pentose D-arabinose, enteric bacteria often recruit the enzymes of the L-fucose pathway by a regulatory mutation. However, Escherichia coli B can grow on D-arabinose without the requirement of a mutation, using some of the L-fucose enzymes and a D-ribulokinase that is distinct from the L-fuculokinase of the L-fucose pathway. To study this naturally occurring D-arabinose pathway, we cloned and partially characterized the E. coli B L-fucose-D-arabinose gene cluster and compared it with the L-fucose gene cluster of E. coli K-12. The order of the fucA, -P, -I, and -K genes was the same in the two E. coli strains. However, the E. coli B gene cluster contained a 5.2-kb segment located between the fucA and fucP genes that was not present in E. coli K-12. This segment carried the darK gene, which encodes the D-ribulokinase needed for growth on D-arabinose by E. coli B. The darK gene was not homologous with any of the L-fucose genes or with chromosomal DNA from other D-arabinose-utilizing bacteria. D-Ribulokinase and L-fuculokinase were purified to apparent homogeneity and partially characterized. The molecular weights, substrate specificities, and kinetic parameters of these two enzymes were very dissimilar, which together with DNA hybridization analysis, suggested that these enzymes are not related. D-Arabinose metabolism by E. coli B appears to be the result of acquisitive evolution, but the source of the darK gene has not been determined.     

The O-antigen gene cluster of Escherichia coli O55:H7 and identification of a new UDP-GlcNAc C4 epimerase gene

Escherichia coli O55 is an important antigen which is often associated with enteropathogenic E. coli clones. We sequenced the genes responsible for its synthesis and identified genes for O-antigen polymerase, O-antigen flippase, four enzymes involved in GDP-colitose synthesis, and three glycosyltransferases, all by comparison with known genes. Upstream of the normal O-antigen region there is a gne gene, which encodes a UDP-GlcNAc epimerase for converting UDP-GlcNAc to UDP-GalNAc and is essential for O55 antigen synthesis. The O55 gne product has only 20 and 26% identity to the gne genes of Pseudomonas aeruginosa and E. coli O113, respectively. We also found evidence for the O55 gene cluster's having evolved from another gene cluster by gain and loss of genes. Only three of the GDP-colitose pathway genes are in the usual location, the other two being separated, although nearby. It is thought that the E. coli O157:H7 clone evolved from the O55:H7 clone in part by transfer of the O157 gene cluster into an O55 lineage. Comparison of genes flanking the O-antigen gene clusters of the O55:H7 and O157:H7 clones revealed one recombination site within the galF gene and located the other between the hisG and amn genes. Genes outside the recombination sites are 99.6 to 100% identical in the two clones, while most genes thought to have transferred with the O157 gene cluster are 95 to 98% identical.