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Fungal and Parasitic Infections

Large-Scale Investigation of Leishmania Interaction Networks with Host Extracellular Matrix by Surface Plasmon Resonance Imaging

Marie Fatoux-Ardore, Franck Peysselon, Anthony Weiss, Patrick Bastien, Francine Pratlong, Sylvie Ricard-Blum
J. L. Flynn, Editor
Marie Fatoux-Ardore
aUMR 5086 CNRS—Université Lyon 1, Institut de Biologie et Chimie des Protéines, Lyon, France
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Franck Peysselon
aUMR 5086 CNRS—Université Lyon 1, Institut de Biologie et Chimie des Protéines, Lyon, France
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Anthony Weiss
bSchool of Molecular Bioscience, University of Sydney, NSW, Australia
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Patrick Bastien
cUniversité Montpellier 1, Centre National de Référence des Leishmanioses, UMR MIVEGEC (CNRS 5290, IRD 224, UM1, UM2), Département de Parasitologie-Mycologie, CHRU de Montpellier, Montpellier, France
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Francine Pratlong
cUniversité Montpellier 1, Centre National de Référence des Leishmanioses, UMR MIVEGEC (CNRS 5290, IRD 224, UM1, UM2), Département de Parasitologie-Mycologie, CHRU de Montpellier, Montpellier, France
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Sylvie Ricard-Blum
aUMR 5086 CNRS—Université Lyon 1, Institut de Biologie et Chimie des Protéines, Lyon, France
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J. L. Flynn
Roles: Editor
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DOI: 10.1128/IAI.01146-13
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  • FIG 1
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    FIG 1

    Binding of live Leishmania promastigotes to a selection of host extracellular biomolecules by SPRi. Shown are sensorgrams resulting from the binding of procyclic promastigotes to heparin (A), plasminogen (B), and angiogenesis regulators ECM-1 (C), vascular endothelial growth factor (VEGF) (D), anastellin (E), endostatin (F), and fibroblast growth factor-2 (FGF-2) (G) spotted onto a Gold affinity chip. Binding was analyzed by SPRi in a Biacore Flexchip system (flow rate, 150 μl/min; running buffer, PBS–0.05% Tween 20; concentration of parasites, ∼40 × 106/ml). (A, B, C, and E) L. donovani (LEM 3318); (D, F, and G) L. infantum (LEM 4705).

  • FIG 2
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    FIG 2

    Binding of selectively desulfated heparins to live log-phase Leishmania promastigotes. The level of binding of the promastigotes to heparin was normalized to 100% for all the experiments. The length of each bar is proportional to the averaged binding level of the four strains of each Leishmania species compared to the level of binding to heparin. (Inset) Structure of a disaccharide of heparin.

  • FIG 3
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    FIG 3

    Extracellular interaction landscapes of logarithmic-phase Leishmania promastigotes. (A) Leishmania interaction repertoires ranked according to the percentage of strains that bind to a given host molecule. CS, chondroitin sulfate; DS, dermatan sulfate; TSP-1, thrombospondin-1; TGase-2, transglutaminase-2. (B) Specificity in Leishmania repertoires dependent on tissue tropism.

  • FIG 4
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    FIG 4

    Interaction of two strains of Leishmania major with human tropoelastin. Shown are sensorgrams resulting from the binding of L. major LEM 4905 (A) and L. major LEM 2983 (B) to tropoelastin spotted onto a Gold affinity chip. Binding was analyzed by SPR imaging in a Biacore Flexchip system (flow rate, 150 μl/min; running buffer, PBS–0.05% Tween 20; concentration of parasites, ∼40 × 106/ml).

  • FIG 5
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    FIG 5

    Digestion of tropoelastin by Leishmania promastigotes. L. tropica LEM 2023 (A), L. guyanensis LEM 4570 (B), and L. infantum LEM 3352 (C) promastigotes were incubated with 10 μg of tropoelastin for 2 and 4 h with (+) or without (−) 15 mM 1,10-phenanthroline. The supernatants were collected by centrifugation and were analyzed by SDS-PAGE (12% acrylamide), followed by staining with Coomassie blue. Arrowheads indicate degradation products.

  • FIG 6
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    FIG 6

    Binding of selectively desulfated heparins to live stationary-phase Leishmania promastigotes. The level of binding of the promastigotes to heparin was normalized to 100% for all the experiments. The length of each bar is proportional to the average binding level of each Leishmania species compared to the level of binding to heparin.

