Fucosylated inhibitors of recently identified bangle lectin from Photorhabdus asymbiotica


A recently described bangle lectin (PHL) from the bacterium Photorhabdus asymbiotica was identified as a mainly fucose-binding protein that could play an important role in the host-pathogen interaction and in the modulation of host immune response. Structural studies showed that PHL is a homo-dimer that contains up to seven l-fucose-specific binding sites per monomer. For these reasons, potential ligands of the PHL lectin: α-l-fucopyranosyl-containing mono-, di-, tetra-, hexa- and dodecavalent ligands were tested. Two types of polyvalent structures were investigated – calix[4]arenes and dendrimers. The shared feature of all these structures was a C-glycosidic bond instead of the more common but physiologically unstable O-glycosidic bond. The inhibition potential of the tested structures was assessed using different techniques – hemagglutination, surface plasmon resonance, isothermal titration calorimetry, and cell cross-linking. All the ligands proved to be better than free l-fucose. The most active hexavalent dendrimer exhibited affinity three orders of magnitude higher than that of standard l-fucose. To determine the binding mode of some ligands, crystal complex PHL/fucosides 24 were prepared and studied using X-ray crystallography. The electron density in complexes proved the presence of the compounds in 6 out of 7 fucose-binding sites.


Reversible interactions between the oligosaccharide chain of glycolipids/glycoproteins and carbohydrate-binding protein receptors (lectins) mediate critical biological recognition at the cell level. They are of vital importance in many normal and pathological processes ranging from fertilization, immune response and intercellular communication to viral/bacterial invasion and tumour metastasis1. The identification of lectin-carbohydrate interactions in human diseases raises the possibility of the development of carbohydrate-based therapeutic products. Anti-adhesion therapy has been used for a few decades and the protective effect of anti-adhesive saccharides has been demonstrated in several studies2. For example, mannose has been used to treat urinary tract infections caused by E. coli. Moreover, the anti-adhesive agents do not kill pathogens or arrest their growth thus development of resistance to anti-adhesive agents will be less likely as compared to antibiotics2,3. Therefore, anti-adhesion therapy is a very attractive approach that not only prevents infections at an early stage but is also an important alternative treatment for bacterial infection considering the alarming increase of drug-resistant bacteria.

P. asymbiotica was originally considered to be exclusively an insect pathogen until 1989, when it was isolated from a patient in the USA for the first time4. Later on, further human infections were reported in Australia5 and nowadays, occurrence of this bacterium is not restricted to American and Australian continents but starts to be globally spread6,7,8,9,10. P. asymbiotica is regarded as an emerging human pathogen causing either locally invasive or disseminated soft-tissue infections11,12,13,14. Relatively low number of infections has been reported so far, however this is likely to be caused by misidentification by automated microbiological techniques, which do not contain Photorhabdus sp. in their databases12. Recently, the two subspecies of P. asymbiotica (P. asymbiotica subsp. asymbiotica and P. asymbiotica subsp. australis) were suggested to be elevated into a higher taxon, creating the P. asymbiotica and P. australis species, respectively15.

As a part of our ongoing programme focused on host-pathogen interactions at the molecular level, we identified a lectin in the P. asymbiotica genome designated PHL, which we prepared in a fully functional recombinant form16. We also determined its carbohydrate specificity and investigated the structural details of ligand binding. Moreover, PHL demonstrated the ability to act as a host-cell recognizing agent through interacting with the haemolymph of Galleria mellonella (order Lepidoptera, family Pyralidae) and human blood components and also to modulate immune system of both hosts16. Lectins of other bacteria were confirmed as virulence factors, e.g. PA-IL and PA-IIL from opportunistic Pseudomonas aeruginosa or FimH from uropathogenic Escherichia coli (UPEC) strains17,18. The PHL lectin can play equally important role as above-mentioned examples and therefore, PHL could be a usable therapeutic target. Interestingly, other two saccharide-binding proteins were detected in Photorhabuds genus – PLL and PllA, both from P. luminescens19,20. PLL is a fucose-specific lectin and a sequence homologue of PHL. However, PLL is not able to modulate the host innate immune response as PHL and probably plays a role in bacteria-nematode interactions. The PllA lectin is a galactose-specific lectin and homologue of PA-IL (LecA) from Pseudomonas aeruginosa, which is known for its utilization in P. aeruginosa infection and biofilm formation in lung of patients with cystic fibrosis21. Due to the PllA specificity, the certain role both in the symbiosis with their nematode hosts and in insect infection is speculated.

The lectin PHL interacts selectively, mainly with l-fucose and oligosaccharides containing terminal l-fucose residue, and prefers an α-glycosidic bond over a β-linkage. Surface plasmon resonance (SPR) in a competition assay revealed that methyl α-l-fucopyranoside, with an IC50 of 40 μM, was about a 10-fold stronger ligand than free l-fucose. Nevertheless, the IC50 value is in the low μM range, suggesting a relatively weak binding of methyl α-l-fucopyranoside to PHL. The preparation of a core l-fucose derivative bearing suitable functional groups for the construction of multivalent systems could enhance this affinity substantially.

