David Goyard

Veronica Baldoneschi

Annabelle Varrot

Michele Fiore

Anne Imberty

Barbara Richichi

Olivier Renaudet

Cristina Nativi

¥ M Fiore

≠ A Imberty

Multivalent Glycomimetics with Affinity and Selectivity toward Fucose-Binding Receptors from Emerging Pathogens

come

INTRODUCTION

Many microorganisms use lectins as adhesins to interact with host glycoconjugates and to trigger the first step of infection. Among carbohydrate epitopes, fucose has a prominent role since it is an ubiquitous ligand that can be the target of microorganisms receptors on human tissue, [START_REF] Becker | Fucose: biosynthesis and biological function in mammals[END_REF] but also in plant cell walls. [START_REF] Anderson | Metabolic click-labeling with a fucose analog reveals pectin delivery, architecture, and dynamics in Arabidopsis cell walls[END_REF] Most opportunistic pathogens are indeed able to spread from soil, to plants and humans. In mammals' tissues, fucose is a marker of inflammation [START_REF] Gringhuis | Fucose-specific DC-SIGN signalling directs T helper cell type-2 responses via IKK and CYLD-dependent Bcl3 activation[END_REF] and fucosylated glycoconjugates are overexpressed in airways of cystic fibrosis patients. [START_REF] Glick | Activity of fucosyltransferases and altered glycosylation in cystic fibrosis airway epithelial cells[END_REF] Pathogens responsible for lung infection in those patients, produce soluble lectins which are involved in adhesion of pathogens to fucosylated glycoconjugates such as human histo-blood group epitopes. [START_REF] Heggelund | Histo-blood group antigens as mediators of infections[END_REF] Soluble fucose-binding lectins have been characterized in Pseudomonas aeruginosa, [START_REF] Garber | Specificity of the fucose-binding lectin of Pseudomonas aeruginosa[END_REF] Burkholderia ambifaria [START_REF] Audfray | The fucosebinding lectin from opportunistic pathogen Burkholderia ambifaria binds to both plant and human oligosaccharidic epitopes[END_REF] and airborne fungus Aspergillus fumigatus. [START_REF] Houser | A soluble fucose-specific lectin from Aspergillus fumigatus conidia -Structure, specificity and possible role in fungal pathogenicity[END_REF] Among them, one structural family is of high interest: B. ambifaria BambL and A. fumigatus AFL (FleA) are made by the repeat of similar small β-sheets of approx. 40 amino acids (Figure 1, panels A and B). Both lectins are able to bind 6 fucose residues with high affinity. [START_REF] Audfray | The fucosebinding lectin from opportunistic pathogen Burkholderia ambifaria binds to both plant and human oligosaccharidic epitopes[END_REF][START_REF] Houser | A soluble fucose-specific lectin from Aspergillus fumigatus conidia -Structure, specificity and possible role in fungal pathogenicity[END_REF] The AFL sequence is longer, consisting in six repeats in tandem [START_REF] Houser | A soluble fucose-specific lectin from Aspergillus fumigatus conidia -Structure, specificity and possible role in fungal pathogenicity[END_REF] while BambL is formed by trimerisation of a two-repeat peptides. [START_REF] Kerr | FleA Expression in Aspergillus fumigatus Is Recognized by Fucosylated Structures on Mucins and Macrophages to Prevent Lung Infection[END_REF] In this family, the affinity for fucose is in the micromolar range, an unusually strong binding for protein-carbohydrate interaction. In order to hamper pathogen adhesion to host cells, fucose mimetics become attracting tools to design synthetic ligands to intercept fucose-binding bacteria. Monomeric fucoside derivatives have been tested against AFL demonstrating that (2E)-hexenyl α-L-fucopyranoside is a potent functional inhibitor of FleA. [START_REF] Kerr | FleA Expression in Aspergillus fumigatus Is Recognized by Fucosylated Structures on Mucins and Macrophages to Prevent Lung Infection[END_REF] With respect to native glycans, glycomimetics are designed to be more resistant to glycosidases, to present a higher bioavailability and to be tailored to decorate scaffolds of different chemical constitutions. [START_REF] Cecioni | Glycomimetics versus multivalent glycoconjugates for the design of high affinity lectin ligands[END_REF][START_REF] Ernst | From carbohydrate leads to glycomimetic drugs[END_REF] In this context, we recently reported the synthesis of a fucose-based glycomimetic 1 (Figure 2) presenting a non-natural aryl -O-fucosyl linkage. [START_REF] Richichi | Synthesis of a selective inhibitor of a fucose binding bacterial lectin from Burkolderia ambifaria[END_REF] The fucosyl derivative 1 binds to BambL lectin with an affinity comparable to that of the natural ligand since its fucoside moiety establishes the same contact with BambL active site as the natural ligand (Figure 1C). Moreover, it presents an unprecedentedly described specificity for BambL binding site and does not bind to other fucosespecific lectins such as P. aeruginosa LecB or human DC-SIGN. Because lectins are multimeric, higher affinity can be obtained by synthesizing multivalent glycocompounds. [START_REF] Chabre | Combining glycomimetic and multivalent strategies toward designing potent bacterial lectin inhibitors[END_REF][START_REF] Bernardi | Multivalent glycoconjugates as anti-pathogenic agents[END_REF] For example, calixarene-based fucosylated clusters display strong affinity for LecB (45 nM) and provide an almost complete protection against P. aeruginosa in a mice infection model. [START_REF] Boukerb | Anti-adhesive properties of glycoclusters against Pseudomonas aeruginosa lung infection[END_REF] Less efforts have been devoted to competing with β-propeller lectins. Fucosylated glycocluster exhibiting 4, 6 or 8 residues were synthesized based on mannosecentered and branched-phosphodiester scaffolds with best affinities of approximately 43 nM. [START_REF] Ligeour | Synthesis of antenna and mannose-centered fucosylated glycoclusters and their binding studies with Burkholderia ambifaria lectin (BambL)[END_REF] Additionally, hexavalent cyclotriveratrylene [START_REF] Galanos | Cyclotriveratrylene-based glycoclusters as high affinity ligands of bacterial lectins from Pseudomonas aeruginosa and Burkholderia ambifaria[END_REF] and penta-and decavalent pillarenes [START_REF] Buffet | Pillar[5]arene-based glycoclusters: synthesis and multivalent binding to pathogenic bacterial lectins[END_REF]20 have also shown very strong affinity for BambL (ca. 20 nM). Multivalent compounds obtained by dynamic assembly in the presence of BambL also blocked the entry in epithelial cells with high efficiency. [START_REF] Machida | Dynamic cooperative glycan assembly blocks the binding of bacterial lectins to epithelial cells[END_REF] The selectivity and the simple synthesis of ligand 1 motivated our interest in synthesizing a multivalent system containing the fucose mimetic 1. This compound was diastereoselectively obtained relying on an efficient inverse electron-demand Diels-Alder reaction. It is stable in the 4-8 pH range and presents a carboxylic hook to introduce a spacer and/or to be linked to a scaffold.

To achieve this purpose, we have selected cyclopeptide-based scaffolds which have been proved useful for the construction of tetra-, [START_REF] Fiore | Tetravalent glycocyclopeptide with nanomolar affinity to wheat germ agglutinin Org[END_REF] hexa- [START_REF] Pifferi | Synthesis of mannosylated glycodendrimers and evaluation against BC2L-A lectin from Burkholderia cenocepacia[END_REF] and hexadecavalent [START_REF] Berthet | High affinity glycodendrimers for the lectin LecB from Pseudomonas aeruginosa[END_REF][START_REF] Thomas | Divergent and convergent synthesis of GalNAc-conjugated dendrimers using dual orthogonal ligations[END_REF] glycoconjugates with nanomolar affinity for diverse vegetal and bacterial lectins. Herein we report the synthesis of a cyclopeptide-based hexavalent structure displaying fucose mimetic 1 (conjugate 2) and the binding studies with BambL, AFL and LecB by isothermal titration calorimetry (ITC). For comparison, the analogue 4, conjugated with aminooxylated fucose 3 has also been prepared, as previously described. [START_REF] Bossu | Synthesis of O-(6-deoxy-α-and β-L-galactopyranosyl) hydroxylamines (α-and β-L-fucopyranosylhydroxylamines)[END_REF][START_REF] Dulery | Alpha and beta L-Fucopyranosyl oxyamines: key intermediates for the preparation of fucose-containing glycoconjugates by oxime ligation[END_REF]

RESULTS AND DISCUSSION

Because the target lectins contain six binding sites, we have synthesized two cyclopeptide scaffolds containing 14 amino acid residues (Scheme 1) instead of the usual cyclodecapeptide as previously described. [START_REF] Daskhan | Multivalent glycocyclopeptides: toward nano-sized glycostructures[END_REF][START_REF] Galan | Multivalent glyco(cyclo)peptides[END_REF] In a recent study, we have indeed demonstrated that the expansion of the cycle does not disturb the conformation stability of the scaffold which still shows a characteristic β-sheet profile as proved by circular dichroism. [START_REF] Pifferi | Synthesis of mannosylated glycodendrimers and evaluation against BC2L-A lectin from Burkholderia cenocepacia[END_REF] The cyclopeptide 5 has been synthesized on solid-phase following a previously described procedure. [START_REF] Pifferi | Synthesis of mannosylated glycodendrimers and evaluation against BC2L-A lectin from Burkholderia cenocepacia[END_REF] After Boc-deprotection of 5 upon treatment with TFA, the Boc-NH-PEG-CO2H linker has been conjugated by standard amide coupling with PyBOP to the lysine side chains and the Boc groups have been successively removed by treatment with TFA to provide scaffold 6. Hexavalent compound 2 was next prepared from 6 by PyBOP-promoted coupling with fucose mimetic 1. The reaction occurred quantitatively at room temperature in 30 min and provided 2 in 50% yield after semi-preparative HPLC purification. Finally, conjugate 4 was synthesized following an oxime strategy. Boc-protected serine residues were first conjugated to 6 then successively deprotected and treated with sodium periodate to generate oxo-aldehyde functions. The resulting compound was finally ligated with aminooxy fucose 3 [START_REF] Pifferi | Synthesis of mannosylated glycodendrimers and evaluation against BC2L-A lectin from Burkholderia cenocepacia[END_REF][START_REF] Berthet | High affinity glycodendrimers for the lectin LecB from Pseudomonas aeruginosa[END_REF] in water with 0.1% TFA (pH 2.2) to provide the oxime-linked compound 4 in quantitative yield after purification by HPLC.

