Jerry Pinkner

Molecular basis of two subfamilies of immunoglobulin-like chaperones.

The initial encounter of a microbial pathogen with the host often involves the recognition of host receptors by different kinds of bacterial adhesive organelles called pili, fimbriae, fibrillae or afimbrial adhesins. The development of over 26 of these architecturally diverse adhesive organelles in various Gram‐negative pathogens depends on periplasmic chaperones that are comprised of two immunoglobulin‐like domains. All of the chaperones possess a highly conserved sheet in domain 1 and a conserved interdomain hydrogen‐bonding network. Chaperone‐subunit complex formation depends on the anchoring of the carboxylate group of the subunit into the conserved crevice of the chaperone cleft and the subsequent positioning of the COOH terminus of subunits along the exposed edge of the conserved sheet of the chaperone. We discovered that the chaperones can be divided into two distinct subfamilies based upon conserved structural differences that occur in the conserved sheet. Interestingly, a subdivision of the chaperones based upon whether they assemble rod‐like pili or non‐pilus organelles that have an atypical morphology defines the same two subgroups. The molecular dissection of the two chaperone subfamilies and the adhesive fibers that they assemble has advanced our understanding of the development of virulence‐associated organelles in pathogenic bacteria.

Ramifications of kinetic partitioning on usher-mediated pilus biogenesis

The biogenesis of diverse adhesive structures in a variety of Gram‐negative bacterial species is dependent on the chaperone/usher pathway. Very little is known about how the usher protein translocates protein subunits across the outer membrane or how assembly of these adhesive structures occurs. We have discovered several mechanisms by which the usher protein acts to regulate the ordered assembly of type 1 pili, specifically through critical interactions of the chaperone–adhesin complex with the usher. A study of association and dissociation events of chaperone–subunit complexes with the usher in real time using surface plasmon resonance revealed that the chaperone–adhesin complex has the tightest and fastest association with the usher. This suggests that kinetic partitioning of chaperone–adhesin complexes to the usher is a defining factor in tip localization of the adhesin in the pilus. Furthermore, we identified and purified a chaperone–adhesin–usher assembly intermediate that was formed in vivo. Trypsin digestion assays showed that the usher in this complex was in an altered conformation, which was maintained during pilus assembly. The data support a model in which binding of the chaperone–adhesin complex to the usher stabilizes the usher in an assembly‐competent conformation and allows initiation of pilus assembly.

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.

Down-regulation of the kps region 1 capsular assembly operon following attachment of Escherichia coli type 1 fimbriae to D-mannose receptors.

ABSTRACT A differential-display PCR procedure identified the capsular assembly gene kpsD after Escherichia coli type 1 fimbrial binding to mannose-coated Sepharose beads. Limiting-dilution reverse-transcribed PCRs confirmed down-regulation of the kpsD gene, and Northern blot and lacZ fusion analyses showed down-regulation of the kpsFEDUCS region 1 operon. KpsD protein levels fell, and an agglutination test showed less K capsular antigen on the surface following the bacterial ligand-receptor interaction. These data show that binding of type 1 fimbriae (pili) to d-mannose receptors triggers a cross talk that leads to down-regulation of the capsule assembly region 1 operon in uropathogenic E. coli.