Here we present a comprehensive study covering interactions of the lectin PHL with a pool of l-fucose-based potential ligands with different valences and topologies, assessed by hemagglutination, SPR, isothermal titration calorimetry (ITC), and cell cross-linking assays. All constructs are stable against both acidic and enzymatic hydrolysis due to the presence of C-glycosidic bonds. In most of the experiments, dendrimers emerged as the best ligands, especially the hexafucosylated dendrimer 9. During SPR measurement, dendrimer 9 had a potency per fucose unit that was over 1,600 times as effective as l-fucose and 52 times as respective saccharide-building unit azide 1. The structure of PHL complexes with fucosides 24 was solved using X-ray crystallography, and compounds were found in 6 out of 7 fucose-binding sites of at least one PHL monomer unit.

Result and Discussion

Ligands tested

In total 10 compounds with α-l-fucopyranosyl unit(s) were selected from our library of recently synthesized potential ligands towards various lectins (Fig. 1). Azide 122 is an analogue of methyl α-l-fucopyranoside, and also serves for the preparation of polyvalent systems. The previous study showed increased affinity to di- and tri-saccharides for PHL. Therefore, pseudo-C-disaccharides 2423 as monovalent ligands differing in the structure of the C-aglycone part were included in the study to analyse the potential influence of the second saccharide-like unit on the ligand binding. 1,4-bis(fucopyranosyl)butane 523 is a divalent mimetic in which the distance between the C1 atoms of both α-l-fucopyranosyl units is derived from the structure of blood group Lewis b tetrasaccharide. Tetravalent constructs 6 and 7 contain a calix[4]arene scaffold in a 1,3-alternate conformation that ensures sufficient solubility in water. Both 6 and 724 were prepared by CuAAC click reaction25,26 from azide 1 and the respective tetrapropargyl calix[4]arene. Finally, a set of dendrimers 81022 with valences ranging from 4 to 12 are more flexible and complex ligands than calix[4]arene derivatives, thus a significant increase in the complexity of the binding event can be expected. The affinity improvements in multivalent carbohydrate−lectin interactions have been attributed to the so-called “cluster glycoside effect”27,28,29.

Figure 1

Structure of ten tested α-l-fucopyranosyl compounds.

Hemagglutination inhibition assay

PHL as a fucose-specific lectin was shown to agglutinate papain-treated RBCs of blood group O16 with the surface-exposed terminal trisaccharide α-l-Fucp-(1 → 2)-β-d-Galp-(1 → 3/4)-d-GlcpNAc. The set of potential ligands 110 with different levels of fucosylation was tested, and their inhibition potency on hemagglutination by PHL was assessed (Table 1, Fig. 2). DMSO-containing blank did not affect the hemagglutination in any way. To assess the contribution of valency to the affinity increase, an affinity improvement factor β was calculated as the relationship MICbasic unit/(valency × MICligand).

Table 1 Hemagglutination inhibition assay with PHL.
Figure 2

Representatives of α-l-fucopyranosyl compounds with a higher potency than α-Me-Fuc and their influence on hemagglutination of RBC O caused by PHL. Two-fold serially diluted carbohydrate compounds 1, 510 were able to inhibit hemagglutination at the minimal concentrations highlighted in yellow. The hemagglutination of RBC O caused by PHL without an inhibitor is shown in the picture in the upper right corner.

Simple monovalent fucosides 24 failed to substantially inhibit the hemagglutination caused by PHL, while azide 1 and difucoside 5 were 16-fold better inhibitors than l-fucose and twice as good as methyl α-l-fucopyranoside. If the structures of simple ligands 15 are compared, it seems that the nonpolar substituent at the anomeric position of l-fucose provides an advantage. The activities of tetravalent calixarenes 6 and 7 are identical, giving a potency of 31.3 without regard to the presence or absence of a tert-butyl group on the upper rim of the skeleton. Tetravalent dendrimer 8 was as efficient inhibitor of hemagglutination as calixarenes 6 and 7, although it occupies a different topology. Hexa- and dodecafucosylated dendrimers 9 and 10 were proven to be the best inhibitors. They were more than 100-fold better ligands than natural l-fucose. However, according to the β factor calculated with respect to azide 1 (βAzide1), all multivalent compounds were comparable with their building unit.

Surface plasmon resonance

To analyse the competitive inhibition of PHL binding to a multivalent surface, the surface plasmon resonance (SPR) technique was employed. A sensor chip presenting α-l-fucopyranoside residues was treated with a constant concentration of PHL, while an increasing concentration of the ligands was used to determine IC50 (Table 2, Fig. 3). Only the ligands comparable with or better than α-Me-Fuc in the hemagglutination assay were investigated by SPR. To assess the contribution of valency to the affinity increase, an affinity improvement factor β was calculated as the relationship IC50,basic unit/(valency × IC50,ligand)30.

Table 2 Inhibition activity of studied ligands towards PHL assessed by SPR.
Figure 3

IC50 determination of fucoligands towards PHL from SPR measurements. (a) A logarithmic plot of inhibition curves calculated from SPR experiments vs. % of inhibition. Individual types of line correspond to monovalent (densely dashed), divalent (dashed), tetravalent (dotted), hexavalent (dash-dotted) and dodecavalent (solid) ligands. (b) A logarithmic plot of IC50 determined for individual mono/multivalent ligands. Measurements were performed in triplicates and standard deviations were calculated. Individual types of line correspond to monovalent (densely dashed), divalent (dashed), tetravalent (dotted), hexavalent (dash-dotted) and dodecavalent (solid) ligands.