Monovalent compound 1 was previously demonstrated to be efficiently bound by BambL with affinity in the same order than the one measured for α-methyl-L-fucoside (αMeFuc) with a Kd close to 1 µM. [START_REF] Richichi | Synthesis of a selective inhibitor of a fucose binding bacterial lectin from Burkolderia ambifaria[END_REF] In the present work, we tested the efficiency of compound 1 to bind AFL and compared it to αMeFuc.

R O O R O R O R O R O O O NH 3 + CF 3 CO 2 - 6 5 2 4 a) b) c) R =
Compound 1 was previously demonstrated to not bind to bacterial lectin LecB and human lectin DC-SIGN. [START_REF] Richichi | Synthesis of a selective inhibitor of a fucose binding bacterial lectin from Burkolderia ambifaria[END_REF] The precise specificity of 1 for BambL and AFL makes it a specific inhibitor for the fucose-binding site present in this family of β-propeller lectin. When analyzing the thermodynamics contribution, the binding mode of 1 does not present the entropy barrier, classically observed in protein-carbohydrate interactions. AFL and BambL interaction with αMeFuc is enthalpy driven with an unfavorable entropy term, while binding to compound 1 does not involve any significant entropy. Multivalent compounds 2 and 4 were evaluated in titration microcalorimetry experiments to determine the stoichiometry and thermodynamic parameters of their binding with LecB, BambL and AFL (Figure 3, Table 2). Compounds 1 and αMeFuc were used as monovalent references to calculate the relative potency of 2 and 4 respectively. Titrations were performed in the direct injection mode (i.e. ligand in syringe and protein in cell). The analysis of the thermodynamic contributions showed that for both compounds, binding is driven by enthalpy but is counterbalanced by a strong entropy barrier resulting from the loss of flexibility induced upon lectin binding. Interestingly, the selectivity of monovalent compound 1 toward BambL and AFL was still observed with hexavalent compound 2, indicating that the fucose mimetic cannot bind to LecB even at high local concentration. On the contrary, hexafucosylated compound 4 exhibits a Kd of 165 nM for LecB, which confirms that the lack of binding of 2 does not come from an unfavorable geometry of the scaffold. In view of the large increase of affinity observed for hexavalent ligands, molecular modeling was performed in order to evaluate if a single hexameric compound could reach the six binding site of one BambL trimer. As displayed in Figure 4, glycocluster 4 in extended conformation brings the fucose-analog extremities at distances that are compatible with the fucose binding site on BambL. However, the orientation of the fucose mimetics does not bring their extremities in a favorable location compared to the geometry of the glycoclusters. It is therefore unlikely that the six fucose-analogs would bind at the same time to the same BambL and cross-linking to different proteins is probably occurring.

We describe for the first time glycomimetics with nM affinity for lectins from pathogen organisms, and with precise specificity towards a class of lectins. At the present, the six-bladed fucose-specific propeller that is targeted by these compounds has been described in two human pathogenic microorganisms, i.e. B. ambifaria and A. fumigatus. The lectin has also been characterized from organisms that interact with plants cell wall, such as RSL from bacterium Ralstonia solanacearum, AOL from fungus Aspergillus oryzae, both plant pathogens, and AAL mushroom Aleuria aurantia. We previously demonstrated that RSL binds to αFuc1,2Gal

disaccharide, an epitope present in both human blood group oligosaccharides, and plant cell wall xyloglucan. Such soluble lectins could be therefore involved in adhesion to different substrates.

B. ambifaria and A. fumigatus are both lung pathogens that cause serious infections in immunosuppressed patients, and those with cystic fibrosis. In order to identify other possible target for glycomimetic compounds mimicking fucose, a search based on BambL sequence has been perfored in NIH Microbial database. Sequences with similar length and strong identity scores could be identified in several bacteria, some of them being considered as emergent pathogens (Figure 4). B. stabilis, B. pyrrocinia, and B. cepacia are members of BCC, the Burkholderia cepacia complex, a group of closely related species that are found in various environments, including hospitals, responsible for grave infection in cystic fibrosis patients, and often multi-resistant to antibiotics. [START_REF] Vandamme | Classification and identification of the Burkholderia cepacia complex: Past, present and future[END_REF] R. picketti and R. mannitolilytica are also emergent pathogens, now causing more frequent and more severe respiratory infections in CF patients, with most Ralstonia identified strains being multi-drug resistant. [START_REF] Lipuma | The changing microbial epidemiology in cystic fibrosis[END_REF] As displayed in Figure 4, the amino acids involved in fucose binding in this family of lectins are conserved in all described pathogens. It can be therefore expected that the glycomimetics described here, and the ones that could be developed from them in the future will be active on several emergent lung pathogens. Strategies for the use of such anti-infectious compounds are getting more urgent since many strains present increasing multi-resistance to antibiotics.

In summary, we report here a convergent approach that brings together the concepts of glycomimetic and of multivalency for reaching both high affinity and high specificity in targeting the interaction between pathogens and human tissues. Such approach was used previously with PGDSVSVTSWLVGSAIHIRVYAT-SGSTTTEWCWDGN-SWTKGGYTATN A.fum_2 KLGSPVAATSKEL---KHIRVYTLTEGNTLQEFAYDSGTGWYNGGLGGAK well characterized glycomimetics for blocking the interaction of human DC-SIGN with HIVglycoprotein [START_REF] Sattin | Inhibition of DC-SIGN-mediated HIV infection by a linear trimannoside mimic in a tetravalent presentation[END_REF] and the interaction of cholera-toxin with glycolipids. [START_REF] Arosio | A synthetic divalent cholera toxin glycocalix[4]arene ligand having higher affinity than natural GM1 oligosaccharide[END_REF] Nevertheless, this is the first report for specific targeting of the fucose-specific -propeller lectin present in both fungal and bacterial pathogens involved in lung infection in immunocompromised patients and cystic fibrosis ones. The affinities for BambL and AFL of the glycomimetic hexavalent clusters are in the same range as the fucoclusters as evaluated via ITC studies with dissociation constants in the nanomolar range. The increase in affinity in comparison with the monovalent epitope is very high, with a gain of almost 2000 for binding to AFL, indicating that the cyclopeptide raft scaffold provides efficient multivalency for binding to this lectin family. The non-natural aryl -O-fucosyl linkage of the glycomimetic adopts a constrained flat conformation that could fit only in the crevasseshaped binding site of this lectin family, resulting in very precise specificity. Since genes coding for highly similar lectin have been identified in the genome of several emergent pathogens, the present study can be the basis of anti-infectious therapy.

EXPERIMENTAL SECTION

General method: Reaction progress was monitored by reverse-phase HPLC-MS using C18 columns, at 1.3 mL min -1 (Nucleosil 120 Å, 3 μm C18 particles, 30 x 4.6 mm 2 ) with UV monitoring at 214 nm and 250 nm and with ESI-MS in positive mode, using a linear A-B gradient (5 to 40% B in 15 min, buffer A: 0.09% CF3COOH in water; buffer B: 0.09% CF3COOH in acetonitrile). Analytical HPLC analyses were performed by using C18 columns and a linear gradient (5 to 40% B in 25 min), with UV monitoring at 214 nm and 250 nm.

Synthesis of compound 2. To a solution of 6 (30 mg, 0.01 mmol) in dry DMF (3 ml), 1 [START_REF] Houser | A soluble fucose-specific lectin from Aspergillus fumigatus conidia -Structure, specificity and possible role in fungal pathogenicity[END_REF] (22 mg, 0.072 mmol), PyBOP (37 mg, 0.072 mmol) and DIPEA (31 µl, 0.18 mmol) were added. The mixture was stirred at rt and after 30 min, analytical HPLC found the quantitative conversion of 6 in 2. The solvent was evaporated and the peptide was precipitated in Et2O, centrifuged and washed three times with Et2O. The crude was purified by semi-preparative HPLC (gradient: 5% to 70% B in 30 min) to obtain 2 (20 mg, 0.005 mmol, 50%). Analytical HPLC: Rt = 14.5 min; MALDI-TOF-MS m/z calcd. for C182H264N26O62S6 2+ [M+2H] 2+ 1999.33 found 2000.00.

Synthesis of compound 4. To a solution of 6 (10 mg, 0.003 mmol) in H2O (2 ml), NaIO4 (38 mg, 0.18 mmol) was added and the mixture was stirred at rt. After 30 min, the mixture was purified by semi-preparative HPLC (gradient: 5% to 40% B in 30 min) to give a crude, which was used in the next reaction without further purification. A solution of crude and 3 (5 mg, 0.027 mmol) was stirred at rt in a mixture of H2O + 0.1% TFA (2 ml). After 30 min, analytical HPLC found the quantitative conversion of the intermediate in 4. The mixture was purified by semipreparative HPLC (gradient: 5% to 100% B in 30 min) to give pure 4 (11 mg, 0.003 mmol, >90%). MALDI-TOF-MS m/z calcd. for C146H246N32O68 2+ [M+2H] 2+ 1768.34 found 1768.40.

ASSOCIATED CONTENT

Figure 1 .

 1 Figure 1. Crystal structures of (A) BambL and (B) AFL, complexed with fucose or fucoside (PDB codes 4AHA and 3ZW2). (C) Model of compound 1 in intramonomeric site of BambL with amino acids of interest. Hydrogen bonds are represented as blue dotted lines.

Figure 2 .

 2 Figure 2. Structure of the aryl fucose mimetic 1, the hexavalent conjugate 2, the aminooxylated fucoside 3 and of the hexavalent conjugate 4.

Scheme 1 .