  • FIG 7
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    FIG 7

    (A) Number of extracellular molecules targeted by live stationary-phase Leishmania promastigotes. *, biomolecules interacting with the 3 Leishmania species. (B and C) Degrees (numbers of interactions in the extracellular interaction network) for the set of all biomolecules spotted onto the arrays (B) and for the set of biomolecules interacting with at least one Leishmania species (C). Open areas represent a degree of 0 partners in the extracellular matrix; shaded areas, degrees of 1 to 9; filled areas, degrees of ≥10. (D) The 22 partners of Leishmania logarithmic-phase promastigotes identified by SPRi (18 new partners; 4 known partners). Molecules are color-coded according to the Leishmania species with which they interact (blue, L. major; yellow, L. braziliensis; red, L. infantum). Symbols: octagons, proteins; rounded squares, bioactive fragments (matricryptins); triangles, multimers; diamonds, glycosaminoglycans. The symbol sizes differ according to the degree of Leishmania partners in the extracellular interaction network built from MatrixDB (http://matrixdb.ibcp.fr). Small symbols indicate a degree of 0; medium symbols, degrees of 1 to 9; large symbols, degrees of ≥10).

Tables

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  • TABLE 1

    Interactions between host glycosaminoglycans and live log-phase promastigotes identified by SPRi

    Spotted moleculeNo. of strains binding/total no. of strainsaBinding (% of all Leishmania strains tested)
    Cutaneous tropismMucocutaneous tropism L. braziliensisVisceral tropism
    L. tropicaL. majorL. guyanensisL. donovaniL. infantum
    Chondroitin sulfate0/40/41/41/40/40/48.3
    Dermatan sulfate0/40/41/41/40/40/48.3
    Heparan sulfate4/42/44/42/41/42/462.5
    Low-mol-wt heparin1/41/41/40/40/40/412.5
    High-mol-wt heparin4/43/44/43/43/43/483.3
    2-O-desulfated heparin4/42/44/42/43/43/475.0
    6-O-desulfated heparin1/42/44/43/43/42/462.5
    N-desulfated heparin3/40/44/41/42/43/454.2
    N-desulfated, re-N-acetylated heparin2/42/43/42/40/41/441.7
    Low-mol-wt hyaluronan2/40/42/42/43/40/437.5
    High-mol-wt hyaluronan2/41/42/42/43/41/445.8
    • ↵a Binding was confirmed in two independent SPRi experiments for each strain, except for one strain of L. braziliensis, where the results of only one experiment were available due to technical problems. Four strains of six Leishmania species were tested.

  • TABLE 2

    Interactions between host proteins and live log-phase promastigotes identified by SPRi

    Spotted moleculeNo. of strains binding/total no. of strainsaBinding (% of all Leishmania strains tested)
    Cutaneous tropismMucocutaneous tropism L. braziliensisVisceral tropism
    L. tropicaL. majorL. guyanensisL. donovaniL. infantum
    Secreted
        Plasminogen4/44/44/44/44/44/4100.0
        β2-Microglobulin0/42/40/42/40/40/416.7
        Amyloid β peptide (1-42)3/42/40/42/40/43/441.7
        Tropomyosin3/40/42/41/40/40/425.0
    ECM proteoglycans
        Brevican4/40/40/41/43/40/433.3
    ECM proteins and fragments
        Extracellular matrix protein-14/44/44/44/43/42/487.5
        Thrombospondin-1 (+2 mM CaCl2)0/40/41/40/40/40/44.2
        Tropoelastin4/44/44/44/44/44/4100.0
        Vitronectin0/40/40/41/42/40/412.5
        Anastellin (FN III1-C)4/44/44/44/43/43/491.7
        Guinea pig TGase-20/43/40/40/40/40/412.5
        Human TGase-20/40/41/42/41/41/420.8
        C-terminal domain (NC1) of collagen XVIII
            Wild type0/41/41/40/40/40/48.3
            R156/268A mutant1/40/40/40/40/40/44.2
    Collagens
        Collagen I1/41/23/42/40/40/431.8
        Collagen II1/43/41/42/40/41/433.3
        Denatured collagen II1/20/225.0
        Collagen III0/40/41/40/40/40/44.2
        Denatured collagen III0/21/225.0
        Collagen IV0/40/41/41/40/40/48.3
        Collagen V1/41/42/43/40/40/429.2
        Collagen VI2/40/43/41/40/43/437.5
        Denatured collagen VI0/22/22/266.7
    Ectodomains of membrane proteins
        Syndecan-20/40/40/41/40/40/44.2
        Glypican-22/40/40/40/40/40/48.3
        Neuroglycan0/41/40/43/40/40/416.7
        Tumor endothelial marker 8 (TEM8)b4/44/44/44/44/44/4100.0
    • ↵a Binding was confirmed in two independent SPRi experiments for each strain, except for one strain of L. braziliensis, where the results of only one experiment were available due to technical problems. Four strains of six Leishmania species were tested.

