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Biomedical subjects

S J Hultgren

Publications and source records attributed to S J Hultgren.

At least 73 records · Page 4Linked to original sources
PubMed1994

PapD chaperone function in pilus biogenesis depends on oxidant and chaperone-like activities of DsbA.

Adhesive P pili of uropathogenic Escherichia coli were not assembled by a strain that lacks the periplasmic disulfide isomerase DsbA. This defect was mostly attributed to the immunoglobulin-like pilus chaperone PapD, which possesses an unusual intrasheet disulfide bond between the last two beta-strands of its CD4-like carboxyl-terminal domain. The DsbA-dependent formation of this disulfide bond was critical for PapD's proper folding in vivo. Interestingly, the absence of the disulfide bond did not prevent PapD from folding in vitro or from forming a complex with the pilus adhesin in vitro. We suggest that DsbA maintains nascently translocated PapD in a folding-competent conformation prior to catalyzing disulfide bond formation, acting both as an oxidant and in a chaperone-like fashion. Disulfide bond formation in pilus subunits was also mediated by DsbA even in the absence of PapD. However, the ability of pilus subunits to achieve native-like conformations in vivo depended on PapD. These results suggest that a productive folding pathway for subunits requires sequential interactions with DsbA and the PapD chaperone.

Bacterial Proteins↗
PubMed1994

Chaperone-assisted self-assembly of pili independent of cellular energy.

Assembly of P pili on the surface of pyelonephritic Escherichia coli proceeds from periplasmic chaperone-subunit complexes. The outer membrane protein PapC, which has been termed a molecular usher, is thought to be the site of assembly, where the chaperone dissociates from the subunits as they are incorporated into the pilus across the outer membrane. The kinetics of assembly and the energy requirements of the "secretion" events at the outer membrane were investigated using a pulse-chase analysis in which preformed labeled periplasmic chaperone-subunit complexes were assembled into pili in synchrony by the induction of PapC. Provided that a sufficient amount of PapC was present and functional in the outer membrane, the incorporation of the major PapA subunit into pili was shown to be completed in less than 5 min. Our results also indicated that the targeting of PapC to the outer membrane may be a rate-limiting factor for pilus assembly. Following the arrival of PapC, the formation of pili seemed to proceed spontaneously and was not sensitive to a pH shift or an inhibitor of the electrochemical gradient across the cytoplasmic membrane. We suggest that the secretion of pili across the outer membrane may be independent of cellular energy and thermodynamically driven.

Bacterial Outer Membrane Proteins↗
PubMed1994

Stable fiber-forming and nonfiber-forming chaperone-subunit complexes in pilus biogenesis.

The P pilus is a composite fiber consisting of a thin adhesive tip fibrillum joined to the pilus rod that mediates specific adherence of uropathogenic Escherichia coli to human uroepithelial cells via the PapG tip adhesin. P pilus assembly depends upon the periplasmic chaperone PapD. The interaction of PapD with different pilus subunits was investigated to gain further insight into pilus assembly. PapA, the major subunit of the pilus rod, formed two periplasmic complexes (DA2 and DA) with PapD. PapK, an adaptor protein that joins the tip fibrillum to the pilus rod, formed only one complex with PapD (DK). Only "fiber forming" or homopolymeric subunits, PapA in the rod and PapE in the tip fibrillum, were able to form subunit-subunit interactions in the periplasm. Subunits that are present in single or low copy in the pilus (PapK and PapG) did not form periplasmic intersubunit interactions. A pulse-chase analysis revealed that a chaperone-PapA complex is a true periplasmic intermediate in pilus assembly.

Bacterial Proteins↗
PubMed1994

The PapG tip adhesin of P fimbriae protects Escherichia coli from neutrophil bactericidal activity.

