Jerome S. Pinkner

Type 1 pilus‐mediated bacterial invasion of bladder epithelial cells

Most strains of uropathogenic Escherichia coli (UPEC) encode filamentous adhesive organelles called type 1 pili. We have determined that the type 1 pilus adhesin, FimH, mediates not only bacterial adherence, but also invasion of human bladder epithelial cells. In contrast, adherence mediated by another pilus adhesin, PapG, did not initiate bacterial internalization. FimH‐mediated invasion required localized host actin reorganization, phosphoinositide 3‐kinase (PI 3‐kinase) activation and host protein tyrosine phosphorylation, but not activation of Src‐family tyrosine kinases. Phosphorylation of focal adhesin kinase (FAK) at Tyr397 and the formation of complexes between FAK and PI 3‐kinase and between α‐actinin and vinculin were found to correlate with type 1 pilus‐mediated bacterial invasion. Inhibitors that prevented bacterial invasion also blocked the formation of these complexes. Our results demonstrate that UPEC strains are not strictly extracellular pathogens and that the type 1 pilus adhesin FimH can directly trigger host cell signaling cascades that lead to bacterial internalization.

Structural basis of tropism of Escherichia coli to the bladder during urinary tract infection

The first step in the colonization of the human urinary tract by pathogenic Escherichia coli is the mannose‐sensitive binding of FimH, the adhesin present at the tip of type 1 pili, to the bladder epithelium. We elucidated crystallographically the interactions of FimH with D‐mannose. The unique site binding pocket occupied by D‐mannose was probed using site‐directed mutagenesis. All but one of the mutants examined had greatly diminished mannose‐binding activity and had also lost the ability to bind human bladder cells. The binding activity of the mono‐saccharide D‐mannose was delineated from this of mannotriose (Man(α1–3)[Man(α1–6)]Man) by gener‐ating mutants that abolished D‐mannose binding but retained mannotriose binding activity. Our structure/function analysis demonstrated that the binding of the monosaccharide α‐D‐mannose is the primary bladder cell receptor for uropathogenic E. coli and that this event requires a highly conserved FimH binding pocket. The residues in the FimH mannose‐binding pocket were sequenced and found to be invariant in over 200 uropathogenic strains of E. coli. Only enterohaemorrhagic E. coli (EHEC) possess a sequence variation within the mannose‐binding pocket of FimH, suggesting a naturally occurring mechanism of attenuation in EHEC bacteria that would prevent them from being targeted to the urinary tract.

Receptor binding studies disclose a novel class of high‐affinity inhibitors of the Escherichia coli FimH adhesin

Mannose‐binding type 1 pili are important virulence factors for the establishment of Escherichia coli urinary tract infections (UTIs). These infections are initiated by adhesion of uropathogenic E. coli to uroplakin receptors in the uroepithelium via the FimH adhesin located at the tips of type 1 pili. Blocking of bacterial adhesion is able to prevent infection. Here, we provide for the first time binding data of the molecular events underlying type 1 fimbrial adherence, by crystallographic analyses of the FimH receptor binding domains from a uropathogenic and a K‐12 strain, and affinity measurements with mannose, common mono‐ and disaccharides, and a series of alkyl and aryl mannosides. Our results illustrate that the lectin domain of the FimH adhesin is a stable and functional entity and that an exogenous butyl α‐ d‐mannoside, bound in  the  crystal  structures,  exhibits  a  significantly better affinity for FimH (Kd = 0.15 µM) than mannose (Kd = 2.3 µM). Exploration of the binding affinities of α‐ d‐mannosides with longer alkyl tails revealed affinities up to 5 nM. Aryl mannosides and fructose can also bind with high affinities to the FimH lectin domain, with a 100‐fold improvement and 15‐fold reduction in affinity, respectively, compared with mannose. Taken together, these relative FimH affinities correlate exceptionally well with the relative concentrations of the same glycans needed for the inhibition of adherence of type 1 piliated E. coli. We foresee that our findings will spark new ideas and initiatives for the development of UTI vaccines and anti‐adhesive drugs to prevent anticipated and recurrent UTIs.

Vaccination with FimH Adhesin Protects Cynomolgus Monkeys from Colonization and Infection by Uropathogenic Eschevichia coli

Escherichia coli FimH adhesin mediates binding to the bladder mucosa. In mice, a FimH vaccine protects against bacterial challenge. In this study, 4 monkeys were inoculated with 100 microgram of FimCH adhesin-chaperone complex mixed with MF59 adjuvant, and 4 monkeys were given adjuvant only intramuscularly. After 2 doses (day 0 and week 4), a booster at 48 weeks elicited a strong IgG antibody response to FimH in the vaccinated monkeys. All 8 monkeys were challenged with 1 mL of 108 E. coli cystitis isolate NU14. Three of the 4 vaccinated monkeys were protected from bacteruria and pyuria; all control monkeys were infected. These findings suggest that a vaccine based on the FimH adhesin of E. coli type 1 pili may have utility in preventing cystitis in humans.

