Structural and Functional Characterization of the FimH Adhesin of Uropathogenic Escherichia coli and Its Novel Applications

    

    

    Figure 1: Schematic and simple illustration of the chaperone/usher pathway
to assemble the Fimbriae; in the first stage, fimbrial subunits (A, F, G, H)
enter the periplasmic space through the Sec system located in the inner
membrane. Connection of the FimC chaperone to the subunits creates a
soluble and stable form, and finally, the subunits are assembled through the
FimD usher in the outer membrane and transferred to the cell surface. OM:
Outer membrane, IM: Inner membrane.
    

FimH structure

FimH protein is the precursor of 300 amino acids. It is then processed to 279 mature forms of amino acids (Figure 3) [12]. It is composed of two domains, the mannose-binding lectin (Ld, 1–156 amino acids) and the fimbria-incorporating pilin (Pd, 160–273 amino acids) connected via a short linker. At the tip of Ld, there is a β-barrel-shaped mannose-binding pocket (MBP) [13]. The MBP of FimH is stable amongst sequenced E. coli strains causing UTIs. Although some residues outside the MBP (positions 27, 62, 66, and 163) under positive pressure in UPEC isolates compared with intestinal strains [14]. The high affinity of FimH to mannose causes this deep pocket. It has three loops, the loops 2 and 3 are involved in polar interactions with mannose and a water molecule, and loop 1, like a clamp, dissolves the mannose [15]. The interior of the MBP in FimH mostly consists of amino acids with hydrophilic side chains, and its exterior has amino acids with hydrophobic side chains [16]. The hydrophobic ridge directs the sugar into the binding pocket and facilitates polar interactions in the pocket. The hydrophobic nature of the pocket ridge distinguishes type 1 fimbriae in E. coli from type 1 adhesins in Salmonella sp. [17]. The tyrosine gate located at the entrance of the binding pocket, TYR48, and TYR137, plays a pivotal role in the strength and specificity of the adhesion-receptor interaction [16,18]. Although Ld has an affinity for all high-mannose glycans in the nanomolar range, the difference among glycans is based on their capacity for interaction with the tyrosine gate [19]. The Ligand-Induced Binding Site (LIBS) epitope is located at the bottom of the Ld, away from MBP [20]. The interaction of mannose with FimH under static conditions leads to the exposure of the LIBS epitope. The binding of monoclonal antibodies to the LIBS epitope, similar to shear stress, leads to a significant increase in FimH affinity to mannose. The existence of LIBS in the interdomain region shows that FimH is an allosterically regulated protein [13].

Conformation Switch of FimH

Interdomain interaction regulates the FimH affinity through the allosteric mechanism. The interaction of Pd with Ld keeps Ld in the low-affinity state, while if Pd is removed or has a weak interaction with Ld, a high-affinity state arises [21]. By increasing the flow of urine and inducing shear stress, FimH conformation changes from mediumto high-affinity, resulting in bacteria with stronger and long-lived binding to mannose ligands resist against micturition [22]. But, under static conditions, breakage and regeneration of short-lived bonds between FimH and mannose cause colonization of mannosylated surfaces by the bacteria [23]. The MBP is wider and looser in the lowaffinity state than in the high-affinity state. As a result, the mannose affinity in the low-affinity conformation is much lower than that of the high-affinity one [24]. Also, inter-domain interactions contribute to UPEC motility in the urothelial surface, which can significantly affect UTIs pathogenesis [25]. Changes in the structure and function of Ld that affect tensile mechanical force created by the shear stress are similar to a molecular fingertrap. This type of FimH activation is the basis of the catch bond mechanism of type 1 fimbriae underflow [26]. Catch bonds are a new type of receptor-ligand interactions, which increases by tensile force, while traditional slip bonds collapse with growing force [13]. The effect of external force on FimH destabilizes the hydrogen bonds between the linker region and Ld. As a result, with the extension of the linker, the binding stability of the MBP increases, and its conformation changes to the high-affinity state. Under such circumstances, the MBP and linker regions are located on opposite sides of Ld [27,28]. The advantages of this adhesin include resistance to soluble inhibitors and rapid colonization of the surface. For example, THP -the most abundant protein in mammalian urineprotects the urothelial surface by binding to FimH, but the long-lived binding of FimH to mannose-coated surfaces under shear stress conditions neutralizes the inhibitory function of THP. The catch bond interactions are a conserved mechanism, and hence, a physiological phenomenon in and it is suggested that it can be used as a powerful and smart adhesion factor in technological applications [29].