Compared with Escherichia coli ORN103, a nonfimbriated K-12 strain, P-fimbriated E. coli ORN103/pPAP5 was found to interact poorly with human neutrophils and resist their bactericidal activity in vitro. PapG, the Gal alpha(1-->4)Gal binding moiety located at the distal end of the P fimbrial filament, appeared to be responsible for this effect because an isogenic PapG- mutant, E. coli ORN103/pPAP24, exhibited binding interactions with neutrophils that were similar to nonfimbriated E. coli ORN103. Although no direct evidence is available, the poor adherence mediated by PapG could be related to its electrostatic properties because the isolated PapG protein had a pI of 5.2, which indicated that in the physiological pH range it possessed a net negative charge. Antibodies against PapG overcame the protective effect of PapG and markedly enhanced the interactions of P-fimbriated E. coli with neutrophils resulting in bacterial killing. When a P-fimbriated clinical E. coli strain or its isogenic PapG- derivative was injected into the peritoneal cavities of mice, a similar number of neutrophils was recruited to the site of injection. After 2 h, the number of P-fimbriated E. coli organisms that survived the neutrophil influx in the mouse peritoneum was approximately four times more than the number of surviving PapG- bacteria. This result demonstrates that the PapG protein, which is strategically located at the distal region of the P-fibrillum structure, protects E. coli from the bactericidal action of neutrophils.

Animals↗
PubMed1993

Structural basis of pilus subunit recognition by the PapD chaperone.

The assembly of different types of virulence-associated surface fibers called pili in Gram-negative bacteria requires periplasmic chaperones. PapD is the prototype member of the periplasmic chaperone family, and the structural basis of its interactions with pilus subunits was investigated. Peptides corresponding to the carboxyl terminus of pilus subunits bound PapD and blocked the ability of PapD to bind to the pilus adhesin PapG in vitro. The crystal structure of PapD complexed to the PapG carboxyl-terminal peptide was determined to 3.0 A resolution. The peptide bound in an extended conformation with its carboxyl terminus anchored in the interdomain cleft of the chaperone via hydrogen bonds to invariant chaperone residues Arg8 and Lys112. Main chain hydrogen bonds and contacts between hydrophobic residues in the peptide and the chaperone stabilized the complex and may play a role in determining binding specificity. Site-directed mutations in Arg8 and Lys112 abolished the ability of PapD to bind pilus subunits and mediate pilus assembly in vivo, an indication that the PapD-peptide crystal structure is a reflection of at least part of the PapD-subunit interaction.

Amino Acid Sequence↗
PubMed1993

FimC is a periplasmic PapD-like chaperone that directs assembly of type 1 pili in bacteria.

Biogenesis of the type 1 pilus fiber in Escherichia coli requires the product of the fimC locus. We have demonstrated that FimC is a member of the periplasmic chaperone family. The deduced primary sequence of FimC shows a high degree of homology to PapD and fits well with the derived consensus sequence for periplasmic chaperones, predicting that it has an immunoglobulin-like topology. The chaperone activity of FimC was demonstrated by purifying a complex that FimC forms with the FimH adhesion. A fimC1 null allele could be complemented by the prototype member of the chaperone superfamily, PapD, resulting in the production of adhesive type 1 pili. The general mechanism of action of members of the chaperone superfamily was demonstrated by showing that the ability of PapD to assemble both P and type 1 pili was dependent on an invariant arginine residue (Arg-8), which forms part of a conserved subunit binding site in the cleft of PapD. We suggest that the conserved cleft is a subunit binding feature of all members of this protein family. These studies point out the general strategies used by Gram-negative bacteria to assemble adhesins into pilus fibers, allowing them to promote attachment to eukaryotic receptors.

Adhesins, Escherichia coli↗
PubMed1993

Outer-membrane PapC molecular usher discriminately recognizes periplasmic chaperone-pilus subunit complexes.

P pili are highly ordered composite structures consisting of thin fibrillar tips joined end-to-end to rigid helical rods. The production of these virulence-associated structures requires a periplasmic chaperone (PapD) and an outer membrane protein (PapC) that is the prototype member of a newly recognized class of proteins that we have named "molecular ushers." Two in vitro assays showed that the preassembly complexes that PapD forms with the three most distal tip fibrillar proteins (PapG, PapF, and PapE) bound to PapC. The relative affinity of each complex for PapC was found to correlate with the final position of the subunit type in the tip fibrillum. In contrast, the complexes PapD forms with the major component of the pilus rod, PapA, or the pilus rod initiating protein, PapK, did not recognize PapC. The in vitro data argue that differential targeting of chaperone-subunit complexes to PapC may be part of a mechanism to ensure the correctly ordered assembly of adhesive composite pili.

Adhesins, Escherichia coli↗
PubMed1993

Neutrophil activation by nascent FimH subunits of type 1 fimbriae purified from the periplasm of Escherichia coli.

