Vasilios Kalas

Positively selected FimH residues enhance virulence during urinary tract infection by altering FimH conformation.

Significance The evolution of multidrug resistance in pathogenic bacteria, including uropathogenic Escherichia coli (UPEC), that cause most urinary tract infections is becoming a worldwide crisis. UPEC use a variety of virulence factors and adhesins, including the mannose-binding FimH adhesin, to colonize and invade bladder tissue, often forming intracellular biofilms and quiescent reservoirs that can contribute to recurrent infections recalcitrant to treatment. Using two prototypical UPEC strains, we discovered that positively selected residues outside of the FimH mannose-binding pocket affect transitions between low- and high-affinity FimH conformations, which extraordinarily impacts FimH function during pathogenesis. Thus, this work elucidates mechanistic and functional insights into pathoadaptation and evolutionary fine-tuning of critical virulence interactions. Chaperone–usher pathway pili are a widespread family of extracellular, Gram-negative bacterial fibers with important roles in bacterial pathogenesis. Type 1 pili are important virulence factors in uropathogenic Escherichia coli (UPEC), which cause the majority of urinary tract infections (UTI). FimH, the type 1 adhesin, binds mannosylated glycoproteins on the surface of human and murine bladder cells, facilitating bacterial colonization, invasion, and formation of biofilm-like intracellular bacterial communities. The mannose-binding pocket of FimH is invariant among UPEC. We discovered that pathoadaptive alleles of FimH with variant residues outside the binding pocket affect FimH-mediated acute and chronic pathogenesis of two commonly studied UPEC strains, UTI89 and CFT073. In vitro binding studies revealed that, whereas 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-like, FimCGH complexes. Structural studies have shown that FimH adopts an elongated conformation when complexed with FimC, but, when incorporated into the pilus tip, FimH can adopt a compact conformation. We hypothesize that the propensity of FimH to adopt the elongated conformation in the tip corresponds to its mannose binding affinity. Interestingly, FimH variants, which maintain a high-affinity conformation in the FimCGH tip-like structure, were attenuated during chronic bladder infection, implying that FimH’s ability to switch between conformations is important in pathogenesis. Our studies argue that positively selected residues modulate fitness during UTI by affecting FimH conformation and function, providing an example of evolutionary tuning of structural dynamics impacting in vivo survival.

Antivirulence Isoquinolone Mannosides: Optimization of the Biaryl Aglycone for FimH Lectin Binding Affinity and Efficacy in the Treatment of Chronic UTI

Uropathogenic E. coli (UPEC) employ the mannose‐binding adhesin FimH to colonize the bladder epithelium during urinary tract infection (UTI). Previously reported FimH antagonists exhibit good potency and efficacy, but low bioavailability and a short half‐life in vivo. In a rational design strategy, we obtained an X‐ray structure of lead mannosides and then designed mannosides with improved drug‐like properties. We show that cyclizing the carboxamide onto the biphenyl B‐ring aglycone of biphenyl mannosides into a fused heterocyclic ring, generates new biaryl mannosides such as isoquinolone 22 (2‐methyl‐4‐(1‐oxo‐1,2‐dihydroisoquinolin‐7‐yl)phenyl α‐d‐mannopyranoside) with enhanced potency and in vivo efficacy resulting from increased oral bioavailability. N‐Substitution of the isoquinolone aglycone with various functionalities produced a new potent subseries of FimH antagonists. All analogues of the subseries have higher FimH binding affinity than unsubstituted lead 22, as determined by thermal shift differential scanning fluorimetry assay. Mannosides with pyridyl substitution on the isoquinolone group inhibit bacteria‐mediated hemagglutination and prevent biofilm formation by UPEC with single‐digit nanomolar potency, which is unprecedented for any FimH antagonists or any other antivirulence compounds reported to date.

