Structural basis of the interaction of the pyelonephritic E. coli adhesin to its human kidney receptor.

PapG is the adhesin at the tip of the P pilus that mediates attachment of uropathogenic Escherichia coli to the uroepithelium of the human kidney. The human specific allele of PapG binds to globoside (GbO4), which consists of the tetrasaccharide GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc linked to ceramide. Here, we present the crystal structures of a binary complex of the PapG receptor binding domain bound to GbO4 as well as the unbound form of the adhesin. The biological importance of each of the residues involved in binding was investigated by site-directed mutagenesis. These studies provide a molecular snapshot of a host-pathogen interaction that determines the tropism of uropathogenic E. coli for the human kidney and is critical to the pathogenesis of pyelonephritis.

Structural basis of chaperone function and pilus biogenesis.

Many Gram-negative pathogens assemble architecturally and functionally diverse adhesive pili on their surfaces by the chaperone-usher pathway. Immunoglobulin-like periplasmic chaperones escort pilus subunits to the usher, a large protein complex that facilitates the translocation and assembly of subunits across the outer membrane. The crystal structure of the PapD-PapK chaperone-subunit complex, determined at 2.4 angstrom resolution, reveals that the chaperone functions by donating its G(1) beta strand to complete the immunoglobulin-like fold of the subunit via a mechanism termed donor strand complementation. The structure of the PapD-PapK complex also suggests that during pilus biogenesis, every subunit completes the immunoglobulin-like fold of its neighboring subunit via a mechanism termed donor strand exchange.

Periplasmic chaperone recognition motif of subunits mediates quaternary interactions in the pilus.

The class of proteins collectively known as periplasmic immunoglobulin-like chaperones play an essential role in the assembly of a diverse set of adhesive organelles used by pathogenic strains of Gram-negative bacteria. Herein, we present a combination of genetic and structural data that sheds new light on chaperone-subunit and subunit-subunit interactions in the prototypical P pilus system, and provides new insights into how PapD controls pilus biogenesis. New crystallographic data of PapD with the C-terminal fragment of a subunit suggest a mechanism for how periplasmic chaperones mediate the extraction of pilus subunits from the inner membrane, a prerequisite step for subunit folding. In addition, the conserved N- and C-terminal regions of pilus subunits are shown to participate in the quaternary interactions of the mature pilus following their uncapping by the chaperone. By coupling the folding of subunit proteins to the capping of their nascent assembly surfaces, periplasmic chaperones are thereby able to protect pilus subunits from premature oligomerization until their delivery to the outer membrane assembly site.

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.

A sequence at the inside end of IS50 down regulates transposition.

Deletion analysis has shown that the segment at the IS50 inside (I) end that is needed for efficient transposition is approximately 19 bp long. Dam methylation at two 5' GATC sequences within this segment decreases I-end transposition activity. A third 5' GATC sequence is present at bp 21-24 of the I end. The comparisons presented here show that extension of the I end from 19 to 24 bp decreases its transposition activity in dam cells 5- to 50-fold, depending on the overall transposon structure.

Saturation mutagenesis of the inside end of insertion sequence IS50.

A 19-bp segment at the inside (I) end of IS50 (Tn5) is needed for efficient transposition. The importance of each position was assayed by making at least one base substitution at each position by either chemical-or oligodeoxyribonucleotide-directed mutagenesis. Mutant I ends were paired with a wild-type (wt) segment from the outside (O) end of IS50 and the transposase (tnp) gene was placed either between the ends or 1200 bp from the O end. The frequency of transposition of the resultant elements to bacteriophage lambda was measured. At least one substitution at each of the 19 I-end positions decreased transposition activity to less than 25% of wt, and most substitutions (25 of 28) decreased it to less than 5% of wt from one or both donor plasmids. These results show that each position in the I end is important during transposition.

Factors affecting transposition activity of IS50 and Tn5 ends.

The partially matched I and O ends of IS50 (the insertion sequence of the transposon Tn5) are needed for transposition, probably as the sites upon which the cis-acting transposase and host proteins act. To better understand how transposition is regulated we made a series of IS50-related elements in which the positions of the ends and of the transposase gene were varied systematically. Assays of these elements showed that the I and O ends differ inherently in transposition activity. Other workers showed that methylation, at DNA N6-adenine methyltransferase (Dam) recognition sites within the I end and the transposase tnp gene promoter, inhibits transposase synthesis and also I end activity. We show that the effect of Dammediated methylation on an I end depends on the end's orientation relative to the tnp gene. Further, in dam+ cells oriented like -tnp----in relation to the first and second ends) are (O, I) greater than (O, O) greater than or equal to (I, O) greater than (I, I). In dam- cells the relative activities are (O, I) = (I, O) = (I, I) greater than (O, O). Our results are consistent with a model orginally developed for IS10, in which hemi-methylation resulting from passage of a replication fork regulates transposition.