FimH Receptors

The primary natural receptor of FimH is an uroplakin Ia glycoprotein with high levels in the urinary tract epithelial cells [30]. Other FimH receptors include Tamm-Horsfall glycoprotein (THP), β1 and α3 integrins, CD48, collagen, laminin, fibronectin, and abiotic surfaces [17,18]. FimH detects a wide range of high-mannose glycoproteins. Hence, proximal tubular cells of the kidney, buccal cells, epithelial cells of the bladder, intestine, lung, and different inflammatory cells are the target of the type 1 fimbriae. Also, FimH is attached to yeast mannans and interferes with the agglutination of yeast cells [30,31]. Based on the adherence of wild and recombinant strains to three substrate types, FimH is phenotypically classified as M, MF, and MFP. M phenotype strains can be only bound to the substrate rich in mannose residues such as mannan yeast (MN). MF phenotype strains attach to complex oligosaccharides such as human plasma fibronectin. MFP phenotype strains bind to MN, fibronectin, and synthetic peptides lacking saccharide compounds [32]. FimH lectin alleles are not identical in the ability to recognize terminal mannose structures. Typically, FimH subunits are capable of adhering through trimannose residues, but only FimH of UPEC isolates can attach to mono-mannose (M1) residues. As a result, phenotypic differences can lead to variations in host tissue tropism [32]. Some of the M1-binding E. coli strains are also efficient to identify complex oligosaccharides with no terminally exposed mannose residues [33].

FimH Mutations

Generally, bacteria modify the structure of the adhesins- e g, FimH, to adapt to the new niche and improve binding under different conditions, static or shear stress [11]. The main point in evolutionary adaptation is to optimize the adhesin binding properties [28]. Accumulation of amino acid replacements in FimH adhesin increases tropism for urothelium [34]. But, UPEC strains with mutant variants of FimH have a relatively well bind to M1 due to the presence of point mutations [35]. Two phenotypes of slow M1-binding (M1L) and high M1-binding (M1H) are observed in FimH. M1L variants are more common in the intestinal isolates, but M1H variants are more commonly expressed in UPEC strains. They recognize cellular receptors and show less resistance to mannosylated compositions [2]. Probably, the conformational stability of the protein loop causes the strong binding of mutants to M1 that carries residues that interact with the receptor [33]. They change allosteric catch bond features of the protein observed in most FimH mutations, and increase binding to M1 residues. These mutations are called pathoadaptive and are responsible for increasing the virulence of the UPEC strains in vivo [20]. The pathoadaptive mutations are usually Single-Nucleotide Polymorphisms (SNPs) and modify the function of the protein by the replacement of amino acid. These changes are essential for the success of the pathogen [36]. The mutations occur in MBP and the interdomain region. Mutations in the interdomain area increase the Ld affinity to mannose up to 300 times [22,29,35]. Pocket zipper/ clamp loop (in MBP), the β-bulge, the α-switch, and the interdomain swing, insertion, and linker loops, display an active role in the signal transition [37]. Mutant variants benefit from stable binding even under low flow conditions such as the upper urinary tract. Consequently, the allosteric catch-bond mechanism is necessary for the survival and transmission of pathogens [38]. On the other hand, strong adhesion to M1 can prevent the spread of UPEC biofilms inflow conditions. Also, these mutations increase the sensitivity to soluble mannosylated inhibitors [39]. Point mutations, along with mobile genetic elements, help to fast adaptation to E. coli strains and thus to spread infection by changing environmental conditions. Specific FimH variants could be indicative of unique E. coli adaptation in the inflammatory state and could be novel objects for the molecular characterization of such strains [34]. The mutations can also lead to the loss of multiple FimHmannose interactions, and changes in the hydrogen bonding network and pocket geometry, which ultimately lead to adverse mannosebinding [17].