Comparing the IC50 values and potency, the results from SPR are in good agreement with the finding obtained by hemagglutination. Azide 1 and fucoside 3 exhibit 32-fold and 38-fold, respectively, stronger affinity to PHL than its counterpart l-fucose; in fact, an 86-fold increase in affinity was found for difucoside 5. The affinity improvement factors of tetravalent calixarenes 6 and 7 as well as dendrimer 8 were comparable with βAzide1 value reaching approximately 15. The hexavalent dendrimer 9 was the best ligand of the lectin PHL with IC50 = 37 nM, βAzide1 = 52 and with potency 9,800-fold and 310-fold higher than l-fucose and azide 1, respectively. Although dodecavalent dendrimer 10 was also an efficient ligand (IC50 = 80 nM), its affinity improvement factor was substantially lower (βAzide1 = 12) than that of the hexavalent dendrimer 9. This observation could be caused by unwanted side effects such as cross-linking or aggregation induced by the sterically demanding structure of 10. Nevertheless, the data support a strong glycoside clustering effect.

Isothermal titration calorimetry

The binding of PHL to different α-l-fucoside compounds was further characterized by isothermal titration calorimetry (ITC), enabling determination of the complete thermodynamic profile of the molecular interactions (Table 3, Fig. 4). As for calixarenes, both compounds caused a cross-linking/aggregation of PHL accompanied by the formation of visible precipitates. Because of this, it was not possible to evaluate the data, despite the obvious interaction. The dendrimers also cross-linked/aggregated the protein as with calixarenes, however the curves provided clear outcomes with an affinity in the low micromolar range. The dissociation constants (KD) of all dendrimers correlate with their valency – the higher number of fucose, the more decreased KD. This implies that dodecafucosylated dendrimer 10 was assessed as the best inhibitor, with KD almost 4 times better than the second-best hexavalent dendrimer 9 and 33 times better than azide 1 (βAzide1 = 3). The stoichiometry value n corresponds to the number of terminal fucoses per ligand molecule. The n value decreases from 4 to 1 as the number of fucose units increases from 1 to 12 (Table 3).

Table 3 Thermodynamic profiles for interaction between PHL and ligands determined by isothermal titration calorimetry at 25 °C.
Figure 4

(a) Thermodynamic profiles for interaction between PHL and fucoligands determined by isothermal titration calorimetry at 25 °C. (b) Selected ITC curves of PHL (50 μM) titration by azide 1 (5 mM), difucoside 5 (5 mM), dendrimer 9 (1.5 mM) and dendrimer 10 (1.5 mM). 20 injections of 2.0 μl of fucoclusters were added every 240 s to a PHL-containing cell. The lower plots show the total heat released as a function of total ligand concentration for the titration shown in the upper panels. Due to low affinity of PHL to monovalent compounds, n, ΔH and ΔS values may suffer from higher inaccuracy.

As for monovalent (azide 1, fucoside 3) and divalent (difucoside 5) compounds, all of them exhibited a low affinity towards PHL (in the millimolar range), as is usually observed for lectin/saccharide interactions. They also correspond with the same β factor.

P. asymbiotica cross-linking – Bacterial aggregation properties

Because of the aggregation of PHL by calixarenes and dendrimers during SPR and ITC experiments, we decided to compare tetravalent compounds at a higher level. We performed a series of in vitro aggregation assays to reveal their aggregation properties towards P. asymbiotica subsp. australis (Table 4, Fig. 5). Prior to each assay, we confirmed the absence of aggregates in two negative controls – bacterial cells in PBS buffer and in the presence of azide 1 as a monomer. With all chosen compounds, we observed bacterial aggregates of a variable size that confirmed that they are capable of interacting with a cell surface. This effect may be caused by interaction of the multivalent inhibitors with PHL or other receptors. The concentration of the first appearance of aggregates varied according to the nature of the ligand used. The highest efficiency (0.625 mM) was observed for the tetravalent dendrimer 8, as we expected. Calixarene 6 proved to be only half as good as dendrimer 8, and aggregated cells at a concentration of 1.25 mM. An unexpected observation was that calixarene 7 had the lowest aggregation potential. In contrast to other techniques (hemagglutination, SPR and ITC), where calixarenes 6 and 7 provided similar results, during cross-linking experiments calixarene 7 was four times worse than calixarene 6. At the same time, both tetrameric calixarenes 6 and 7 are in a 1,3-alternate conformation with the two-faced presentation of the fucopyranosyl epitopes on opposite regions probably occupying a similar spatial orientation. The only difference is the tert-Bu group on the upper rim in calixarene 6, which might contribute to nonpolar interactions with whole P. asymbiotica cells.

Table 4 Determination of minimal concentration of multivalent fucoclusters able to aggregate P. asymbiotica subsp. australis cells.
Figure 5

Representative images of optical microscopy observation (200x) of bacterial cell aggregation for Photorhabdus asymbiotica subsp. australis with different fucosylated clusters. Bacterial cells in PBS buffer were used as a negative control, shown in the bottom right image. Two-fold serially diluted carbohydrate compounds 6, 7 and 8 were able to agglutinate the cells at minimal concentrations of 1.25 mM, 5 mM and 0.625 mM, respectively. The background of all pictures was subtracted in the software GIMP.