Small-molecule inhibitors target Escherichia coli amyloid biogenesis and biofilm formation

Curli are functional extracellular amyloid fibers produced by uropathogenic Escherichia coli (UPEC) and other Enterobacteriaceae. Ring-fused 2-pyridones, such as FN075 and BibC6, inhibited curli biogenesis in UPEC and prevented the in vitro polymerization of the major curli subunit protein CsgA. The curlicides FN075 and BibC6 share a common chemical lineage with other ring-fused 2-pyridones termed pilicides. Pilicides inhibit the assembly of type 1 pili, which are required for pathogenesis during urinary tract infection. Notably, the curlicides retained pilicide activities and inhibited both curli-dependent and type 1-dependent biofilms. Furthermore, pretreatment of UPEC with FN075 significantly attenuated virulence in a mouse model of urinary tract infection. Curli and type 1 pili exhibited exclusive and independent roles in promoting UPEC biofilms, and curli provided a fitness advantage in vivo. Thus, the ability of FN075 to block the biogenesis of both curli and type 1 pili endows unique anti-biofilm and anti-virulence activities on these compounds.

THE CHAPERONE-ASSISTED MEMBRANE RELEASE AND FOLDING PATHWAY IS SENSED BY TWO SIGNAL TRANSDUCTION SYSTEMS

The assembly of interactive protein subunits into extracellular structures, such as pilus fibers in the Enterobacteriaceae, is dependent on the activity of PapD‐like periplasmic chaperones. The ability of PapD to undergo a β zippering interaction with the hydrophobic C‐terminus of pilus subunits facilitates their folding and release from the cytoplasmic membrane into the periplasm. In the absence of the chaperone, subunits remained tethered to the membrane and were driven off‐pathway via non‐productive interactions. These off‐pathway reactions were detrimental to cell growth; wild‐type growth was restored by co‐expression of PapD. Subunit misfolding in the absence of PapD was sensed by two parallel pathways: the Cpx two‐component signaling system and the σE modulatory pathway.

Chaperone priming of pilus subunits facilitates a topological transition that drives fiber formation

Periplasmic chaperones direct the assembly of adhesive, multi-subunit pilus fibers that play critical roles in bacterial pathogenesis. Pilus assembly occurs via a donor strand exchange mechanism in which the N-terminal extension of one subunit replaces the chaperone G(1) strand that transiently occupies a groove in the neighboring subunit. Here, we show that the chaperone primes the subunit for assembly by holding the groove in an open, activated conformation. During donor strand exchange, the subunit undergoes a topological transition that triggers the closure of the groove and seals the N-terminal extension in place. It is this topological transition, made possible only by the priming action of the chaperone that drives subunit assembly into the fiber.

Rationally designed small compounds inhibit pilus biogenesis in uropathogenic bacteria

A chemical synthesis platform with broad applications and flexibility was rationally designed to inhibit biogenesis of adhesive pili assembled by the chaperone–usher pathway in Gram-negative pathogens. The activity of a family of bicyclic 2-pyridones, termed pilicides, was evaluated in two different pilus biogenesis systems in uropathogenic Escherichia coli. Hemagglutination mediated by either type 1 or P pili, adherence to bladder cells, and biofilm formation mediated by type 1 pili were all reduced by ≈90% in laboratory and clinical E. coli strains. The structure of the pilicide bound to the P pilus chaperone PapD revealed that the pilicide bound to the surface of the chaperone known to interact with the usher, the outer-membrane assembly platform where pili are assembled. Point mutations in the pilicide-binding site dramatically reduced pilus formation but did not block the ability of PapD to bind subunits and mediate their folding. Surface plasmon resonance experiments confirmed that the pilicide interfered with the binding of chaperone–subunit complexes to the usher. These pilicides thus target key virulence factors in pathogenic bacteria and represent a promising proof of concept for developing drugs that function by targeting virulence factors.

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.

Fiber Formation across the Bacterial Outer Membrane by the Chaperone/Usher Pathway

Gram-negative pathogens commonly exhibit adhesive pili on their surfaces that mediate specific attachment to the host. A major class of pili is assembled via the chaperone/usher pathway. Here, the structural basis for pilus fiber assembly and secretion performed by the outer membrane assembly platform--the usher--is revealed by the crystal structure of the translocation domain of the P pilus usher PapC and single particle cryo-electron microscopy imaging of the FimD usher bound to a translocating type 1 pilus assembly intermediate. These structures provide molecular snapshots of a twinned-pore translocation machinery in action. Unexpectedly, only one pore is used for secretion, while both usher protomers are used for chaperone-subunit complex recruitment. The translocating pore itself comprises 24 beta strands and is occluded by a folded plug domain, likely gated by a conformationally constrained beta-hairpin. These structures capture the secretion of a virulence factor across the outer membrane of gram-negative bacteria.