FimH Typing

Identification and typing of bacterial pathogens are particularly important in epidemiological studies, especially on E. coli, which is a common microorganism with high genetic diversity [40]. Multiple methods are applied to characterize E. coli strains, including Pulsed- Field Gel Electrophoresis (PFGE), Multilocus Sequence Typing (MLST), Restriction Fragment Length Polymorphism (RFLP), and some types of PCR-based methods. PFGE is the gold standard in outbreak researches since its high discriminatory power or MLST is a useful and popular tool for global or long-term epidemiological studies [41,42]. Although these methods have high reproducibility and discriminatory power, they have disadvantages such as the need for high knowledge, time-consuming, high costs, and complexity of the work process; therefore, fast, simple, and cheap typing methods with reliable results can be very helpful [40].

Mutations that cause alterations in function occur as SNPs, which is a change in a nucleotide that causes amino acid replacements into essential pathogen proteins [36]. Most UPEC strains encode the FimH gene. It is located in an entirely recombinant region on the bacterial chromosome and is under positive pressure for functional mutations that alter the bacterial binding properties by the production of SNPs [43]. The high frequency of this gene among the UPEC strains, presence of sequence variants of this gene in GenBank, and the high heterogeneity in the gene sequences made it suitable for E. coli genotyping [44]. The FimH typing is a sequence-based and straightforward method for epidemiological studies [45]. Typically, FimH typing is performed by amplifying the FimH gene, followed by Sanger sequencing and alignment of the typing region with FimH variants contained in a database. However, this type can be performed quickly and easily on the Whole-Genome Sequencing (WGS) data [46]. The fumC/FimH typing is a rapid sequence-based typing method for UPEC isolates that have high discriminatory power. It could be cost-effective to evaluate large clinical isolate collections [43,47].

Furthermore, users can employ web tools designed to obtain FimH allelic information from simple Sanger sequences or WGS data [46]. The discriminative power of this method is suitable to investigate epidemiological events that occur over short periods or in a limited geographic area. This type can sometimes be used alongside other methods such as MLST and PFGE or even replace techniques such as Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR [44]. ERIC sequences are short, highly preserved 126-base pair noncoding regions found in Enterobacteriaceae. This method is a simple and cost-effective genotyping technology for discriminating different types of strains. The ERIC sequences are known in an enormous number of bacterial genomes, comprising E. coli [48].

Novel Therapeutic Target

The weakness of current therapeutic approaches to effectively combat resistant E. coli infections highlights the need for new therapeutic strategies [49]. Today, attention is drawn to FimHmediated adhesion as a potential target to treat UTIs [22]. Applying mannosides as a FimH inhibitor, preventing assembly and biogenesis of pilus by application of pilicides, using recombinant vaccines, and employing anti-FimH antibodies are the new approaches to treatment [34,50]. Over the last three decades, a large number of FimH inhibitors are introduced. The majority of studies are focused on the efficacy of tyrosine gate affinity [51]. Mannose-containing oligosaccharides inhibit FimH-mediated binding. This inhibition is based on the structural similarity between mannose and mannosylated receptors on urothelial surfaces. D-mannose binds to bacterial FimH to prevent attachment of bacteria to mannosylated receptors in the urinary tract, and ultimately flushing of urethral of unattached bacteria [52].