X-ray structure of PHL/fucosides complex

The PHL lectin forms a seven-bladed β-propeller assembling into a homo-dimer. Both units contain two types of binding sites situated between the blades and have different amino acid compositions and binding preferences16. As mixing PHL with the branched compounds leads to protein precipitation and thus prevents co-crystallization, the direct study of complexes was not possible. Soaking ligand-free PHL crystals with a low concentration of individual branched compounds did not result in complex formation while soaking with their higher concentrations led to a crystal decomposition. Therefore, we soaked the PHL crystals with monomeric fucoside 2, 3 and 4, respectively. The structure of these complexes was determined using X-ray diffraction (Table 5).

Table 5 Data collection and refinement statistics for PHL complexes.

Crystal structures confirmed that all the studied fucosides were able to bind the lectin PHL. In each complex, the electron density was clear enough to determine the position of the fucosyl part of the compounds in 6 out of 7 fucose-binding sites of at least one PHL monomer unit – A or B (Table 6, Fig. 6), while site 2 is occupied by Met60 of the neighbouring monomer, as was observed previously16. This demonstrates the binding potency of studied fucosides and experimentally confirmed a presence of at least 6 binding sites per monomer. In previous studies, only 2 to 5 binding sites were detected16,31. The second saccharide unit was clearly assigned for all three fucosides 24 in sites 1 and 3. For fucoside 3 and 4, the second saccharide was also distinguishable in sites 4-7 of at least one protein monomer (Table 6). This allows us to compare the binding mode for C-glycoside-based molecules and published previously O-glycoside structures. The fucose moiety is coordinated in the same way as was shown for α-Me-Fuc and blood group H trisaccharide16. Briefly, the O3, O4 and O5 of fucose are coordinated by the backbone atoms of two neighbouring loops and in sites 1, 3, and 5–7 also by the side chain of nearby threonine. The C6 methyl group and nonpolar surface of fucose are stabilized by a CH-π interaction with two tryptophan side chains. Considering fucosides 24, the position of the bridging methylene group is close to the α-Me-Fuc O1 atom, which is not directly coordinated by the protein in the complex. Additional direct interactions between the protein and the studied molecules are infrequent. The O4 of the d-arabino-hexopyranosyl unit in fucoside 2 and 4 is coordinated by a glycine backbone nitrogen in sites 1, 3, 5, and 6, while the O6 atom of fucoside 3 is linked to a tryptophan backbone oxygen in sites 4 and 6. When comparing the overall orientation of the second hexopyranosyl unit of fucosides 24, it is similar to the galactose unit of blood group H trisaccharide, with minor distortions in individual binding sites influenced e.g. by crystal packing. The remaining parts of fucosides 2 and 3 were not resolved in the electron density, suggesting their negligible importance for ligand binding.

Table 6 Binding site occupancy of PHL complexes with fucosides 24.
Figure 6

Structure of PHL with fucosides 24 and α-Me-Fuc from 5MXE structure for comparison. (A) Overall structure of chain A of PHL complexes. Individual binding sites labelled, label in italics for empty site. Fucosides shown as sticks. (B) Binding site 3 with coordinated ligand. Key residues labelled. Colour code: fucoside 2 – green, fucoside 3 – cyan, fucoside 4 – magenta, α-Me-Fuc – yellow, CH-π interacting tryptophans – grey, H-bond interacting residues – blue.


In the previous study16, we identified an interesting lectin from emerging human pathogen P. asymbiotica harbouring seven potential fucose-binding sites per monomer and able to modulate innate immune system of human. Infection strategies used by pathogens often involve highly specific protein/carbohydrate interactions and therefore design of suitable inhibitors preventing this interaction is needed. Thus, we tested inhibition potency of l-fucose-based compounds with different valences and topologies. All these structures share the same feature of bearing C-glycosidic bond instead of the common but physiologically unstable O-glycosidic bond.

α-l-fucopyranosyl-containing mono-, di-, tetra-, hexa- and dodecavalent ligands were investigated by hemagglutination, ITC, SPR, X-ray crystallography, and cell cross-linking. The binding mode of monovalent ligands was studied via crystal complexes with PHL where the electron density proved the presence of the fucosyl part of the compounds in all accessible fucose-binding sites. The second saccharide unit of studied fucosides was only marginally coordinated by PHL binding sites what, together with affinity comparable to monosaccharides, suggests a low importance of this type of compounds for inhibitor design. On the other hand, the affinity towards azide 1 is thirteen times higher than towards l-fucose and comparable to previously studied propargyl-α-l-fucoside31. This suggests, together with data from the previously studied PHL complexes, that introduction of a linker with delocalized π-electrons has not negligible contributory effect to binding affinity and increases also binding site availability/occupancy (4–5 binding sites for azide 1/propargyl-α-l-fucoside vs. 2–3 sites for α-Me-Fuc). This effect was already reported for the PA-IIL lectin and p-nitrophenyl-α-l-fucoside32. The affinity of PHL for multivalent ligands reached low micromolar values, which corresponds to an affinity three orders of magnitude higher than that of standard l-fucose and two orders of magnitude higher than that of monovalent azide 1. We have further demonstrated that all of the tested compounds were able to inhibit the PHL binding towards both artificial and natural fucosylated surfaces. Generally, the potency of ligands depended on the valency and types of polyvalent structures (calix[4]arenes and dendrimers). Even though the relation between the increasing number of fucoses per cluster and increasing affinity, the most active compound was a hexavalent dendrimer exhibited an IC50 of 37 nM and KD of 13 µM. Several tetravalent compounds of different type were also tested for their ability to agglutinate P. asymbiotica bacteria. The cell clumps at different concentrations of tested compounds were observed further supporting the previously reported presence of lectins on surface as was also proved for other bacteria (e.g. P. aeruginosa, E. coli)31,33,34,35.