    • ↵b See Materials and Methods.

  • TABLE 3

    Interactions between live logarithmic-phase promastigotes and endostatin or proangiogenic growth factors identified by SPRi

    Spotted moleculeExpt seriesaNo. of strains binding/total no. of strainsBinding (% of all Leishmania strains tested)
    Cutaneous tropismMucocutaneous tropism L. braziliensisVisceral tropism
    L. tropicaL. majorL. guyanensisL. donovaniL. infantum
    EndostatinA4/43/32/2NDb4/4ND100.0
    B1/10/11/11/11/11/183.3
    FGF-2A4/43/32/2ND4/4ND100.0
    B0/10/10/10/11/11/133.3
    VEGFA4/43/32/2ND4/4ND100.0
    B1/10/11/11/11/11/183.3
    • ↵a Two SPRi experiments were run in series A and two in series B.

    • ↵b ND, not determined.

  • TABLE 4

    Interactions between host molecules and live logarithmic- and stationary-phase promastigotes identified by SPRi

    Spotted moleculeBindinga by promastigotes of
    Cutaneous tropism (L. major LEM 2462)Mucocutaneous tropism (L. braziliensis LEM 2585)Visceral tropism (L. infantum LEM 4705)
    Logarithmic phaseStationary phaseLogarithmic phaseStationary phaseLogarithmic phaseStationary phase
    Glycosaminoglycans
        Chondroitin sulfate−+−−−−
        Dermatan sulfate−−−+−+
        Heparan sulfate++++−+
        Low-mol-wt heparin+−−+−+
        High-mol-wt heparin++++++
        High-mol-wt hyaluronan−−−−+−
    Secreted
        Plasminogen++++++
        β-2 Microglobulin+−+−−−
        Amyloid β peptide (1-42)++++−+
    ECM proteoglycan
        Brevican−−−+−−
    ECM proteins and fragments
        Extracellular matrix protein-1++++−+
        Thrombospondin-1 (+2 mM CaCl2)−−−+−−
        Tropoelastin+−+−++
        Endostatin−−++++
        Fibronectin−−−+−−
        Laminin-111−−−+−−
        Anastellin (FN III1-C)+−++−+
        Guinea pig TGase-2+−−−−−
        C-terminal domain (NC1) of collagen XVIII++−+−+
    Growth factors
        FGF-2−−−−+−
        VEGF++−+++
    Collagens
        Collagen IND−−−−−
        Collagen II++−+++
        Collagen III−−−−−+
        Denatured collagen III−−++−−
        Collagen V−−++−+
        Collagen VI−+−−−+
        Denatured collagen VI++++−−
    Ectodomains of membrane proteins
        Neuroglycan−−−+−−
        Tumor endothelial marker 8++++++
    • ↵a Binding (+) was confirmed in two independent SPRi experiments for each strain. −, no binding; ND, not determined.

Additional Files

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  • Supplemental material

    Files in this Data Supplement:

    • Supplemental file 1 -

      Fig. S1. Representative sensorgram of the specific binding Leishmania promastigotes to GST-tagged TEM8 in SPRi experiments. Fig. S2. Leishmania promastigotes before and after recirculation over protein and glycosaminoglycan arrays. Fig. S3. Digestion of fibronectin by Leishmania promastigotes. Fig. S4. Antiangiogenic matricryptins did not inhibit the growth of Leishmania tropica. Fig. S5. Endostatin supplemented in zinc and mutant endostatin did not inhibit the growth of L. tropica promastigotes. Table S1. Leishmania species and strains tested for their ability to bind mammalian membrane and extracellular biomolecules by SPRi. Table S2. Mammalian biomolecules (proteins and glycosaminoglycans) spotted on the arrays. Table S3. List of host biomolecules spotted on the arrays which did not bind to procyclic Leishmania promastigotes in SPRi experiments.

      PDF, 421K

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Large-Scale Investigation of Leishmania Interaction Networks with Host Extracellular Matrix by Surface Plasmon Resonance Imaging
Marie Fatoux-Ardore, Franck Peysselon, Anthony Weiss, Patrick Bastien, Francine Pratlong, Sylvie Ricard-Blum
Infection and Immunity Jan 2014, 82 (2) 594-606; DOI: 10.1128/IAI.01146-13

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Large-Scale Investigation of Leishmania Interaction Networks with Host Extracellular Matrix by Surface Plasmon Resonance Imaging
Marie Fatoux-Ardore, Franck Peysselon, Anthony Weiss, Patrick Bastien, Francine Pratlong, Sylvie Ricard-Blum
Infection and Immunity Jan 2014, 82 (2) 594-606; DOI: 10.1128/IAI.01146-13
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