Previous studies of type 1 fimbriae of Escherichia coli have implicated FimH, a minor subunit, as the determinant of its mannose binding property. Structure-function analysis of FimH has not been possible because of the difficulty in obtaining adequate amounts of the subunit from type 1 fimbriae. We have obtained nascent FimH that has not been incorporated into the fimbrial structure from the periplasm of an E. coli strain expressing the cloned fimH gene. Nascently translocated FimH was initially degraded in the periplasm; however, when co-expressed with FimC, a putative fimbrial chaperone, the FimH molecules were stabilized and readily isolated from the periplasmic extract by fractionation on a sodium dodecyl sulfate-polyacrylamide gel followed by electroelution of the FimH band from the gel. The eluted protein was purified to homogeneity by affinity chromatography on a mannose-Sepharose column. Purified FimH displayed the same mannose-inhibitable binding to human neutrophils as type 1 fimbriated bacteria, including triggering an oxidative burst with concomitant release of reactive oxygen metabolites. In addition, inert microspheres coated with FimH, but not those coated with bovine serum albumin, were phagocytosed by neutrophils. These data provide direct evidence that FimH is the determinant on type 1 fimbriae which is responsible for mediating mannose-specific adherence and that isolated FimH is a potent activator of human neutrophils.

Adhesins, Escherichia coli↗
PubMed1993

A novel secretion apparatus for the assembly of adhesive bacterial pili.

The biogenesis of most types of bacterial pili requires two specialized proteins: a chaperone that caps the pilus subunits in the periplasm, and an outer membrane usher that receives the subunits and serves as an assembly platform. This secretion and assembly machinery is proposed to be a novel export apparatus found widely in Gram-negative pathogens.

Bacterial Adhesion↗
PubMed1993

PapD and superfamily of periplasmic immunoglobulin-like pilus chaperones.

The formation of a P pilus requires a molecular chaperone in the periplasm and a molecular usher in the outer membrane. Each pilus is composed of six different types of proteins that are assembled into a composite fiber in a defined order. The correct folding of subunits into domains that can serve as assembly modules requires an association with the periplasmic chaperone. PapD is the prototype member of the family of bacterial pilus chaperones that have a three-dimensional structure consistent with an immunoglobulin fold. In general, proteins with an immunoglobulin fold structure have molecular recognition functions in eukaryotic cells that are often integrated with effector functions. PapD has also a recognition function, binding nascently translocated pilus subunits and maintaining them in assembly-competent conformations. The association of the chaperone with the subunit triggers the targeting of the latter to an outer membrane usher. The usher serves as a molecular gatekeeper, allowing the ordered incorporation of the pilus subunits into the pilus structure from the periplasmic chaperone complexes. The two immunoglobulin-like domains of PapD are oriented to form a cleft that contains the subunit binding site. This is a different binding paradigm from that used by either antibodies or the growth hormone receptor. The blend of genetics, biochemistry, X-ray crystallography, and carbohydrate chemistry in the study of pili biogenesis will continue to give insight into some of the most basic intellectual challenges in molecular biology concerning how proteins fold into domains that serve as modules for the formation of larger assemblies, and relating these processes to microbial pathogenesis.

Adhesins, Escherichia coli↗
PubMed1992

P pili in uropathogenic E. coli are composite fibres with distinct fibrillar adhesive tips.

Escherichia coli is a frequent cause of several common bacterial infections in humans and animals, including urinary tract infections, bacteraemia and bacteria-related diarrhoea and is also the main cause of neonatal meningitis. Microbial attachment to surfaces is a key event in colonization and infection and results mainly from a stereochemical fit between microbial adhesins and complementary receptors on host cells. Bacterial adhesins required for extracellular colonization by Gram-negative bacteria are often minor components of heteropolymeric fibres called pili which must be oriented in an accessible manner in these structures to be able to bind to specific receptor architectures. P pili mediate the binding of uropathogenic E. coli to a digalactoside receptor determinant present in the urinary tract epithelium. We report here that the adhesin is a component of distinct fibrillar structures present at the tips of the pili. These virulence-associated tip fibrillae are thin, flexible polymers composed mostly of repeating subunits of PapE that frequently terminate with the alpha-D-galactopyranosyl-(1-4)-beta-D-galactopyranose or Gal alpha (1-4)Gal binding PapG adhesin.