Based on the findings presented in this work, we consider these compounds as a milestone on the way to design efficient inhibitors targeting P. asymbiotica.


Ligand preparation

All ligands were prepared as 50 mM stock solutions. Lyophilized azide 1, fucosides 2–4, difucoside 5, and dendrimers 8–10 were dissolved in the deionized water, whereas calixarenes 6–7 were dissolved in DMSO.

Protein production and purification

The PHL lectin was produced in Escherichia coli Tuner(DE3)/pET25b_phl cells as previously published16. In short, the cells were grown in LB broth medium containing 100 μM ampicillin at 37 °C. After reaching an OD600 of 0.5, gene expression was induced with 0.2 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). Cells were incubated for an additional 20 hours at 18 °C, harvested by centrifugation at 12,000 g for 10 min and resuspended in buffer A (20 mM Tris/HCl, 300 mM NaCl, pH 7.5). Harvested cells were stored at −20 °C prior to protein purification.

For protein purification, cells were disintegrated by sonication (VCX 500, Sonics & Materials, Inc., USA) and the cytosolic fraction containing soluble PHL was collected by centrifugation at 21,000 g at 4 °C for 1 hour and filtrated through a 0.45 μm pore size filter (Carl Roth, Germany). Recombinant protein PHL was purified with isocratic elution on a d-mannose-agarose (Sigma-Aldrich, USA) resin equilibrated with buffer A by affinity chromatography using an ÄKTA FPLC system (GE Healthcare, UK) and used for further studies. For agglutination assays, purified PHL was dialyzed against PBS buffer (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.4).

Hemagglutination inhibition assay

Human red blood cells (RBCs) O were processed according to previously published work16. In brief, RBCs O treated with sodium citrate were washed four times by PBS buffer (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.4), diluted to 50% by PBS with 0.005% (w/w) sodium azide and treated by 0.1% papain for the duration of 30 minutes.

Hemagglutination inhibition assay was performed for specificity and semi-quantitative affinity of the PHL interaction with the compounds. All carbohydrate inhibitors in 50 mM starting concentration were serially diluted in the PBS buffer and used for a determination of the lowest inhibiting concentration. As a blank, the PBS buffer supplemented with the appropriate amount of DMSO was used in order to exclude potential interference with the interaction. The individual samples were mixed with the lectin in concentration 2 mg/ml in a 5 μl:5 μl ratio. Thereafter, 10 μl of 10% RBCs O in PBS buffer was added, thoroughly mixed and incubated for 10 minutes at room temperature36. Following incubation, the mixture was again mixed and the reaction was observed on microscope slides using Levenhuk 2L NG microscope with Levenhuk D2L digital camera (Levenhuk, USA). Imagines were obtained via software ToupView for Windows (Levenhuk). The positive (PHL without inhibitor) and negative control (reaction without PHL) were prepared and processed in the same way using the appropriate volume of PBS buffer instead of the not included components. The lowest concentration of inhibitor able to inhibit agglutination was determined and compared with the standard (l-fucose). Minimal inhibitory concentrations (MIC) of synthesised inhibitors were determined from two independent measurements.

Surface plasmon resonance

Surface plasmon resonance (SPR) experiments were performed on a BIAcore T200 instrument (GE Healthcare, UK) at 25 °C, using buffer A supplemented with 0.05% Tween 20 as a running buffer. α-l-fucoside was immobilized onto CM5 sensor chip (GE Healthcare, UK) covered with a carboxymethylated dextran matrix. The sensor chip surface was activated with N-ethyl-N-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide solution and then coated with streptavidin using the manufacturer’s standard protocol using HBS buffer (10 mM HEPES, 150 mM NaCl, 0.05% Tween 20, pH 7. 5). Unreacted groups were blocked with 1 M ethanolamine-HCl, pH 8.5. Biotinylated carbohydrate (biotinilated monomeric probes, Lectinity, Russia) was injected onto particular measuring channel and pure biotin on blank channel at a flow rate of 5 μl/min.

SPR inhibition measurements were carried out on measuring channel with l-fucoside at a flow rate of 5 µl/min. PHL was diluted to a concentration of 20 μg/ml by the buffer A supplemented with 0.05% Tween 20, mixed 1:1 (v/v) with various concentrations of inhibitors (500–5 μM) in the same buffer and injected onto the sensor chip. The response of lectin bound to the sugar surface at equilibrium was plotted against the concentration of inhibitor in order to determine IC50 (concentration of inhibitor resulting in 50% inhibition of binding). As IC50 is not a constant and depends on the experimental set-up, a parameter called potency was used for characterization. The potency of a tested inhibitor is the ratio of IC50 of a chosen standard inhibitor (in this case l-fucose) and the inhibitor in question. Pure PHL lectin was used as a control (0% inhibition) and channel with pure biotin served as a blank. IC50 was determined from triplicates.