Molecular basis of usher pore gating in Escherichia coli pilus biogenesis

Significance Gram-negative bacteria use chaperone-usher pathway (CUP) pili to colonize host tissues and mediate biofilm formation. CUP ushers are outer membrane (OM) pore proteins that catalyze pilus assembly and mediate pilus extrusion in a gated fashion to maintain OM homeostasis. Using antibiotic sensitivity and electrophysiology experiments, specific residues of two ushers, FimD of type 1 pili and PapC of P pili, were identified as crucial for proper usher gating. Further, deletion of the P pilus anchoring/terminator subunit resulted in an open usher conformation, indicating this subunit may be a target for antivirulence compounds to potentiate antibiotic treatment. Identification of mechanisms that can be used to increase bacterial sensitivity to antibiotics is crucial for development of novel compounds to fight infections. Extracellular fibers called chaperone-usher pathway pili are critical virulence factors in a wide range of Gram-negative pathogenic bacteria that facilitate binding and invasion into host tissues and mediate biofilm formation. Chaperone-usher pathway ushers, which catalyze pilus assembly, contain five functional domains: a 24-stranded transmembrane β-barrel translocation domain (TD), a β-sandwich plug domain (PLUG), an N-terminal periplasmic domain, and two C-terminal periplasmic domains (CTD1 and 2). Pore gating occurs by a mechanism whereby the PLUG resides stably within the TD pore when the usher is inactive and then upon activation is translocated into the periplasmic space, where it functions in pilus assembly. Using antibiotic sensitivity and electrophysiology experiments, a single salt bridge was shown to function in maintaining the PLUG in the TD channel of the P pilus usher PapC, and a loop between the 12th and 13th beta strands of the TD (β12–13 loop) was found to facilitate pore opening. Mutation of the β12–13 loop resulted in a closed PapC pore, which was unable to efficiently mediate pilus assembly. Deletion of the PapH terminator/anchor resulted in increased OM permeability, suggesting a role for the proper anchoring of pili in retaining OM integrity. Further, we introduced cysteine residues in the PLUG and N-terminal periplasmic domains that resulted in a FimD usher with a greater propensity to exist in an open conformation, resulting in increased OM permeability but no loss in type 1 pilus assembly. These studies provide insights into the molecular basis of usher pore gating and its roles in pilus biogenesis and OM permeability.

Evolutionary fine-tuning of conformational ensembles in FimH during host-pathogen interactions

Evolutionary selection fine-tunes conformational ensembles of FimH for optimal bladder colonization by E. coli during UTI. Positive selection in the two-domain type 1 pilus adhesin FimH enhances Escherichia coli fitness in urinary tract infection (UTI). We report a comprehensive atomic-level view of FimH in two-state conformational ensembles in solution, composed of one low-affinity tense (T) and multiple high-affinity relaxed (R) conformations. Positively selected residues allosterically modulate the equilibrium between these two conformational states, each of which engages mannose through distinct binding orientations. A FimH variant that only adopts the R state is severely attenuated early in a mouse model of uncomplicated UTI but is proficient at colonizing catheterized bladders in vivo or bladder transitional-like epithelial cells in vitro. Thus, the bladder habitat has barrier(s) to R state–mediated colonization possibly conferred by the terminally differentiated bladder epithelium and/or decoy receptors in urine. Together, our studies reveal the conformational landscape in solution, binding mechanisms, and adhesive strength of an allosteric two-domain adhesin that evolved “moderate” affinity to optimize persistence in the bladder during UTI.

Adhesive pili of the chaperone-usher family

The chaperone–usher (CU) pathway constitutes one of the most prevalent mechanisms for the assembly of adhesive pili on the surface of Gram-negative bacteria. Studies at the interface of genetics, biochemistry, and structural biology have detailed the functions of the chaperone and the usher, providing a step-by-step understanding of pilus biogenesis by this sophisticated molecular machine. Further work has elucidated the molecular basis of specificity in host recognition by CU pili. Snapshots in CU pilus assembly and CU pilus-mediated pathogenesis have unveiled necessary molecular details for the design and application of promising antibiotic compounds that may soon prevent and treat acute, chronic, and recurrent bacterial infections in humans.