 1 Scheme 1. Reagents and conditions: a) i: TFA/CH 2 Cl 2 /H 2 O (47.5/47.5/5), rt, 2h; ii: Boc-NH-PEG-CO 2 H, PyBOP, DIPEA, DMF, rt, 30 min; iii: TFA/CH 2 Cl 2 /H 2 O (47.5/47.5/5), rt, 2h, 6 (69% over three steps); b) 1, PyBOP, DIPEA, DMF, rt, 30 min, 2 (50%); c) i: BocSer(tBu)OH, PyBOP, DIPEA, DMF, rt, 2 h; ii: TFA/TIS/H 2 O (95/2.5/2.5), rt, 2h; iii: NaIO 4 , H 2 O, rt, 30 min; iv: 3, H 2 O, 0.1% TFA, 4 (> 90%).

Figure 3 .

 3 Figure 3. Isothermal titration calorimetry data: thermograms and corresponding titration curves obtained by injections of compounds 2 and 4 into solutions of BambL (left) and AFL (right). Molar ratio is defined as the number of glycocluster molecules per lectin monomer.

Figure 4 .

 4 Figure 4. A. Model of compound 4 built with extended conformation of linkers. The cyclopeptide was built by extended a related model (see Ref. 30) obtained from NMR studies and molecular modeling. B. Model of BambL with compound 1 in the six fucose binding sites (adapted from Ref. 13). C. Superimposition of the two models with same scale for size evaluation.

Figure 4 .

 4 Figure 4. Multiple sequence alignment of BambL related sequences identified in the genomes of Burkholderia and Raltsonia pathogenic species. The two blocks correspond to blade repeats and the four amino acids directly involved in fucose binding are highlighted. Accession number are BAX62607.1 for B. stabilis, WP_034182107.1 for B. pyrrocinia, WP_059237490.1 for B. cepacia, WP_024976975.1 for R. picketti and WP_063392503.1 for R. mannitolilytica. Sequences of the two first blades of AFL (A.fum, amino acids 10 to 103) are included for comparison.

B

  .amb_1 ---MQTAAISWGTT--PSIRVYTAN-GNKITERCYDGS-NWYTGAFNQ B.stab_1 ---MQTAAISWGTT--PSIRVYTAN-GNKITERCYDGA-NWYTGAFNQ B.pyr_1 ---MQTAAISWGTT--PSIRVYTAN-GNKITERCYDGS-NWYTGAFAQ B.cep_1 ---MQTAAISWGST--PSIRVYTAN-GNKITERCYDGQ-NWYTGAFNQ R.pic_1 MSNVQTAATSWGTV--PSIRVYTAN-GGKITERCYDGK-GWYTGAFNE R.man_1 MSNVQTAATSWGTV--PSIRVYTAN-GGKITERCYDGK-GWYTGAFNE A.fum_1 LFRTGIAAVNSTN----HLRVYFQDVYGSIRESLYEG-

Table 1 .

 1 Microcalorimetry data for monovalent compounds interacting with BambL and AFL

			Boc			Boc			Boc
	Gly Boc	Lys Ala Boc	Lys Ala Boc	Lys Pro
	Pro	Lys	Ala	Lys	Ala	Lys	Gly	Gly	Lys Ala	Lys Ala	Lys Pro
											Pro	Lys	Ala	Lys	Ala	Lys	Gly
	Compound		Lectin			K d (µM)		ΔG (kJ/mol)	ΔH (kJ/mol)	-TΔS (kJ/mol)
					BambL a			1.54		-33.2	-28.9	-4.3
	1				AFL				10.3		-28.5	-28.7	0.2
	αMeFuc			BambL a AFL			0.99 43.3		-34.3 -24.9	-50.1 -39.2	15.8 14.3
	a from 8

Table 2 .

 2 Microcalorimetry data for multivalent compounds interacting with LecB, BambL and AFL each sugar is 314 times more efficient than the monovalent reference.Studies with BambL show that compound 2 binds with a Kd of 13.8 nM resulting in an improvement factor of 112 when compared to 1 and a relative potency of 19 per sugar residue.

	Compound	Lectin	K d (nM)	N a	ΔG (kJ/mol)	ΔH (kJ/mol)	T*ΔS (kJ/mol)	α
		LecB			No binding
	2	BambL	13.8±3.8	1.01±0.06	-45	-162.5±12.5	-117.5	112
		AFL	44±10	0.68±0.01	-42.1	-115.5±0.5	-73.7	234
		LecB	165±5	0.60±0.01	-38.7	-133.5±5.5	-94.6	2.6
	4	BambL	16.8±2.2	1.53±0.1	-44.5	-165±3	-120.5	59.1
		AFL	18.5±8.5	0.77±0.01	-44.4	-154.5±3.5	-109.5	1886 1
	a The stoichiometry has been calculated as ratio of multivalent compound for the whole lectin (four binding sites for
	LecB, six fucose binding sites for trimer of BambL and monomer of AFL). Coefficient α is the relative potency of the
	glycocluster compared to its monomeric epitope
	ITC measurements also showed that hexavalent compounds 2 and 4 bind very strongly to

both BambL and AFL. Surprisingly, they both exhibit similar Kd values despite their different epitopes. Compound 4 shows a Kd of 18.5 nM on AFL making it 1886 times more potent than αMeFuc meaning that to accommodate the clusters' flexible arms in the appropriate direction. Altogether these data indicate that compounds 2 and 4 are the best known ligands for these lectins BambL and AFL to date.

ACKNOWLEDGMENTS

This study was supported by CNRS and Université Grenoble Alpes. O.R. acknowledges the European Research Council Consolidator Grant "LEGO" (647938). This work has been supported by the French ANR project Glyco@Alps (ANR-15-IDEX-02) and Labex ARCANE (ANR-11-LABX-003) and association Vaincre la Mucoviscidose. Fondazione Cassa di Risparmio di Firenze is acknowledged for financial support. Authors thank Mr. Alvaro Brittoli (University of Florence) for having provided a sample of compound 1.

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: Isothermal Titration Calorimetry; Synthesis, RP-HPLC and ESI-MS of compounds 2, 4, 5', 6, 8 (PDF).

AUTHOR INFORMATION

Corresponding Authors

*E-mail: cristina.nativi@unifi.it *E-mail: anne.imberty@cermav.cnrs.fr *E-mail: olivier.renaudet@ujf-grenoble.fr

Notes

The authors declare no competing financial interest.

Keywords: glycoconjugates • biological activity • lectines • antipathogen agents • glycopeptides


Shuangshuang Zheng

Thorsten Eierhoff
email: eierhoff@hhu.de

Sahaja Aigal

Annette Brandel

Roland Thuenauer

Sophie De Bentzmann

Anne Imberty

Winfried Römer
email: winfried.roemer@bioss.uni-freiburg.de

The Pseudomonas aeruginosa lectin

Keywords: Bacterial infection, Host-pathogen interactions, Lectin, P. aeruginosa, Signal transduction

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen, which causes severe infections of the respiratory tract, urinary tract, skin and eyes [START_REF] Engel | Molecular Pathogenesis of Acute Pseudomonas aeruginosa Infections[END_REF]. Moreover, P. aeruginosa causes chronic infections in the lungs of patients with cystic fibrosis (CF) leading to damage of the lung epithelium, decline in lung function and death [START_REF] Lyczak | Lung infections associated with cystic fibrosis[END_REF]. Internalisation of P. aeruginosa by host cells significantly contributes to its pathogenicity [START_REF] Chi | Interaction of Pseudomonas aeruginosa with A549 pneumocyte cells[END_REF][START_REF] Fleiszig | Pseudomonas aeruginosa invades corneal epithelial cells during experimental infection[END_REF][START_REF] Fleiszig | Relationship between cytotoxicity and corneal epithelial cell invasion by clinical isolates of Pseudomonas aeruginosa[END_REF]. P. aeruginosa interacts with various host cell receptors including α5β1 integrin [START_REF] Roger | Fibronectin and alpha5beta1 integrin mediate binding of Pseudomonas aeruginosa to repairing airway epithelium[END_REF], CF transmembrane conductance regulator (CFTR) [START_REF] Pier | Cystic fibrosis transmembrane conductance regulator is an epithelial cell receptor for clearance of Pseudomonas aeruginosa from the lung[END_REF] and the glycosphingolipids (GSLs) asialo GM1 and asialo GM2 [START_REF] De Bentzmann | Asialo GM1 is a receptor for Pseudomonas aeruginosa adherence to regenerating respiratory epithelial cells[END_REF][START_REF] Emam | Glycolipid-Dependent, Protease Sensitive Internalization of Pseudomonas aeruginosa Into Cultured Human Respiratory Epithelial Cells[END_REF][START_REF] Krivan | Pseudomonas aeruginosa and Pseudomonas cepacia isolated from cystic fibrosis patients bind specifically to gangliotetraosylceramide (asialo GM1) and gangliotriaosylceramide (asialo GM2)[END_REF], to ensure efficient adhesion and cell entry. Although asialo GM1 and asialo GM2 have been shown not to be directly involved in initial Type-4-pili-mediated adhesion [START_REF] Emam | Laboratory and clinical Pseudomonas aeruginosa strains do not bind glycosphingolipids in vitro or during type IV pilimediated initial host cell attachment[END_REF], it is widely accepted that host cell GSLs can be hijacked by microbial factors leading to signalling and internalisation events [START_REF] Aigal | Plasma membrane reorganization: A glycolipid gateway for microbes[END_REF]. However, the interactions of P. aeruginosa ligands with GSLs and their pathophysiological consequences are poorly understood. In recent years, the two P. aeruginosa lectins LecA and LecB have attracted attention as drug targets, also because of exciting new discoveries about their diverse roles in impairing host cell physiology [START_REF] Cott | Pseudomonas aeruginosa lectin LecB inhibits tissue repair processes by triggering β-catenin degradation[END_REF][START_REF] Kühn | Considering autophagy, β-Catenin and E-Cadherin as innovative therapy aspects in AML[END_REF][START_REF] Kühn | The interplay of autophagy and β-Catenin signaling regulates differentiation in acute myeloid leukemia[END_REF][START_REF] Schneider | Lectins from opportunistic bacteria interact with acquired variable-region glycans of surface immunoglobulin in follicular lymphoma[END_REF][START_REF] Eierhoff | A lipid zipper triggers bacterial invasion[END_REF][START_REF] Novoa | A LecA ligand identified from a galactoside-conjugate array inhibits host cell invasion by Pseudomonas aeruginosa[END_REF][START_REF] Hauck | Discovery of two classes of potent glycomimetic inhibitors of Pseudomonas aeruginosa LecB with distinct binding modes[END_REF][START_REF] Boukerb | Antiadhesive Properties of Glycoclusters against Pseudomonas aeruginosa Lung Infection[END_REF]. LecA (also known as PA-IL) is a homotetrameric, multifunctional virulence factor with a preferential binding affinity for α-D-galactosylated glycans, in particular for the GSL globotriaosylceramide (galactose α1-4 galactose β1-4 glucosyl ceramide, also known as Gb3, CD77 or Pk blood group antigen) [START_REF] Gilboa-Garber | Inhibition of broad spectrum hemagglutinin from Pseudomonas aeruginosa by D-galactose and its derivatives[END_REF][START_REF] Gilboa-Garber | Pseudomonas aeruginosa lectins[END_REF][START_REF] Gilboa-Garber | Purification of the galactose-binding hemagglutinin of Pseudomonas aeruginosa by affinity column chromatography using sepharose[END_REF][START_REF] Lanne | Binding of the galactose-specific Pseudomonas aeruginosa lectin, PA-I, to glycosphingolipids and other glycoconjugates[END_REF][START_REF] Chen | Studies on the binding site of the galactose-specific agglutinin PA-IL from Pseudomonas aeruginosa[END_REF][START_REF] Blanchard | Structural basis of the preferential binding for globo-series glycosphingolipids displayed by Pseudomonas aeruginosa lectin I[END_REF]. It is involved in the attachment of the bacterium to human lung epithelial cells and biofilm formation [START_REF] Gilboa-Garber | Pseudomonas aeruginosa lectins[END_REF][START_REF] Sommer | New approaches to control infections: anti-biofilm strategies against gram-negative bacteria[END_REF][START_REF] Chemani | Role of LecA and LecB lectins in Pseudomonas aeruginosa-induced lung injury and effect of carbohydrate ligands[END_REF][START_REF] Mewe | Pseudomonas aeruginosa lectins I and II and their interaction with human airway cilia[END_REF]. Furthermore, LecA induces alveolar destruction during bacterial infection in vivo [START_REF] Chemani | Role of LecA and LecB lectins in Pseudomonas aeruginosa-induced lung injury and effect of carbohydrate ligands[END_REF]. Recently, we have demonstrated that LecA acts as an invasion factor for P. aeruginosa besides its known role as an adhesion factor. Thereby, LecA interacts with Gb3, triggering membrane engulfment of P. aeruginosa at the initial stage of entry into human lung epithelial cells [START_REF] Eierhoff | A lipid zipper triggers bacterial invasion[END_REF]. So far, it is unknown whether LecA further acts on other stages of the invasion process, e.g. the induction of host cell signalling.