Isothermal titration calorimetry

PHL protein in buffer A was equilibrated at room temperature at least for 30 min before ITC measurement. All ITC experiments were performed using ITC200 calorimeter (Malvern Panalytical, UK) at 25 °C. Carbohydrate ligands dissolved in buffer A were used at different concentrations. Protein in the calorimeter cell (50 μM) was titrated by consecutive additions (2 μl) of the ligand (1.5–5 mM) in the syringe while stirring at 1000 rpm. Integrated heat effects were analysed by nonlinear regression using a single-site binding model and global fitting in Origin 7 software (Microcal Instruments)37. The thermodynamic parameters of synthesised inhibitors were determined from two independent measurements.

Crystallization and data collection

PHL was concentrated to 13.6 mg/ml using an ultrafiltration unit with a 10-kDa cut-off membrane (Vivaspin 20, Sartorius, Germany) and crystallized under previously described conditions16. The crystals were obtained under the following conditions: 4 μl sitting drop, protein solution mixed with precipitant (3.7 M NaCl, 100 mM Hepes, pH 7.5) in ratios 1:1, 3:5, 1:3 and 1:7. The drops were set against 0.5 ml of the same equilibration precipitant. To determine the PHL structure complexed with fucosides 24, the crystals of PHL were soaked in a 10 mM solution of compounds 24 for 1 hour. The crystals were cryo-protected using 40% PEG 400 and frozen in liquid nitrogen. The diffraction data of PHL complexed with saccharides were collected at the BESSY II electron storage ring (Berlin-Adlershof, Germany)38.

Structure determination

Images were processed using XDSAPP39 and converted to structure factors using the program package CCP4 v.6.540, with 5% of the data reserved for Rfree calculation. The structures of PHL complexes were solved by molecular replacement with Phaser41 using the monomeric coordinates of the ligand-free PHL structure (5MXE). Refinement of the molecule was performed using REFMAC542 alternated with manual model building in Coot v.0.843. Sugar-derived ligand coordinates were established using JLigand44 included in CCP4 program package. Ligands and other compounds that were present were placed manually using Coot. Water molecules were added by Coot and checked manually. The addition of alternative conformations, where necessary, resulted in final structures that were validated using the MolProbity45 (http://molprobity.biochem.duke.edu) validation server and were deposited in the PDB as entries 6FHX, 6FHY and 6FLU. Molecular drawings were prepared using Pymol (Schrödinger, Inc.).

P. asymbiotica growth

P. asymbiotica subsp. australis was inoculated from stock cells stored at −80 °C into a standard LB media and were grown in 30 °C under an orbital shaking overnight. Bacterial growth was tracked by measuring the optical density of the broth at 600 nm (OD600) using a spectrophotometer (Biochrom Ltd., BioPhotometer WGA CO 8000 Biowave). Cells were harvested when reaching an OD600 = 0.5. The culture was centrifuged at 4,300 g and room temperature for 2 minutes (Minispin, Eppendorf, Germany) and cells were then washed three times with PBS buffer. Cells were diluted in PBS to OD600 of 2.0 and kept in a fridge until performing the aggregation assay.

Cell aggregation assay

P. asymbiotica subsp. australis cells and fucosylated compounds in a concentration ranging from 50 mM to 3.125 mM were gently mixed in a 9:1 (v/v) ratio. The reaction mixtures were left at room temperature for 10 minutes. 5 µl of reaction mixture was then transferred onto a microscope slide and observed under a fluorescence microscope (OLYMPUS IX81 Microscope IX81F-3 with IX2-UCB-2 Controller and X-Cite 120PC Q; Olympus and Excelitas Technologies, Japan, resp. USA). As a negative control, bacterial cells with/without azide 1 as a monomer were used. Three independent measurements were performed.


  1. 1.

    Varki, A. Biological roles of glycans. Glycobiology 27, 3–49 (2017).

    CAS  Article  Google Scholar 

  2. 2.

    Sharon, N. Carbohydrates as future anti-adhesion drugs for infectious diseases. Biochim. Biophys. Acta 1760, 527–537 (2006).

    CAS  Article  Google Scholar 

  3. 3.

    Imberty, A., Wimmerová, M., Mitchell, E. P. & Gilboa-Garber, N. Structures of the lectins from Pseudomonas aeruginosa: insight into the molecular basis for host glycan recognition. Microbes Infect. 6, 221–228 (2004).

    CAS  Article  Google Scholar 

  4. 4.

    Farmer, J. J. et al. Xenorhabdus luminescens (DNA hybridization group 5) from human clinical specimens. J. Clin. Microbiol. 27, 1594–1600 (1989).

    PubMed  PubMed Central  Google Scholar 

  5. 5.

    Peel, M. M. et al. Isolation, identification, and molecular characterization of strains of Photorhabdus luminescens from infected humans in Australia. J. Clin. Microbiol. 37, 3647–3653 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Mulley, G. et al. From insect to man: Photorhabdus sheds light on the emergence of human pathogenicity. PloS One 10, e0144937 (2015).

    Article  Google Scholar 

  7. 7.

    Gerrard, J. G. et al. Nematode symbiont for Photorhabdus asymbiotica. Emerg. Infect. Dis. 12, 1562–1564 (2006).