Adhesins, Escherichia coli↗
PubMed1992

Conserved immunoglobulin-like features in a family of periplasmic pilus chaperones in bacteria.

Detailed structural analyses revealed a family of periplasmic chaperones in Gram-negative prokaryotes which are structurally and possibly evolutionarily related to the immunoglobulin superfamily and assist in the assembly of adhesive pili. The members of this family have similar structures consistent with the overall topology of an immunoglobulin fold. Seven pilus chaperone sequences from Escherichia coli, Haemophilus influenzae and Klebsiella pneumoniae were aligned and their consensus sequence was superimposed onto the known three-dimensional structure of PapD, a representative member of the family. The molecular details of the conserved and variable structural motifs in this family of periplasmic chaperones give important insight into their structure, function, mechanism of action and evolutionary relationship with the immunoglobulin superfamily.

Adhesins, Escherichia coli↗
PubMed1992

Interactive surface in the PapD chaperone cleft is conserved in pilus chaperone superfamily and essential in subunit recognition and assembly.

The assembly of adhesive pili in Gram-negative bacteria is modulated by specialized periplasmic chaperone systems. PapD is the prototype member of the superfamily of periplasmic pilus chaperones. Previously, the alignment of chaperone sequences superimposed on the three dimensional structure of PapD revealed the presence of invariant, conserved and variable amino acids. Representative residues that protruded into the PapD cleft were targeted for site directed mutagenesis to investigate the pilus protein binding site of the chaperone. The ability of PapD to bind to fiber-forming pilus subunit proteins to prevent their participation in misassembly interactions depended on the invariant, solvent-exposed arginine-8 (R8) cleft residue. This residue was also essential for the interaction between PapD and a minor pilus adaptor protein. A mutation in the conserved methionine-172 (M172) cleft residue abolished PapD function when this mutant protein was expressed below a critical threshold level. In contrast, radical changes in the variable residue glutamic acid-167 (E167) had little or no effect on PapD function. These studies provide the first molecular details of how a periplasmic pilus chaperone binds to nascently translocated pilus subunits to guide their assembly into adhesive pili.

Adhesins, Escherichia coli↗
PubMed1991

Immunoglobulin-like PapD chaperone caps and uncaps interactive surfaces of nascently translocated pilus subunits.

Molecular chaperones are found in the cytoplasm of bacteria and in various cellular compartments in eukaryotes to maintain proteins in nonnative conformations that permit their secretion across membranes or assembly into oligomeric structures. Virtually nothing, however, has been reported about a similar requirement for molecular chaperones in the periplasm of Gram-negative bacteria. We used the well-characterized P pilus biogenesis system in Escherichia coli as a model to elucidate the mechanism of action of a periplasmic chaperone, PapD, which is specifically required for P pilus biogenesis. PapD probably associates with at least six P pilus subunits after their secretion across the cytoplasmic membrane, but PapD is not incorporated into the pilus. We used purified periplasmic complex that PapD forms with the PapG adhesin to investigate the function of interactions between the chaperone and its targets. We demonstrated that PapD binds to PapG to form a stable, discrete bimolecular complex and that, unlike cytoplasmic chaperones, the periplasmic PapD chaperone maintained PapG in a native-like conformation. Bound PapD in the complex was displaced by free PapD in vitro; however, the in vivo release of subunits to the nascent pilus is probably driven by an ATP-independent mechanism involving the outer membrane protein PapC. In addition, the binding of PapD to PapG in vitro prevented aggregation of PapG. We propose that the function of PapD and other periplasmic pilus chaperones is to partition newly translocated pilus subunits into assembly-competent complexes and thereby prevent nonproductive aggregation of the subunits in the periplasm. These data provide important information for understanding the mechanism of action of this general class of chaperones that function in the periplasmic space.

Adhesins, Escherichia coli↗
PubMed1991

Biogenesis of the bacterial pilus.

The assembly of surface structures in gram-negative bacteria requires specialized secretion and chaperone systems localized on both sides of the cytoplasmic membrane. Major advances have been made over the last year in understanding how these systems form part of a general strategy used by bacteria to cap and target interactive subunits imported into the periplasmic space to outer membrane uncapping and assembly sites.

Bacterial Outer Membrane Proteins↗