Research

Urinary Tract InfectionUPEC genomics/geneticsChaperone/Usher Pathway PiliEnterococcal pathogenesis/catheter-associated UTIBacterial amyloid (Curli) biogenesisBacterial Community Interactions (Biofilms and Microbiome)Drug and Vaccine Development

Chaperone/Usher Pathway Pili

Extracellular fibers called chaperone-usher pathway (CUP) pili are critical virulence factors in a wide range of pathogenic Gram-negative bacteria. Today, with genome sequencing and varied infection models, we now know that this is a wide-spread family of hundreds of different extracellular fibers, many with essential adherence functions in various niches and body habitats and/or in biofilm formation in diverse Gram-negative bacteria including E. coli, Klebsiella, Pseudomonas, Haemophilus, Salmonella and Yersiniae. We use the type 1 pili and P pili systems, which enable UPEC to cause bladder and kidney infections, respectively, as models to understand the assembly of CUP pili. We first discovered that P pili (and later type 1 and S pili) were multicomponent fibers, each consisting of a helical rod joined end to end to a linear tip fibrillum, which contains a two-domain adhesin at its distal end. Elucidating how these structures are built is uncovering basic principles of protein folding, chaperone function and macromolecular assembly.
Collaborators: Gabriel Waksman, Han Remaut, Carl Frieden

CUP Pilus biogenesis: We discovered that the molecular basis of chaperone-assisted pilus subunit folding occurrs by a reaction we termed donor strand complementation (DSC) in which conserved alternating exposed hydrophobic residues on the chaperone’s G1 strand are buried in complementary pockets in the subunit, completing the subunit fold. The crystal structures of each of the five immunoglobulin (Ig)-like P pilins and the Saf pilins in complexes with their respective chaperones revealed that subunits are held in a primed high-energy state having incompletely collapsed hydrophobic cores when bound to the chaperone. These primed complexes are targeted to the outer membrane usher, which is a gated channel that catalyzes pilus assembly by driving subunit polymerization in a reaction we termed donor strand exchange (DSE). All subunits, excluding the adhesin, contain a short N-terminal extension (Nte) comprised of conserved alternating hydrophobic residues (analogous, but not identical, to those on the G1 strand of the chaperone). In DSE, an incoming subunit’s Nte zips into the pockets of the chaperone-bound groove of a nascently incorporated subunit at the growing terminus of the pilus, resulting in chaperone dissociation. This allows the final folding of the subunit with the collapse of the hydrophobic core and the ordering of loop regions, such that every subunit in the pilus completes the Ig fold of its predecessor. The energy for pilus assembly is derived in part by this stepwise folding process. Current studies are elucidating the mechanistic events at the usher, which promote proper ordering, assembly and extrusion of the final pilus fiber to the extracellular surface of the bacteria.

CUP adhesins: Distal CUP pilus adhesins are two-domain proteins, consisting of an N terminal receptor-binding domain and a C terminal pilin domain. The N terminal receptor-binding domain binds to host or environmental moieties, and thus has important influences on bacterial tropism and initiation of infections. The pilin domain links the receptor-binding domain to the tip of the pilus via donor strand exchange. We have solved the structure of multiple CUP pilus adhesins including the FimH, type 1 pilus adhesin, and the PapG, P pilus adhesin. Through the analysis of multiple FimH sequences from E. coli isolates combined with functional binding and bladder infections studies, we discovered that pathoadaptive alleles of FimH with variant residues outside the mannose binding pocket, affect transitions between low and high-affinity FimH conformations and impact FimH-mediated pathogenesis. In vitro binding studies revealed that while all pathoadaptive variants tested displayed the same high affinity for mannose when bound by the chaperone FimC, affinities varied when FimH was incorporated into pilus tip structures. Structural studies have shown that FimH adopts an elongated conformation when complexed with FimC, but when incorporated into the pilus tip, FimH can also adopt a compact conformation. Interestingly, FimH variants, which maintain a high-affinity conformation in the pilus tip were attenuated during chronic bladder infection arguing that FimH’s ability to switch between conformations is important in pathogenesis. Further work to understand possible conformer equilibriums within CUP adhesins, as well as the specificity of the myriad of CUP adhesins encoded by bacteria will give important insights into bacterial tropism and disease initiation.

CUP regulation/cross-talk: Studies on type 1, S and P pili have demonstrated multiple regulatory elements controlling their expression. Global transcriptional regulators are known to affect multiple CUP operons. Further, each CUP operon appears to have specific associated regulators, which impact expression. Both type 1 and P pili are regulated by a process termed phase variation, where individual bacteria have all-or-none expression of pili despite the population being subject to the same environmental conditions. The fim operon is under phase variable expression regulated by site specific recombinases (FimB, FimE, FimX and others), which can switch the orientation of the fim promoter (fimS) by acting on 9bp palindromic sequences flanking fimS, In contrast, pap phase variable switching is governed by differential DAM-DNA methylation at two GATC sites, which alters regulator binding. Additional studies confirm these general themes for the regulation and function of other CUP operons as well as provide evidence that under any given condition each bacterium likely only expresses one CUP system at a time, perhaps to insure fidelity of assembly and outer membrane integrity. This apparent hierarchy of CUP expression argues for cross-regulation between the CUP operons. We are characterizing the expression of all CUP operons in a prototypical UPEC isolate, UTI89, under a variety of growth conditions and in a variety of mutants in general regulators. This work will provide insights into bacterial mechanisms that determine tropism and potential compensatory mechanisms that UPEC may use against chemotherapeutics that target a particular CUP system.

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