    Article  Google Scholar 

  8. 8.

    Peat, S. M. et al. A robust phylogenetic framework for the bacterial genus Photorhabdus and its use in studying the evolution and maintenance of bioluminescence: A case for 16S, gyrB, and glnA. Mol. Phylogenet. Evol. 57, 728–740 (2010).

    CAS  Article  Google Scholar 

  9. 9.

    Kuwata, R., Yoshiga, T., Yoshida, M. & Kondo, E. Mutualistic association of Photorhabdus asymbiotica with Japanese heterorhabditid entomopathogenic nematodes. Microbes Infect. 10, 734–741 (2008).

    CAS  Article  Google Scholar 

  10. 10.

    Thanwisai, A. et al. Diversity of Xenorhabdus and Photorhabdus spp. and their symbiotic entomopathogenic nematodes from Thailand. PLoS ONE 7, e43835 (2012).

    CAS  ADS  Article  Google Scholar 

  11. 11.

    Gerrard, J. G., McNevin, S., Alfredson, D., Forgan-Smith, R. & Fraser, N. Photorhabdus species: bioluminescent bacteria as emerging human pathogens? Emerg. Infect. Dis. 9, 251–254 (2003).

    Article  Google Scholar 

  12. 12.

    Gerrard, J., Waterfield, N., Vohra, R. & ffrench-Constant, R. Human infection with Photorhabdus asymbiotica: an emerging bacterial pathogen. Microbes Infect. 6, 229–237 (2004).

    CAS  Article  Google Scholar 

  13. 13.

    Weissfeld, A. S. et al. Photorhabdus asymbiotica, a pathogen emerging on two continents that proves that there is no substitute for a well-trained clinical microbiologist. J. Clin. Microbiol. 43, 4152–4155 (2005).

    Article  Google Scholar 

  14. 14.

    Costa, S. C. P. et al. Recent insight into the pathogenicity mechanisms of the emergent pathogen Photorhabdus asymbiotica. Microbes Infect. 12, 182–189 (2010).

    CAS  Article  Google Scholar 

  15. 15.

    Machado, R. A. R. et al. Whole-genome-based revisit of Photorhabdus phylogeny: proposal for the elevation of most Photorhabdus subspecies to the species level and description of one novel species Photorhabdus bodei sp. nov., and one novel subspecies Photorhabdus laumondii subsp. clarkei subsp. nov. Int. J. Syst. Evol. Microbiol. 68, 2664–2681 (2018).

  16. 16.

    Jančaříková, G. et al. Characterization of novel bangle lectin from Photorhabdus asymbiotica with dual sugar-binding specificity and its effect on host immunity. PLOS Pathog. 13, e1006564 (2017).

    Article  Google Scholar 

  17. 17.

    Chemani, C. et al. Role of LecA and LecB lectins in Pseudomonas aeruginosa - induced lung injury and effect of carbohydrate ligands. Infect. Immun. 77, 2065–2075 (2009).

    CAS  Article  Google Scholar 

  18. 18.

    Wu, X. R., Sun, T. T. & Medina, J. J. In vitro binding of type 1-fimbriated Escherichia coli to uroplakins Ia and Ib: relation to urinary tract infections. Proc. Natl. Acad. Sci. USA 93, 9630–9635 (1996).

    CAS  ADS  Article  Google Scholar 

  19. 19.

    Kumar, A. et al. A novel fucose-binding lectin from Photorhabdus luminescens (PLL) with an unusual hepta-bladed β-propeller tetrameric structure. J. Biol. Chem. 291, 25032–25049 (2016).

    CAS  Article  Google Scholar 

  20. 20.

    Beshr, G. et al. Photorhabdus luminescens lectin A (PllA): A new probe for detecting α-galactoside–terminating glycoconjugates. J. Biol. Chem. 292, 19935–19951 (2017).

    CAS  Article  Google Scholar 

  21. 21.

    Diggle, S. P. et al. The galactophilic lectin, LecA, contributes to biofilm development in Pseudomonas aeruginosa. Environ. Microbiol. 8, 1095–1104 (2006).

    CAS  Article  Google Scholar 

  22. 22.

    Bertolotti, B. et al. Polyvalent C-glycomimetics based on l-fucose or d-mannose as potent DC-SIGN antagonists. Org. Biomol. Chem. 15, 3995–4004 (2017).

    CAS  Article  Google Scholar 

  23. 23.

    Bertolotti, B. et al. Nonhydrolyzable C-disaccharides, a new class of DC-SIGN ligands. Carbohydr. Res. 435, 7–18 (2016).

    CAS  Article  Google Scholar 

  24. 24.

    Kašáková, M. et al. Selectivity of original C-hexopyranosyl calix[4]arene conjugates towards lectins of different origin. Carbohydr. Res. 469, 60–72 (2018).

    Article  Google Scholar 

  25. 25.

    Rostovtsev, V. V., Green, L. G., Fokin, V. V. & Sharpless, K. B. A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective “Ligation” of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. 41, 2596–2599 (2002).

    CAS  Article  Google Scholar 

  26. 26.

    Tornøe, C. W., Christensen, C. & Meldal, M. Peptidotriazoles on Solid Phase: [1,2,3]-Triazoles by Regiospecific Copper(I)-Catalyzed 1,3-Dipolar Cycloadditions of Terminal Alkynes to Azides. J. Org. Chem. 67, 3057–3064 (2002).