Structure-based discovery of glycomimetic FmlH ligands as inhibitors of bacterial adhesion during urinary tract infection

Significance The emergence of multidrug-resistant bacteria, including uropathogenic Escherichia coli (UPEC), makes the development of targeted antivirulence therapeutics a critical focus of research. During urinary tract infections (UTIs), UPEC uses chaperone–usher pathway pili tipped with an array of adhesins that recognize distinct receptors with sterochemical specificity to facilitate persistence in various tissues and habitats. We used an interdisciplinary approach driven by structural biology and synthetic glycoside chemistry to design and optimize glycomimetic inhibitors of the UPEC adhesin FmlH. These inhibitors competitively blocked FmlH in vitro, in in vivo mouse UTI models, and in ex vivo healthy human kidney tissue. This work demonstrates the utility of structure-driven drug design in the effort to develop antivirulence therapeutic compounds. Treatment of bacterial infections is becoming a serious clinical challenge due to the global dissemination of multidrug antibiotic resistance, necessitating the search for alternative treatments to disarm the virulence mechanisms underlying these infections. Uropathogenic Escherichia coli (UPEC) employs multiple chaperone–usher pathway pili tipped with adhesins with diverse receptor specificities to colonize various host tissues and habitats. For example, UPEC F9 pili specifically bind galactose or N-acetylgalactosamine epitopes on the kidney and inflamed bladder. Using X-ray structure-guided methods, virtual screening, and multiplex ELISA arrays, we rationally designed aryl galactosides and N-acetylgalactosaminosides that inhibit the F9 pilus adhesin FmlH. The lead compound, 29β-NAc, is a biphenyl N-acetyl-β-galactosaminoside with a Ki of ∼90 nM, representing a major advancement in potency relative to the characteristically weak nature of most carbohydrate–lectin interactions. 29β-NAc binds tightly to FmlH by engaging the residues Y46 through edge-to-face π-stacking with its A-phenyl ring, R142 in a salt-bridge interaction with its carboxylate group, and K132 through water-mediated hydrogen bonding with its N-acetyl group. Administration of 29β-NAc in a mouse urinary tract infection (UTI) model significantly reduced bladder and kidney bacterial burdens, and coadministration of 29β-NAc and mannoside 4Z269, which targets the type 1 pilus adhesin FimH, resulted in greater elimination of bacteria from the urinary tract than either compound alone. Moreover, FmlH specifically binds healthy human kidney tissue in a 29β-NAc–inhibitable manner, suggesting a key role for F9 pili in human kidney colonization. Thus, these glycoside antagonists of FmlH represent a rational antivirulence strategy for UPEC-mediated UTI treatment.

Pili and Fimbriae of Gram-Negative Bacteria

Research on the function and assembly of extracellular fibres in Gram-negative pathogenic bacteria has provided insights into some of the most basic principles of molecular biology: how a protein folds into domains that serve as assembly modules for building up large supramolecular structures. Studies of the chaperone–usher pathway (CUP) pili have elucidated a reaction called donor strand complementation, in which the chaperone mediates pilus subunit folding, and a reaction called donor strand exchange, in which subunits of a pilus polymerize into a fibre with the aid of the usher, an outer-membrane-gated channel. CUP pili are ubiquitous in Gram-negative bacteria, with many genomes encoding ten or more types, all containing dedicated adhesins that function in adherence, invasion of host tissues, and biofilm formation on medical devices and in various niches and body habitats. Many Gram-negative bacteria also use specific molecular machinery to direct production of amyloid fibres called curli, which can provide structural, adhesive and protective properties to a biofilm. Frequent and long-term prophylactic use of antimicrobial agents has contributed to a looming worldwide crisis of multi-drug resistance that has spawned the need for new ways of thinking about drug development, including the targeting of bacterial molecular machines that catalyse the biogenesis of virulence-associated extracellular fibres.