Some of them, such as α-D-mannosides with longer alkyl and aryl chains and heptyl mannose have a higher affinity for binding to FimH due to hydrophobic interactions with tyrosine gate [6,53]. FimH antagonists are a new therapeutic opportunity that does not lead to bacterial resistance in comparison with conventional antibiotics since they are not selective pressure on UPEC growth and do not affect bacterial metabolism. They are particular for a certain type of procedure or bacterial species and do not disrupt the host-microbiota [22,54]. The stability of bacterial phenotype, lack of selection of modified FimH variants after long-term exposure to mannose, minimal support for bacterial growth and metabolism, and emphasis of the bladder cell tolerance to the safe use of mannose require paying more attention to mannose to prevent and treat UTIs caused by UPEC [52]. A balance should be found between the ability of FimH antagonists to cross the cell membrane and the degree of lipophilicity in oral absorption [55]. Therefore, designing drugs based on FimH inhibition, quantum chemistry, and thermodynamics of FimH-ligand interactions can be a solution for treatments [51]. In recent years, several monovalent and multivalent FimH antagonists are synthesized. The n-heptyl α-Dmannoside treated with β-cyclodextin is a highly effective heptavalent antagonist when introduced into the bladder of C3H/HeN mice infected with the UTI89 bacterial strain [56]. The interest is higher in optimizing monovalent antagonists than multivalent antagonists. Since the former has little penetration into the gastrointestinal tract, and oral dosing is not possible due to its high molecular weight and high polarization. The advantage of multivalent antagonists is the increase in avidity [9]. In addition to the use of these compounds to treat UTIs, recent efforts to treat Corn’s disease show that FimH antagonists can reduce the population of adherent and invasive E. coli (AIEC) in vivo [54]. Pilicides or substituted 2-pyridones regulate the level of pili expression by inhibiting the pilus assembly process without affecting the biophysical properties of the pilus rod [57]. IBCs are probably the leading cause of recurrent or chronic UTIs and are resistant to antibiotic clearance. Therefore, vaccines are an excellent option to eradicate these intracellular reservoirs of UPEC strains. FimH is extremely immunogenic among E. coli antigenic proteins and provides high protection against UTIs in mice and primates [58]. Also, it is a good target for the UT β Is vaccine since it possesses conserved and surface-exposed epitopes that mimic the humoral immune system [59]. Immunization with FimH vaccines reduces the binding of UPEC strains to human bladder cells in vitro and a 99% reduction in bacterial colonization in a murine model in vivo. This approach may even prevent acute and recurrent UTIs [60]. A study in 2019 showed that the non-pathogenic and recombinant Lactococcus lactis containing FimH prevents the spread of biofilm of uropathogens by biofilm formation in the bladder of mice [59]. The production of antibodies that target epitopes at the binding site of ligand proteins is essential to develop therapeutic antibodies [50]. However, antibodies against FimH may cause high-affinity conformation of the adhesin and increased bacterial binding [24]. But, genetically engineered lowaffinity variants including A10P, R60P, V67K, and V27C/L34C may be useful vaccines. These variants, due to creating strong low-affinity conformation, can also be used to study FimH-mediated bacterial adhesion [61]. Inhibitors act in three ways; the first orthosteric inhibitors compete with the ligand to bind to the receptor. The second allosteric inhibitors exert their effect by binding distinctly to the ligand-binding site. The third type has a non-competitive impact on the ligand-binding place and has a better effect than the first two types [50]. Also, a FimH-based expression system is developed that provides the expression of functional eukaryotic proteins. One of the available methods is the production of recombinant proteins in genetically modified bacteria for biotechnology, therapeutic, and analytical purposes. After the production of recombinant protein, the carrier protein is required to transfer the desired protein to the cell surface. One of the carrier proteins is FimH, which can carry more abundant proteins than other carrier proteins such as LamB, OmpA, and PhoE (Figure 2) [62]. Besides, FimH can be used as a vaccine adjuvant for human cancer immunotherapy due to its water solubility and strong stimulatory effects on the immune system. By the immune system induction and subsequently the promotion of TLR4 and dendritic cells and activation of T cells, FimH prevents tumor cell growth, including melanoma and carcinoma, in mice [63].

    

    

    Figure 2: Diagram of FimH Applications in Different Approaches.
    

    

    

    Figure 3: A schema of FimH-containing fimbrial tip crystal structure.
    

Download PDF

Citation: Neamati F, Moniri R, Khorshidi A and Saffari M. Structural and Functional Characterization of the FimH Adhesin of Uropathogenic Escherichia coli and Its Novel Applications. J Bacteriol Mycol. 2020; 7(5): 1142.

Home

Journal Scope

Editorial Board

Instruction for Authors

Submit Your Article

Contact Us