Crk (CT10 regulator of kinase) family members, including CrkI, CrkII and Crk-like, serve as major signalling integrators in multiple cellular processes, such as cell adhesion, migration, and immune responses [START_REF] Feller | Crk family adaptors-signalling complex formation and biological roles[END_REF]. Amongst others, Crk adapter proteins are major substrates for the Abelson non-receptor tyrosine kinase Abl, but become also phosphorylated by membrane-bound receptors like EGFR and integrins [START_REF] Feller | c-Abl kinase regulates the protein binding activity of c-Crk[END_REF][START_REF] Cui | c-Abl kinase is required for beta 2 integrin-mediated neutrophil adhesion[END_REF][START_REF] Hashimoto | Phosphorylation of CrkII adaptor protein at tyrosine 221 by epidermal growth factor receptor[END_REF]. The family of Abelson non-receptor tyrosine kinases consists of two widely expressed members, Abl (ABL1) and Arg (Abl-related gene; ABL2) [START_REF] Pendergast | The Abl family kinases: mechanisms of regulation and signaling[END_REF][START_REF] Hanks | Genomic analysis of the eukaryotic protein kinase superfamily: a perspective[END_REF]. Abl acts in conjunction with Crk to regulate a number of cellular processes, including cell growth, survival, stress responses, proliferation, adhesion, actin dynamics and cell migration [START_REF] Pendergast | The Abl family kinases: mechanisms of regulation and signaling[END_REF][START_REF] Woodring | Regulation of F-actin-dependent processes by the Abl family of tyrosine kinases[END_REF][START_REF] Van Etten | Cycling, stressed-out and nervous: cellular functions of c-Abl[END_REF][START_REF] Hantschel | Regulation of the c-Abl and Bcr-Abl tyrosine kinases[END_REF][START_REF] Koleske | Essential roles for the Abl and Arg tyrosine kinases in neurulation[END_REF]. It is known that Abl/CrkII interaction is often engaged by human pathogens like Shigella flexneri and Salmonella enterica, for cell invasion, actin tail formation and host cell release [START_REF] Burton | Abl tyrosine kinases are required for infection by Shigella fexneri[END_REF][START_REF] Burton | Abl kinases regulate actin comet tail elongation via an N-WASP-dependent pathway[END_REF][START_REF] Burton | The Caenorhabditis elegans ABL-1 tyrosine kinase is required for Shigella flexneri pathogenesis[END_REF][START_REF] Ly | Abelson tyrosine kinase facilitates Salmonella enterica serovar typhimurium entry into epithelial cells[END_REF]. Data of a previously conducted RNAi screen using Drosophila S2 cells demonstrate that Abl-dependent CrkII phosphorylation promotes the cellular uptake of P. aeruginosa [START_REF] Pielage | RNAi screen reveals an Abl kinase-dependent host cell pathway involved in Pseudomonas aeruginosa internalization[END_REF]. However, the bacterial factors as well as the host cell receptors, which activate Abl/CrkII signalling during P. aeruginosa invasion, remain elusive.

In our study, we identified the host cell GSL Gb3 and the bacterial lectin LecA as key factors for CrkII Y221 phosphorylation during infection of human lung epithelial cells by P. aeruginosa.

Of note, we could demonstrate that the phosphorylation of CrkII is not depending on the presence and activity of Abl kinase. Binding of LecA to Gb3 is probably sensed by c-Src, which mediates the phosphorylation of CrkII, as suggested by Src kinase inhibitor studies.

Results

P. aeruginosa entry into lung epithelial cells is sensitive to tyrosine kinase inhibitors

In their study, Pielage et al. demonstrated that Abl-dependent CrkII phosphorylation supports the cellular internalisation of P. aeruginosa into HeLa and 3T3 fibroblast cells [START_REF] Pielage | RNAi screen reveals an Abl kinase-dependent host cell pathway involved in Pseudomonas aeruginosa internalization[END_REF]. Therefore, we first explored whether the uptake of the P. aeruginosa strain PAO1 into our cell model, the human lung epithelial cell line H1299, is susceptible to tyrosine kinase inhibitors, which have been described to inhibit Abl kinase activity. We used imatinib (STI571, Gleevec ® ), a wellknown Abl kinase inhibitor [START_REF] Goldman | Chronic myeloid leukemia: current treatment options[END_REF][START_REF] Wisniewski | Characterization of potent inhibitors of the Bcr-Abl and the c-kit receptor tyrosine kinases[END_REF], and quantified the uptake of PAO1 into H1299 cells in the presence of the inhibitor. Imatinib lowered PAO1 invasion in a dose-dependent manner (Fig. 1A). For instance, a reduction of around 80 % in invasiveness was achieved at a concentration of 100 µM. In a complementary approach, we used nilotinib (AMN107), which is a 20-to 40-fold more potent Abl kinase inhibitor [START_REF] Saglio | Nilotinib versus imatinib for newly diagnosed chronic myeloid leukemia[END_REF][START_REF] Weisberg | Characterization of AMN107, a selective inhibitor of native and mutant Bcr-Abl[END_REF][START_REF] Verstovsek | AMN107, a novel aminopyrimidine inhibitor of p190 Bcr-Abl activation and of in vitro proliferation of Philadelphia-positive acute lymphoblastic leukemia cells[END_REF][START_REF] Golemovic | AMN107, a novel aminopyrimidine inhibitor of Bcr-Abl, has in vitro activity against imatinib-resistant chronic myeloid leukemia[END_REF][START_REF] O'hare | In vitro activity of Bcr-Abl inhibitors AMN107 and BMS-354825 against clinically relevant imatinib-resistant Abl kinase domain mutants[END_REF]. Nilotinib treatment resulted in an even more pronounced inhibition of PAO1 invasion into H1299 cells (Suppl. Fig. 1A). A concentration of 1 µM of nilotinib was already sufficient to decrease PAO1 host cell entry by 50 %, whereas the reduction in invasion at a concentration of 100 µM was comparable to that achieved with imatinib.

To assess whether Abl kinase is also activated during infection of lung epithelial cells by PAO1, we analysed lysates of PAO1-infected H1299 cells for the phosphorylation level of CrkII at tyrosine 221 (phospho-CrkII Y221 ), which has been described to be phosphorylated by Abl kinase [START_REF] Feller | c-Abl kinase regulates the protein binding activity of c-Crk[END_REF]. We observed that the phospho-CrkII Y221 level was significantly increased in H1299 cells infected with PAO1 compared to uninfected cells (Fig. 1B). We next investigated if one of the virulence factors of P. aeruginosa, the lectin LecA, contributes to CrkII Y221 phosphorylation. LecA is a multifunctional virulence factor, supporting biofilm formation, bacterial attachment and cellular invasion [START_REF] Imberty | Structures of the lectins from Pseudomonas aeruginosa: insights into the molecular basis for host glycan recognition[END_REF][START_REF] Sicard | AFM investigation of Pseudomonas aeruginosa lectin LecA (PA-IL) filaments induced by multivalent glycoclusters[END_REF][START_REF] Bajolet-Laudinat | Cytotoxicity of Pseudomonas aeruginosa internal lectin PA-I to respiratory epithelial cells in primary culture[END_REF][START_REF] Gilboa-Garber | PA-I and PA-II lectin interactions with the ABO (H) and P blood group glycosphingolipid antigens may contribute to the broad spectrum adherence of Pseudomonas aeruginosa to human tissues in secondary infections[END_REF][START_REF] Diggle | The galactophilic lectin, LecA, contributes to biofilm development in Pseudomonas aeruginosa[END_REF][START_REF] Eierhoff | A lipid zipper triggers bacterial invasion[END_REF]. Our previous data adumbrates a role of LecA in signallingdependent entry processes. Therefore, we used a LecA-deletion mutant of PAO1 (ΔlecA, Suppl. Fig. 1B) to test if LecA is involved in CrkII phosphorylation during infection. Interestingly, ΔlecA-infected H1299 cells showed a lower phospho-CrkII Y221 level than wild type PAO1infected cells, indicating that LecA might play a role in CrkII phosphorylation (Fig. 1B). However, probably due to other activating bacterial factors, which could partially compensate the LecA-deletion effect, the level of phospho-CrkII Y221 in ΔlecA-infected H1299 cells did not approach the basal level. These results prompted us to analyse the impact of LecA in more detail in the absence of the multifactorial, bacterial context.

P. aeruginosa lectin LecA triggers CrkII Y221 phosphorylation in human lung epithelial cells

We then aimed for specifically addressing the effect of LecA on CrkII phosphorylation in the absence of additional, putative bacterial activators. Therefore, we used purified LecA for stimulation experiments of H1299 cells. Indeed, incubation of cells with nanomolar concentrations (20-100 nM) of LecA led to a significant increase in CrkII Y221 phosphorylation (Fig. 2A). Time-resolved CrkII Y221 phosphorylation studies showed a remarkable induction of phosphorylation, which peaked at 60 min of LecA treatment (Fig. 2B). Subsequent experiments using imatinib and nilotinib, demonstrate the dependency of LecA-induced phosphorylation of CrkII Y221 on general tyrosine kinase activity (Fig. 2C and Suppl. Fig. 2A). Both inhibitors potently reduced CrkII Y221 phosphorylation, for example by up to 90 % at a concentration of 1 µM of nilotinib. Besides imatinib and nilotinib, we tested the effect of two other tyrosine kinase inhibitors on LecA-induced CrkII Y221 phosphorylation: dasatinib (BMS-354825) and GNF-2. Dasatinib is a highly potent ATP-competitive dual Src/Abl kinase inhibitor [START_REF] Lombardo | Discovery of N-(2chloro-6-methyl-phenyl)-2-(6-(4-(2-hydroxyethyl)-piperazin-1-yl)-2-methylpyrimidin-4ylamino)thiazole-5-carboxamide (BMS-354825), a dual Src/Abl kinase inhibitor with potent antitumor activity in preclinical assays[END_REF], which is thought to bind the active conformation of the Abl kinase domain [START_REF] O'hare | In vitro activity of Bcr-Abl inhibitors AMN107 and BMS-354825 against clinically relevant imatinib-resistant Abl kinase domain mutants[END_REF][START_REF] Shah | Overriding imatinib resistance with a novel ABL kinase inhibitor[END_REF]. GNF-2, which binds to the myristate-binding pocket in the C-lobe of Abl kinase domain, is not known to inhibit any additional kinases [START_REF] Adrián | Allosteric inhibitors of Bcr-abldependent cell proliferation[END_REF][START_REF] Choi | N-myristoylated c-Abl tyrosine kinase localizes to the endoplasmic reticulum upon binding to an allosteric inhibitor[END_REF][START_REF] Zhang | Targeting Bcr-Abl by combining allosteric with ATP-binding-site inhibitors[END_REF]. The same level of inhibition was obtained with dasatinib and GNF-2 (Suppl. Fig. 2B).

To explore whether the binding of LecA to glycosylated receptors [START_REF] Gilboa-Garber | Inhibition of broad spectrum hemagglutinin from Pseudomonas aeruginosa by D-galactose and its derivatives[END_REF][START_REF] Gilboa-Garber | Pseudomonas aeruginosa lectins[END_REF][START_REF] Gilboa-Garber | Purification of the galactose-binding hemagglutinin of Pseudomonas aeruginosa by affinity column chromatography using sepharose[END_REF] is crucial for CrkII phosphorylation, we pre-incubated LecA with p-nitrophenyl-α-D-galactopyranoside (PNPG), a soluble galactose, which competes with galactose-conjugated host cell receptors for LecA binding. PNPG pre-treatment of LecA inhibited binding of LecA to H1299 cells in a dosedependent manner as confirmed by fluorescence microscopy and fluorescence-activated cell sorting (FACS) (Suppl. Fig. 2C-D). Consequently, PNPG prevented CrkII phosphorylation by LecA in a concentration-dependent manner as indicated by a significant reduction of phospho-CrkII Y221 levels (Fig. 2D). Collectively, these results suggest that CrkII phosphorylation in PAO1-infected lung epithelial cells is triggered by binding of LecA to glycosylated host cell receptors.

Gb3 binding is crucial for LecA-induced CrkII phosphorylation

LecA has been reported to bind the α-D-galactose of the host GSL Gb3 [START_REF] Lanne | Binding of the galactose-specific Pseudomonas aeruginosa lectin, PA-I, to glycosphingolipids and other glycoconjugates[END_REF][START_REF] Chen | Studies on the binding site of the galactose-specific agglutinin PA-IL from Pseudomonas aeruginosa[END_REF][START_REF] Gilboa-Garber | PA-I and PA-II lectin interactions with the ABO (H) and P blood group glycosphingolipid antigens may contribute to the broad spectrum adherence of Pseudomonas aeruginosa to human tissues in secondary infections[END_REF]. Therefore, we explored if GSLs in general and Gb3 in particular could serve as lipid receptors for LecAtriggered activation of Abl kinase. To this end, we inhibited the synthesis of glucosylceramide, a precursor of GSLs, in H1299 cells, using DL-threo-1-Phenyl-2-palmitoylamino-3-morpholino-1propanol (DL-PPMP) [START_REF] Emam | Glycolipid-Dependent, Protease Sensitive Internalization of Pseudomonas aeruginosa Into Cultured Human Respiratory Epithelial Cells[END_REF][START_REF] Lee | Improved inhibitors of glucosylceramide synthase[END_REF]. Treatment of H1299 cells with DL-PPMP for seven days prior to LecA incubation completely prevented LecA-induced phosphorylation of CrkII Y221 (Fig. 3A).

To deplete Gb3 specifically from the plasma membrane, H1299 cells were incubated with different concentrations of the B-subunit of Shiga toxin (StxB) to induce Gb3 endocytosis [START_REF] Jacewicz | Pathogenesis of shigella diarrhea. XI. Isolation of a shigella toxin-binding glycolipid from rabbit jejunum and HeLa cells and its identification as globotriaosylceramide[END_REF][START_REF] Lindberg | Identification of the carbohydrate receptor for Shiga toxin produced by Shigella dysenteriae type 1[END_REF][START_REF] Waddell | Induction of verotoxin sensitivity in receptor-deficient cell lines using the receptor glycolipid globotriosylceramide[END_REF][START_REF] Johannes | Shiga toxins-from cell biology to biomedical applications[END_REF]. Subsequent incubation of cells with Cy3-labeled StxB confirmed the dose-dependent depletion of Gb3 (Suppl. Fig. 3A, left panel), whereas the level of phospho-CrkII Y221 was not altered (Suppl. Fig. 3A, right panel). Interestingly, LecA treatment could not raise the amount of phospho-CrkII Y221 beyond the basal level, when cells were pre-treated with 65 nM StxB (Fig. 3B, indicated by a horizontal line).

To prove that Gb3 is indeed the key receptor for LecA-mediated CrkII phosphorylation, we used Chinese hamster ovary (CHO) cells, which do not express Gb3 [START_REF] Keusch | Globotriaosylceramide, Gb3, is an alternative functional receptor for Shiga-like toxin 2e[END_REF]. Accordingly, LecA incubation did not change the phospho-CrkII Y221 levels in these cells (Suppl. Fig. 3B). However, when CHO cells were stably transfected with Gb3 synthase (CHO-Gb3) (Suppl. Fig. 3C), they were sensitized for LecA-dependent phosphorylation of CrkII Y221 in contrast to cells which were transfected with the empty vector (CHO-vector) (Fig. 3C). Taken together, our data suggest that LecA induces CrkII phosphorylation by binding to the host GSL Gb3. We subsequently analysed if Abl kinase is responsible for LecA-mediated Crk Y221 phosphorylation.

Abl kinase is not involved in LecA-triggered CrkII Y221 phosphorylation

Since CrkII is reported to be a substrate of Abl, we asked whether Abl is involved in LecAtriggered phosphorylation of CrkII. Therefore, we performed an Abl knock down by RNAi. siRNA-mediated knock down resulted in up to 90 % decreased levels of Abl compared to cells transfected with scrambled siRNA. Interestingly, LecA was still able to stimulate CrkII phosphorylation in Abl knock down cells, with even increased levels compared to cells transfected with scrambled siRNA (Fig. 4). These results demonstrate that LecA triggers CrkII phosphorylation in an Abl-independent manner. In conclusion, the tyrosine kinase inhibitors that prevented LecA-triggered phosphorylation of CrkII (Fig. 2, Suppl. Fig. 2A,B) seem to affect also other tyrosine kinases beyond Abl, which are involved in CrkII Y221 phosphorylation, e.g. Src family kinases.

Src kinases mediate LecA-induced Abl-independent CrkII phosphorylation in human lung epithelial cells

Lectin/toxin binding to glycosphingolipids has been linked to the activation of Src family kinases (SFKs) and SFK-mediated signalling in mammalian cells [START_REF] Plattner | c-Abl is activated by growth factors and Src family kinases and has a role in the cellular response to PDGF[END_REF][START_REF] Dorey | Phosphorylation and structure-based functional studies reveal a positive and a negative role for the activation loop of the c-Abl tyrosine kinase[END_REF][START_REF] Furstoss | c-Abl is an effector of Src for growth factor-induced c-myc expression and DNA synthesis[END_REF]. SFKs, including Src, Fyn, Yes, Blk, Yrk, Fgr, Hck, Lck and Lyn, are involved in apoptosis, cell cycle progression, cytoskeletal rearrangement, differentiation, development, immune response, nervous system function, and transcription [START_REF] Brown | Regulation, substrates and functions of src[END_REF][START_REF] Thomas | Cellular functions regulated by Src family kinases[END_REF]. We tested whether Src kinases play a role in LecA-triggered CrkII phosphorylation in human lung epithelial cells. Therefore, we used a highly selective SFK inhibitor (KB SRC4), which does not inhibit homologous kinases, such as Abl kinase, in contrast to other SFK inhibitors [START_REF] Brandvold | Development of a highly selective c-Src kinase inhibitor[END_REF]. Treatment of H1299 cells with KB SRC4 prevented LecA-induced phosphorylation of CrkII Y221 completely at 10 µM (Fig. 5A), indicating that Src kinases are crucial upstream factors for CrkII Y221 phosphorylation.

Based on these findings, we then specifically tested the phosphorylation of Src at Y418, which is required for full Src activity. LecA increased phospho-Src Y418 levels in H1299 cells (Fig. 5B) characterizing Src as a signalling mediator amongst other kinases for CrkII phosphorylation. Pre-incubation of cells with Nilotinib prevented CrkII phosphorylation, as observed in previous experiments (Suppl. Fig. 1A), but did not affect LecA-induced Src kinase phosphorylation (Fig. 5C). In summary, our data suggest that the host GSL receptor Gb3 is engaged by P. aeruginosa through its lectin LecA to activate c-Src, which acts upstream or in parallel with Nilotinibsensitive kinases to phosphorylate CrkII Y221 , and to promote bacterial entry into human lung epithelial cells.

Discussion

Abl kinase function is often coupled to CrkII phosphorylation during infection of different human pathogens. However, the molecular mechanisms leading to activation of the Abl/CrkII pathway including pathogenic factors are largely unknown. For instance, Abl regulates the cellular invasion of the Gram-negative bacteria S. flexneri and S. enterica serovar Typhimurium via phosphorylation of CrkII [START_REF] Burton | Abl tyrosine kinases are required for infection by Shigella fexneri[END_REF][START_REF] Ly | Abelson tyrosine kinase facilitates Salmonella enterica serovar typhimurium entry into epithelial cells[END_REF]. The type III secretion systems (T3SS) of these two bacteria are required for host cell invasion by manipulating the actin polymerization machinery, involving the Arp2/3 and N-WASP/WAVE complexes in an Abl-dependent manner [START_REF] Burton | Abl kinases regulate actin comet tail elongation via an N-WASP-dependent pathway[END_REF][START_REF] Ly | Abelson tyrosine kinase facilitates Salmonella enterica serovar typhimurium entry into epithelial cells[END_REF][START_REF] Shi | WAVE2 signaling mediates invasion of polarized epithelial cells by Salmonella typhimurium[END_REF]. However, it is largely unknown whether (effector) proteins, translocated through the T3SS, could directly activate Abl kinase or represent Abl substrates. Solely for the effector proteins CagA of Helicobacter pylori and Tir of enteropathogenic Escherichia coli (EPEC), it was shown that they represent Abl kinase substrates [START_REF] Tammer | Activation of Abl by Helicobacter pylori: a novel kinase for CagA and crucial mediator of host cell scattering[END_REF][START_REF] Bommarius | Enteropathogenic Escherichia coli Tir is an SH2/3 ligand that recruits and activates tyrosine kinases required for pedestal formation[END_REF]. A recent study identified Abl tyrosine kinase coupled to CrkII phosphorylation to be an essential host cellular pathway for P. aeruginosa internalisation [START_REF] Pielage | RNAi screen reveals an Abl kinase-dependent host cell pathway involved in Pseudomonas aeruginosa internalization[END_REF]. It is not known which bacterial factor triggers the activation of Abl-dependent CrkII phosphorylation during the invasion process of P. aeruginosa. Our study highlights that LecA induces phospho-CrkII Y221 by binding to the GSL Gb3 in the host cell plasma membrane. Since there is currently no evidence that LecA is directly translocated into the cytoplasm of host cells or represents a substrate for host cell kinases, LecA could either function at the host cell surface or upon endocytosis. This certainly represents an uncommon mode of activation amongst Gramnegative pathogens.

Proteinaceous membrane receptors, including EGFR, PDGFR, GPCR and β2-integrins, have been described to be implicated in Abl activation and/or direct phosphorylation of CrkII [START_REF] Plattner | c-Abl is activated by growth factors and Src family kinases and has a role in the cellular response to PDGF[END_REF][START_REF] Yang | Non-receptor-tyrosine kinases integrate fast glucocorticoid signaling in hippocampal neurons[END_REF][START_REF] Cui | c-Abl kinase is required for beta 2 integrin-mediated neutrophil adhesion[END_REF][START_REF] Hashimoto | Phosphorylation of CrkII adaptor protein at tyrosine 221 by epidermal growth factor receptor[END_REF]. Here, we demonstrate that the GSL Gb3 mediates LecA-dependent CrkII activation (Fig. 3 and Suppl. Fig. 3), and therefore provides an uncommon and as yet undescribed receptor for a pathogenic bacterium to induce host cell signalling. The molecular mechanism how Gb3, which is located in the outer leaflet of the plasma membrane, communicates with the cytosolic membrane leaflet and induces CrkII phosphorylation on the cytosolic side is still unclear. Previous studies have shown that asymmetrically distributed GSLs with long fatty acyl chains might lead to interdigitation into the opposing leaflet of the bilayer [START_REF] Mehlhorn | Evidence that trans-bilayer interdigitation of glycosphingolipid long chain fatty acids may be a general phenomenon[END_REF][START_REF] Boggs | Do the long fatty acid chains of sphingolipids interdigitate across the center of bilayer of shorter chain symmetric phospholipids?[END_REF]. A recent study demonstrates that lactosyl ceramide (LacCer) species, which are common components of GSLs including Gb3, are indispensable for activation of the sarcoma tyrosine-protein kinase Lyn and that especially long fatty acid chains promote Lyn kinase activation [START_REF] Sonnino | Role of very long fatty acid-containing glycosphingolipids in membrane organization and cell signaling: the model of lactosylceramide in neutrophils[END_REF][START_REF] Iwabuchi | Significance of glycosphingolipid fatty acid chain length on membrane microdomain-mediated signal transduction[END_REF]. In fact, binding of StxB to Gb3 has been shown to activate Lyn kinase [START_REF] Mori | Globotriaosyl ceramide (CD77/Gb3) in the glycolipid-enriched membrane domain participates in B-cell receptor-mediated apoptosis by regulating lyn kinase activity in human B cells[END_REF]. Other groups have shown that differently hydroxylated Gb3 species have varying affinity for StxB, which consequently lead to differences in Src kinase activation [START_REF] Lingwood | Aglycone modulation of glycolipid receptor function[END_REF][START_REF] Lingwood | Glycosphingolipid functions[END_REF]. Src kinases themselves have been reported to regulate Abl kinase or Abl-dependent Crk phosphorylation [START_REF] Plattner | c-Abl is activated by growth factors and Src family kinases and has a role in the cellular response to PDGF[END_REF][START_REF] Furstoss | c-Abl is an effector of Src for growth factor-induced c-myc expression and DNA synthesis[END_REF] and notably, it has also been demonstrated for Lyn to promote cell entry of P. aeruginosa [START_REF] Kannan | Src kinase Lyn is crucial for Pseudomonas aeruginosa internalization into lung cells[END_REF][START_REF] Kannan | Cholesterol-rich membrane rafts and Lyn are involved in phagocytosis during Pseudomonas aeruginosa infection[END_REF]. In light of our observation of a LecA-induced and SFK-mediated CrkII phosphorylation, it will be interesting to investigate if distinct Gb3 species (in terms of hydroxylation, fatty acid chain length and saturation) determine the recruitment and activation of specific SFKs to promote bacterial uptake. Interestingly, although StxB and LecA both bound to Gb3, only LecA induces CrkII phosphorylation. Therefore, in addition to the diverse Gb3 species mentioned above, differences in the avidities between StxB and LecA towards Gb3 may lead to differences in Gb3 clustering behavior and receptor complex geometries in the plasma membrane, which could be decisive for recruiting and activating SFKs upstream of CrkII. Of note, we demonstrate here that CrkII is phosphorylated independently of Abl kinase but sensitive to Nilotinib treatment. This raises the question, which kinase(s) is/are acting upstream. Amongst those kinases that are effectively inhibited by nilotinib beyond c-Abl, are receptor tyrosine kinases [START_REF] Blay | Nilotinib: a novel, selective tyrosine kinase inhibitor[END_REF]. These kinases, such as the Discoidin domain receptor (DDR) [START_REF] Lemeer | Phosphotyrosine mediated protein interactions of the discoidin domain receptor 1[END_REF], have been reported to specifically bind CrkII, and could therefore be interesting candidates to be further explored if they phosphorylate CrkII in a Gb3dependent manner.

Previous studies revealed the multiple functions of LecA as a virulence factor of P. aeruginosa, which is involved in bacterial invasion, adhesion and biofilm formation [START_REF] Eierhoff | A lipid zipper triggers bacterial invasion[END_REF][START_REF] Chemani | Role of LecA and LecB lectins in Pseudomonas aeruginosa-induced lung injury and effect of carbohydrate ligands[END_REF][START_REF] Gilboa-Garber | PA-I and PA-II lectin interactions with the ABO (H) and P blood group glycosphingolipid antigens may contribute to the broad spectrum adherence of Pseudomonas aeruginosa to human tissues in secondary infections[END_REF][START_REF] Diggle | The galactophilic lectin, LecA, contributes to biofilm development in Pseudomonas aeruginosa[END_REF]. Our observation of LecA-induced CrkII activation adds a previously unidentified role of this lectin as a bacterial signalling factor, which regulates the entry of P. aeruginosa into host cells (Suppl. Fig. 4). These diverse roles of LecA are even more striking because LecA has no enzymatic activity, which could be attributed to a certain function.

Considering the multifactorial, cellular context of P. aeruginosa, LecA might not be the only lectin which triggers Abl kinase activity in lung epithelial cells. In fact, ongoing experiments in our lab with the fucose-binding lectin LecB, another lectin expressed by P. aeruginosa, show an elevating effect of LecB on phospho-CrkII Y221 levels in lung epithelial cells (data not shown), but neither the host cell receptor(s) nor the molecular mechanism of Abl activation have been solved yet. Moreover, the fimbrial adhesin PapG of uropathogenic E. coli, which, like LecA, also binds to a galactose-containing glycolipid (a-D-galactopyranosyl-(14)-3-D-galactopyranose) [START_REF] Lund | The PapG protein is the alpha-Dgalactopyranosyl-(1----4)-beta-D-galactopyranose-binding adhesin of uropathogenic Escherichia coli[END_REF] might be an interesting candidate to conceptually proove the potential of GSL-binding lectins to trigger CrkII signalling in epithelial cells.

Our data suggest a new signalling receptor for P. aeruginosa invasion and demonstrates a bivalent role for LecA during the bacterial invasion process. LecA-Gb3 interactions not only trigger initial, bacterial membrane engulfment [START_REF] Eierhoff | A lipid zipper triggers bacterial invasion[END_REF] but may also induce CrkII-dependent signalling that likely promotes efficient bacterial entry into host cells. Hence, this lectin-GSL complex may represent a potential target for drug development. Synthetic, bivalent LecA ligands have already been shown to have a pronounced inhibitory effect on bacterial uptake at low micro-molar concentrations [START_REF] Novoa | A LecA ligand identified from a galactoside-conjugate array inhibits host cell invasion by Pseudomonas aeruginosa[END_REF], probably reflecting the significant impact of this lectin on P. aeruginosa uptake. On the other hand, the identification of host cell factors, like Gb3, c-Src and CrkII, offers promising approaches for the development of drugs, targeting distinct host cell processes in order to face the problem of increasing antibiotic-resistant pathogens. The second generation tyrosine kinase inhibitor nilotinib, which is a well-tolerated drug and 20-to 40-fold more potent compared to imatinib [START_REF] Saglio | Nilotinib versus imatinib for newly diagnosed chronic myeloid leukemia[END_REF][START_REF] Weisberg | Characterization of AMN107, a selective inhibitor of native and mutant Bcr-Abl[END_REF][START_REF] Verstovsek | AMN107, a novel aminopyrimidine inhibitor of p190 Bcr-Abl activation and of in vitro proliferation of Philadelphia-positive acute lymphoblastic leukemia cells[END_REF][START_REF] Golemovic | AMN107, a novel aminopyrimidine inhibitor of Bcr-Abl, has in vitro activity against imatinib-resistant chronic myeloid leukemia[END_REF][START_REF] O'hare | In vitro activity of Bcr-Abl inhibitors AMN107 and BMS-354825 against clinically relevant imatinib-resistant Abl kinase domain mutants[END_REF], might prove to be effective against P. aeruginosa and other pathogens that subvert GSL/tyrosine kinase-dependent pathways.

Materials and Methods

Cell Culture, generation of stable cell lines and depletion of GSLs

The human lung epithelial cell line H1299 (American Type Culture Collection No. CRL-5803) was grown in Roswell Park Memorial Institute (RPMI) 1640 medium. CHO cells were grown in Dulbecco's Modified Eagle Medium (DMEM) medium. For the generation of stable cell lines, CHO cells were transfected with a plasmid, either encoding Gb3-synthase (Homo sapiens alpha 1,4-galactosyltransferase, NCBI reference sequence: NM_017436.5, expressed from vector pcDNA3; "CHO-Gb3") or the empty vector ("CHO-vector", Clontech Cat. No. 632523), both containing a Geneticin resistance marker. After selection with 2 mg/ml Geneticin, positive clones were maintained in DMEM containing 1 mg/ml Geneticin. To check for the expression of Gb3, we used Alexa488-labeled B-subunit of Shiga toxin (StxB-Alexa488), which is known to interact specifically with the GSL Gb3. All media were supplemented with 10 % fetal calf serum (FCS) and 2 mM L-glutamine. For GSL depletion, H1299 cells were passaged for seven days in the presence of 5 μM of glucosylceramide synthase inhibitor DL-PPMP. All cells were grown at 37 °C and 5 % CO2.

Bacterial strains and infections

The P. aeruginosa PAO1 wild type strain (PAO1), the LecA deletion mutant of PAO1 (ΔlecA) and GFP-tagged PAO1 [START_REF] Eierhoff | A lipid zipper triggers bacterial invasion[END_REF] were grown on Luria Broth (LB) agar plates and in LB medium. GFP tagging and depletion of lecA in P. aeruginosa were described in detail in Eierhoff et al. [START_REF] Eierhoff | A lipid zipper triggers bacterial invasion[END_REF]. The GFP-tagged strain was cultured in the presence of Gentamicin (60 μgꞏml -1 ). For infection, overnight cultures of P. aeruginosa were grown in LB medium to stationary phase. H1299 cells, CHO-vector cells and CHO-Gb3 cells were seeded in 6-well plates in growth medium. Next day, cells (70-80 % confluent) were starved in growth medium without FCS for 2 h at 37 °C. Then, cells were incubated with P. aeruginosa for 60 min at 37 °C at a multiplicity of infection (MOI) of 100. Cells were then lysed, and lysates were processed for Western blot analysis.

Invasion assays

Overnight cultures of GFP-tagged P. aeruginosa PAO1 were pelleted and re-suspended in RPMI medium, containing additionally 1 mM CaCl2 and MgCl2. H1299 cells (70-80 % confluent) were pre-treated with Abl kinase inhibitor imatinib (1-100 µM) or nilotinib (1-100 µM) for 30 min at 37 °C, respectively. In the presence of inhibitors, cells were incubated with P. aeruginosa (MOI of 100) for 2 h at 37 °C. Cells were washed three times with Dulbecco's phosphatebuffered saline (DPBS). Extracellular bacteria were inactivated by a 2 h treatment of cells at 37 °C with 400 μgꞏml -1 Amikacin sulfate. Subsequently, cells were washed two times with DPBS and lysed with 0.25 % (vol/vol) Triton X-100 at 37 °C. Cell extracts were plated on Gentamicin-containing (60 μgꞏml -1 ) LB-Miller Agar plates and incubated over night at 37 °C. The next day bacterial colonies were counted. Invasion was calculated as percentage of Amikacin-survived bacteria compared with the total number of bacteria associated with Amikacin-untreated cells. For comparison purposes, mean values of n ≥ 3 independent experiments were normalized to the invasion into inhibitor-untreated H1299 cells.

Lectin stimulation, RNA interference, SDS-PAGE and immunoblot analysis

LecA was expressed and purified from E. coli BL21 (DE3) transformed with the LecA encoding plasmid pET25-pa1l as described earlier [START_REF] Chemani | Role of LecA and LecB lectins in Pseudomonas aeruginosa-induced lung injury and effect of carbohydrate ligands[END_REF]. Labelling of purified LecA with the fluorophore Alexa488 (Thermo Fisher Scientific) was performed according to the manufacturer's protocol. Lyophilized, recombinant LecA was dissolved in DPBS (supplemented with Ca 2+ , Mg 2+ ), and used at a concentration of 100 nM. Cells were seeded to reach 70-80 % confluency, starved for 2 h and stimulated as indicated. Starvation was performed to exclude possible effects of growth factors and cytokines inherently present in FCS, which might interfere with LecA effects. Cells were harvested and lysed in modified radio-immunoprecipitation assay (RIPA) buffer (20 mM Tris (pH 8.0), 0.5 % (w/vol) Na-deoxycholate, 13.7 mM NaCl, 10 % (vol/vol) Glycerol, 0.1 % (w/vol) SDS, 2 mM EDTA, 200 μM Pefabloc, 11 µM Leupeptin, 0.8 µM Aprotinin, 1 mM Na-orthovanadate, 1 % (vol/vol) Phosphatase Inhibitor Cocktail 3 for at least 45 min at 4 °C. Cell lysates were collected and centrifuged to remove cell debris. Knock down of Abl was performed by RNA interference (RNAi). H1299 cells were transfected with 80-100 pmol of scrambled or Abl-specific siRNA (Santa Cruz, Cat. No. sc-37007 and sc-29843) using Lipofectamine (Thermo Fisher Scientific). Knock down efficiency was about 80 % as quantified by densitometric analysis of Abl Western blots.

The protein concentration of each extract was determined by using the Pierce BCA Protein Assay Kit. Equal amounts of proteins were loaded and separated on 8 % Tris-glycine SDS-PAGE gels and then transferred on nitrocellulose membranes. Membranes were blocked in 3 % BSA prepared with TBST (50 mM Tris (pH 7.6), 154 mM NaCl, 0.5 % (vol/vol) Tween 20) and incubated with primary antibodies (1:1000) at 4 °C overnight. Next day, horseradish peroxidase conjugates appropriate for each IgG (1:2000) were used to detect primary antibodies. Blot stripping was performed if necessary. Clarity TM Western ECL Chemiluminescent Substrate was used for detection of bound peroxidase. Luminescence was detected using Vilber Lourmat Fusion FX chemiluminescence imager. α-Tubulin was used as a loading control in all immunoblot experiments. If not indicated otherwise, α-Tubulin normalization of phospho-CrkII was justified by proofed correlation with Crk normalization (Suppl. Fig. 5). For comparison purposes, mean values of CrkII phosphorylation of n ≥ 3 independent experiments were normalized to uninfected or untreated H1299 and CHO cells, respectively.

Immunofluorescence microscopy and FACS

H1299 cells were seeded on glass coverslips. The next day cells (70-80 % confluent) were starved for 2 h at 37 °C with FCS-free medium. Starvation was performed to achieve results that are comparable to Western blot experiments. Meanwhile, LecA (100 nM), which was labeled with Alexa 488, was incubated with PNPG (0.1-5 mM) for 15 min at 37 °C. Cells were incubated with LecA-Alexa488 (with or without PNPG) for 60 min at 37 °C. Afterwards, cells were fixed with 4 % paraformaldehyde, saturated with 50 mM NH4Cl and permeabilized with a buffer containing 0.2 % (w/vol) BSA, 0.02 % saponin (w/vol) in DPBS for 20 min at room temperature. Finally, cells were counterstained with Phalloidin-ATTO647N (1:2000), DAPI (1:5000) and mounted with Mowiol containing DABCO. Samples were imaged on a confocal microscope (Nikon Eclipse Ti-E with Nikon A1R confocal laser scanner, 60 × oil objective, N.A. 1.49). Image acquisition and analysis were performed with NIS-Elements (Nikon). For FACS analysis, H1299 cells were treated with LecA-Alexa488 (with or without PNPG) as described above, then scraped and re-suspended in DPBS.

Antibodies and chemical reagents

The following antibodies were obtained from commercial sources: polyclonal rabbit anti-Crk p38 (phospho Y221) (Abcam, Cat. No. ab51222, RRID:AB_869267), monoclonal mouse anti-Crk (BD Biosciences, Cat. No. 610035, RRID:AB_397451), monoclonal rabbit anti-Src Phospho (pY418) (Abcam, Cat. No. 5595-1, RRID:AB_10999890), monoclonal rabbit anti-Src (32G6) mAb (Cell Signaling Technology, Cat. No. 2123, RRID:AB_10695000), polyclonal rabbit antic-Abl (Cell Signaling Technology, Cat. No. 2862, RRID:AB_2257757), monoclonal mouse antiβ-Actin (Sigma-Aldrich, Cat. No. A5316, RRID:AB_476743), monoclonal mouse anti-α-Tubulin (Sigma-Aldrich, Cat. No. T5168, RRID:AB_477579), horse anti-mouse-HRP (Cell Signaling Technology, Cat. No. 7076, RRID:AB_330924) and goat anti-rabbit-HRP (Cell Signaling Technology, Cat. No. 7074, RRID:AB_2099233). LecA was detected by a polyclonal rabbit anti-LecA antibody. Pierce BCA Protein Assay Kit and Clarity TM Western ECL Chemiluminescent Substrate were purchased from Thermo Scientific and BIO-RAD, respectively. Abl kinase inhibitor imatinib (mesylate) was obtained from Biovision, other inhibitors nilotinib, dasatinib and GNF-2 were from Santa Cruz. Purified DL-PPMP and PNPG were obtained from Santa Cruz and Sigma-Aldrich, respectively. SFK inhibitor KB SRC4 was from TOCRIS bioscience. The proteases Aprotinin, Leupeptin, Pefablock, Sodium orthovanadate and Phosphatase Inhibitor Cocktail 3 were all purchased from Sigma-Aldrich. RPMI 1640 and DMEM, DPBS, FCS and L-Glutamine were all purchased from Gibco. The following chemicals were obtained from Roth: BSA, DABCO, DAPI, EDTA, Geneticin, Glycerol, LB, Mowiol, NaCl, Na-deoxycholate, NH4Cl, paraformaldehyde, SDS, Tris (hydroxymethyl)-aminoethane, Triton X-100 and Tween 20. Amikacin sulfate, Gentamicin, Phalloidin-ATTO647N and Saponin were obtained from Sigma-Aldrich. StxB was purchased from Sigma-Aldrich (Cat. No. SML0562).

Statistical analysis

All data are presented as mean ± standard deviation (SD) values that have been calculated from the results of independent experiments. Statistical testing was performed with Microsoft Excel (Microsoft Office Standard 2010) and GraphPad Prism software (5.04) with data of n ≥ 3 independent experiments. Differences to the controls were analysed for significance by using one-way analysis of variance (ANOVA), with a Dunnett's Multiple Comparison Test compared to the control conditions.

  90. Römer W, Pontani LL, Sorre B, Rentero C, Berland L, Chambon V, et al. Actin dynamics drive membrane reorganization and scission in clathrin-independent endocytosis. Cell. 2010 Feb 19; 140(4):540-53. doi: 10.1016/j.cell.2010.01.010.

FigureFig. 1 :

 1 Figure

Fig. 2 :

 2 Fig. 2: P. aeruginosa lectin LecA triggers CrkII Y221 phosphorylation in human lung epithelial cells. (A) LecA induces CrkII Y221 phosphorylation in a dosedependent manner. (left) H1299 cells were incubated for 60 min in the presence of LecA (0-200 nM). (right) Phospho-CrkII Y221 levels were normalized with respect to untreated cells. (B) CrkII is transiently phosphorylated by LecA. (left) H1299 cells were incubated for 60 min in the presence of LecA (100 nM), then LecA was removed and cells were further incubated for 30 min and 60 min, respectively. (right) Phospho-CrkII Y221 levels were normalized with respect to untreated cells, at the corresponding time points. (C) Phosphorylation of CrkII Y221 is sensitive to imatinib (left) H1299 cells were stimulated for 60 min in the presence of imatinib (0-100 µM) and LecA (100 nM). Imatinib was pre-incubated with cells for 30 min at 37 °C and maintained during stimulation. (right) Phospho-CrkII Y221 levels were normalized with respect to DMSO-treated cells (mock). (D) Inhibition of LecA results in decreased CrkII Y221 phosphorylation. (left) H1299 cells were stimulated for 60 min in the presence of PNPG (0-1 mM) and LecA (100 nM) or PNPG (5 mM) only. LecA was pre-incubated with PNPG for 15 min at 37 °C and maintained during stimulation. (right) Phospho-CrkII Y221 levels were normalized with respect to PNPG-only-treated cells. Total cell lysates were immunoblotted with indicated antibodies. Phospho-CrkII Y221 levels were quantified relative to α-Tubulin. Error bars show SD; n=3 biological experiments. One-way ANOVA and Dunnett's Multiple Comparison Tests were performed. In figures B, C and D, mock samples were compared to 60 min (B), imatinib (0 µM, C) and PNPG (0 mM, D), respectively, by Student's t-test. n.s.: non-significant compared to mock samples. * p<0.05; ** p<0.01; *** p<0.001.

Fig. 3 :

 3 Fig. 3: Glycosphingolipid Gb3 is crucial for LecA-induced CrkII Y221 phosphorylation in mammalian cells. (A) GSLs mediate LecA-dependent Abl kinase activation. (left) H1299 cells, pre-treated without or with DL-PPMP (5 µM), were stimulated for 60 min in the presence of LecA (100 nM). (right) Phospho-CrkII Y221 levels were normalized with respect to untreated cells. (B) Gb3 is crucial for LecA-induced Abl kinase activation. (left) H1299 cells were stimulated with StxB (0-130 nM) for 30 min, then StxB was removed, and cells were further incubated with (+) or without (-) LecA (100 nM) for 60 min. (right) Phospho-CrkII Y221 levels were normalized with respect to only StxB-treated cells. The horizontal line indicates the basal level of phospho-CrkII Y221 after StxB treatment. (C) Gb3 in particular is required for LecA-induced Abl kinase activation in CHO transfected cells. (left) CHO cells stably transfected with Gb3 synthase (CHO-Gb3) or empty vector (CHO-vector) were stimulated with LecA (100 nM) for 60 min. (right) Phospho-CrkII Y221 levels were normalized with respect to empty vector-transfected CHO cells treated with LecA. Total cell lysates were immunoblotted with indicated antibodies. Phospho-CrkII Y221 levels were quantified relative to α-Tubulin. Error bars show SD; n=3 biological experiments. One-way ANOVA and Dunnett's Multiple Comparison Test were performed. In figure A, the mock sample was compared to DL-PPMP (5 µM) (-), in figure C, the CHO-Gb3 sample was compared to the CHO-vector sample, respectively, by Student's t-test. * p<0.05; ** p<0.01; *** p<0.001.

Fig. 4 :

 4 Fig. 4: Abl kinase is not involved in LecA-induced CrkII Y221 phosphorylation in human lung epithelial cells H1299 cells were transfected for 2-3 days with 80-100 pmol of Abl-specific or scrambled siRNA and thereafter incubated with 100 nM LecA for 60 min at 37 °C. Total cell lysates were immunoblotted with indicated antibodies. Phospho-CrkII Y221 levels were quantified relative to Crk. Error bars show SD; n=5 biological experiments. Untreated samples were compared LecAtreated samples, respectively, by Student's t-test. * p<0.05.

Fig. 5 :

 5 Fig. 5: Src family kinases mediate LecA-stimulated CrkII Y221 phosphorylation in human lung epithelial cells. (A) H1299 lung epithelial cells were pre-incubated with KB SRC4 (0-10 µM) for 30 min at 37 °C, then, cells were stimulated with LecA (100 nM) for 60 min in the presence of KB SRC4. Total cell lysates were immunoblotted with indicated antibodies. Phospho-CrkII Y221 levels were quantified relative to α-Tubulin and normalized with respect to DMSO-treated cells (mock). Error bars show SD; n=3 biological experiments. One-way ANOVA and Dunnett's Multiple Comparison Test were performed. The mock sample was compared to the KB SRC4 sample (0 µM) by Student's t-test. n.s.: non-significant compared to mock sample. ** p<0.01. (B) H1299 cells were incubated for 60 min in the presence of LecA (100 nM). Total cell lysates were immunoblotted with indicated antibodies. Phospho-Src levels were quantified relative to β-Actin. Phospho-Src Y418 levels were normalized with respect to untreated cells. Error bars show SD; n=3 biological experiments. Untreated samples were compared to LecA-stimulated samples by Student's t-test. * p<0.05. (C) H1299 cells were pretreated with Nilotinib (1 µM) and subsequently incubated with LecA (100 nM) for 60 min at 37 °C. Total cell lysates were immunoblotted with indicated antibodies.

Acknowledgements

This work was supported by the Excellence Initiative of the German Research Foundation [EXC 294], the Ministry of Science, Research and the Arts of Baden-Württemberg [Az: 33-7532.20], the German Research Foundation grants [RO 4341/2-1] and [RTG 2202], and a starting grant from the European Research Council [Programme "Ideas," ERC-2011-StG 282105]. SZ acknowledges support from the China Scholarship Council. SA was supported from the International Max Planck Research School for Molecular and Cellular Biology.