Article Figures & Data
Figures
- 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
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
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
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
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
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
(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
- TABLE 1
Interactions between host glycosaminoglycans and live log-phase promastigotes identified by SPRi
Spotted molecule No. of strains binding/total no. of strainsa Binding (% of all Leishmania strains tested) Cutaneous tropism Mucocutaneous tropism L. braziliensis Visceral tropism L. tropica L. major L. guyanensis L. donovani L. infantum Chondroitin sulfate 0/4 0/4 1/4 1/4 0/4 0/4 8.3 Dermatan sulfate 0/4 0/4 1/4 1/4 0/4 0/4 8.3 Heparan sulfate 4/4 2/4 4/4 2/4 1/4 2/4 62.5 Low-mol-wt heparin 1/4 1/4 1/4 0/4 0/4 0/4 12.5 High-mol-wt heparin 4/4 3/4 4/4 3/4 3/4 3/4 83.3 2-O-desulfated heparin 4/4 2/4 4/4 2/4 3/4 3/4 75.0 6-O-desulfated heparin 1/4 2/4 4/4 3/4 3/4 2/4 62.5 N-desulfated heparin 3/4 0/4 4/4 1/4 2/4 3/4 54.2 N-desulfated, re-N-acetylated heparin 2/4 2/4 3/4 2/4 0/4 1/4 41.7 Low-mol-wt hyaluronan 2/4 0/4 2/4 2/4 3/4 0/4 37.5 High-mol-wt hyaluronan 2/4 1/4 2/4 2/4 3/4 1/4 45.8 ↵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 molecule No. of strains binding/total no. of strainsa Binding (% of all Leishmania strains tested) Cutaneous tropism Mucocutaneous tropism L. braziliensis Visceral tropism L. tropica L. major L. guyanensis L. donovani L. infantum Secreted Plasminogen 4/4 4/4 4/4 4/4 4/4 4/4 100.0 β2-Microglobulin 0/4 2/4 0/4 2/4 0/4 0/4 16.7 Amyloid β peptide (1-42) 3/4 2/4 0/4 2/4 0/4 3/4 41.7 Tropomyosin 3/4 0/4 2/4 1/4 0/4 0/4 25.0 ECM proteoglycans Brevican 4/4 0/4 0/4 1/4 3/4 0/4 33.3 ECM proteins and fragments Extracellular matrix protein-1 4/4 4/4 4/4 4/4 3/4 2/4 87.5 Thrombospondin-1 (+2 mM CaCl2) 0/4 0/4 1/4 0/4 0/4 0/4 4.2 Tropoelastin 4/4 4/4 4/4 4/4 4/4 4/4 100.0 Vitronectin 0/4 0/4 0/4 1/4 2/4 0/4 12.5 Anastellin (FN III1-C) 4/4 4/4 4/4 4/4 3/4 3/4 91.7 Guinea pig TGase-2 0/4 3/4 0/4 0/4 0/4 0/4 12.5 Human TGase-2 0/4 0/4 1/4 2/4 1/4 1/4 20.8 C-terminal domain (NC1) of collagen XVIII Wild type 0/4 1/4 1/4 0/4 0/4 0/4 8.3 R156/268A mutant 1/4 0/4 0/4 0/4 0/4 0/4 4.2 Collagens Collagen I 1/4 1/2 3/4 2/4 0/4 0/4 31.8 Collagen II 1/4 3/4 1/4 2/4 0/4 1/4 33.3 Denatured collagen II 1/2 0/2 25.0 Collagen III 0/4 0/4 1/4 0/4 0/4 0/4 4.2 Denatured collagen III 0/2 1/2 25.0 Collagen IV 0/4 0/4 1/4 1/4 0/4 0/4 8.3 Collagen V 1/4 1/4 2/4 3/4 0/4 0/4 29.2 Collagen VI 2/4 0/4 3/4 1/4 0/4 3/4 37.5 Denatured collagen VI 0/2 2/2 2/2 66.7 Ectodomains of membrane proteins Syndecan-2 0/4 0/4 0/4 1/4 0/4 0/4 4.2 Glypican-2 2/4 0/4 0/4 0/4 0/4 0/4 8.3 Neuroglycan 0/4 1/4 0/4 3/4 0/4 0/4 16.7 Tumor endothelial marker 8 (TEM8)b 4/4 4/4 4/4 4/4 4/4 4/4 100.0 - TABLE 3
Interactions between live logarithmic-phase promastigotes and endostatin or proangiogenic growth factors identified by SPRi
Spotted molecule Expt seriesa No. of strains binding/total no. of strains Binding (% of all Leishmania strains tested) Cutaneous tropism Mucocutaneous tropism L. braziliensis Visceral tropism L. tropica L. major L. guyanensis L. donovani L. infantum Endostatin A 4/4 3/3 2/2 NDb 4/4 ND 100.0 B 1/1 0/1 1/1 1/1 1/1 1/1 83.3 FGF-2 A 4/4 3/3 2/2 ND 4/4 ND 100.0 B 0/1 0/1 0/1 0/1 1/1 1/1 33.3 VEGF A 4/4 3/3 2/2 ND 4/4 ND 100.0 B 1/1 0/1 1/1 1/1 1/1 1/1 83.3 - TABLE 4
Interactions between host molecules and live logarithmic- and stationary-phase promastigotes identified by SPRi
Spotted molecule Bindinga by promastigotes of Cutaneous tropism (L. major LEM 2462) Mucocutaneous tropism (L. braziliensis LEM 2585) Visceral tropism (L. infantum LEM 4705) Logarithmic phase Stationary phase Logarithmic phase Stationary phase Logarithmic phase Stationary 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 I ND − − − − − 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.
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.
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- Supplemental file 1 -