    Article  Google Scholar 

  27. 27.

    Lundquist, J. J. & Toone, E. J. The cluster glycoside effect. Chem. Rev. 102, 555–578 (2002).

    CAS  Article  Google Scholar 

  28. 28.

    Cecioni, S., Imberty, A. & Vidal, S. Glycomimetics versus multivalent glycoconjugates for the design of high affinity lectin ligands. Chem. Rev. 115, 525–561 (2015).

    CAS  Article  Google Scholar 

  29. 29.

    Bernardi, A. et al. Multivalent glycoconjugates as anti-pathogenic agents. Chem. Soc. Rev. 42, 4709–4727 (2013).

    CAS  Article  Google Scholar 

  30. 30.

    Tabarani, G. et al. DC-SIGN neck domain is a pH-sensor controlling oligomerization: SAXS and hydrodynamic studies of extracellular domain. J. Biol. Chem. 284, 21229–21240 (2009).

    CAS  Article  Google Scholar 

  31. 31.

    Jančaříková, G. et al. Synthesis of α-L-fucopyranoside-presenting glycoclusters and investigation of their interaction with recombinant Photorhabdus asymbiotica lectin (PHL). Chem. - Eur. J., https://doi.org/10.1002/chem.201705853 (2018).

  32. 32.

    Mitchell, E. P. et al. High affinity fucose binding of Pseudomonas aeruginosa lectin PA-IIL: 1.0 Å resolution crystal structure of the complex combined with thermodynamics and computational chemistry approaches. Proteins Struct. Funct. Bioinforma. 58, 735–746 (2004).

    Article  Google Scholar 

  33. 33.

    Boukerb, A. M. et al. Antiadhesive properties of glycoclusters against Pseudomonas aeruginosa lung infection. J. Med. Chem. 57, 10275–10289 (2014).

    CAS  Article  Google Scholar 

  34. 34.

    Yu, G. et al. A Sugar-functionalized amphiphilic pillar[5]arene: Synthesis, self-assembly in water, and application in bacterial cell agglutination. J. Am. Chem. Soc. 135, 10310–10313 (2013).

    CAS  Article  Google Scholar 

  35. 35.

    Gestwicki, J. E., Strong, L. E., Cairo, C. W., Boehm, F. J. & Kiessling, L. L. Cell aggregation by scaffolded receptor clusters. Chem. Biol. 9, 163–169 (2002).

    CAS  Article  Google Scholar 

  36. 36.

    Adamová, L., Malinovská, L. & Wimmerová, M. New sensitive detection method for lectin hemagglutination using microscopy. Microsc. Res. Tech. 77, 841–849 (2014).

    Article  Google Scholar 

  37. 37.

    Wiseman, T., Williston, S., Brandts, J. F. & Lin, L.-N. Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal. Biochem. 179, 131–137 (1989).

    CAS  Article  Google Scholar 

  38. 38.

    Mueller, U. et al. Facilities for macromolecular crystallography at the Helmholtz-Zentrum Berlin. J. Synchrotron Radiat. 19, 442–449 (2012).

    CAS  Article  Google Scholar 

  39. 39.

    Krug, M., Weiss, M. S., Heinemann, U. & Mueller, U. XDSAPP: a graphical user interface for the convenient processing of diffraction data using XDS. J. Appl. Crystallogr. 45, 568–572 (2012).

    CAS  Article  Google Scholar 

  40. 40.

    Winn, M. D. et al. Overview of the CCP 4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

    CAS  Article  Google Scholar 

  41. 41.

    McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    CAS  Article  Google Scholar 

  42. 42.

    Murshudov, G. N. et al. REFMAC 5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011).

    CAS  Article  Google Scholar 

  43. 43.

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    CAS  Article  Google Scholar 

  44. 44.

    Lebedev, A. A. et al. JLigand: a graphical tool for the CCP4 template-restraint library. Acta Crystallogr. D Biol. Crystallogr. 68, 431–440 (2012).

    CAS  Article  Google Scholar 

  45. 45.

    Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    CAS  Article  Google Scholar 

Download references


The project was supported by the Czech Science Foundation (15-17572S, 18-18964S). We acknowledge the Biomolecular Interactions and Crystallization Core Facility of CEITEC supported by the CIISB research infrastructure (LM2015043) funded by MEYS CR for their support with obtaining scientific data presented in this paper. Diffraction data have been collected on BL14.3 at the BESSY II electron storage ring operated by the Helmholtz-Zentrum Berlin (Mueller et al., 2015). We would particularly like to acknowledge the help and support of Franziska Huschmann during the experiment. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We also thank Ben Watson-Jones for language corrections.

Author information




M.W. and J.M. designed the study. M.K., B.B., B.O. synthesized the fucosylated ligands, G.J. and J.H. conducted experiments. J.M., K.P. and M.W. supervised experiments and validated the results. G.J., J.H., J.M. and M.W. wrote the paper.

Corresponding author

Correspondence to Michaela Wimmerová.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Paulíková, G., Houser, J., Kašáková, M. et al. Fucosylated inhibitors of recently identified bangle lectin from Photorhabdus asymbiotica. Sci Rep 9, 14904 (2019). https://doi.org/10.1038/s41598-019-51357-9

Download citation


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing