Repeating structures of the major staphylococcal autolysin are essential for the interaction with human thrombospondin 1 and vitronectin

Abstract

Human thrombospondin 1 (hTSP-1) is a matricellular glycoprotein facilitating bacterial adherence to and invasion into eukaryotic cells. However, the bacterial adhesin(s) remain elusive. In this study, we show a dose-dependent binding of soluble hTSP-1 to Gram-positive but not Gram-negative bacteria. Diminished binding of soluble hTSP-1 to proteolytically pretreated staphylococci suggested a proteinaceous nature of potential bacterial adhesin(s) for hTSP-1. A combination of separation of staphylococcal surface proteins by two-dimensional gel electrophoresis with a ligand overlay assay with hTSP-1 and identification of the target protein by mass spectrometry revealed the major staphylococcal autolysin Atl as a bacterial binding protein for hTSP-1. Binding experiments with heterologously expressed repeats of the AtlE amidase from Staphylococcus epidermidis suggest that the repeating sequences (R1ab-R2ab) of the N-acetyl-muramoyl-L-alanine amidase of Atl are essential for binding of hTSP-1. Atl has also been identified previously as a staphylococcal vitronectin (Vn)-binding protein. Similar to the interaction with hTSP-1, the R1ab-R2ab repeats of Atl are shown here to be crucial for the interaction of Atl with the complement inhibition and matrix protein Vn. Competition assays with hTSP-1 and Vn revealed the R1ab-R2ab repeats of AtlE as the common binding domain for both host proteins. Furthermore, Vn competes with hTSP-1 for binding to Atl repeats and vice versa. In conclusion, this study identifies the Atl repeats as bacterial adhesive structures interacting with the human glycoproteins hTSP-1 and Vn. Finally, this study provides insight into the molecular interplay between hTSP-1 and Vn, respectively, and a bacterial autolysin.

Keywords: Adhesion; Autolysin; Extracellular Matrix Proteins; Pathogenesis; Repeats; Staphylococcus; Thrombospondin; Vitronectin.

FIGURE 1.

Binding of soluble hTSP-1 by bacteria. A, dose-dependent binding of hTSP-1 to Gram-positive S. pneumoniae D39Δcps, S. aureus SA113Δspa, S. epidermidis RP62A, and Gram-negative E. coli 536 and P. aeruginosa strain 6, respectively, was analyzed by flow cytometry. Bacterial suspensions of 5 × 10⁸ bacteria in 100 μl of PBS were incubated with increasing concentrations of hTSP-1 (0–50 μg/ml) and analyzed for surface-bound hTSP-1 using a specific mouse anti-TSP-1 antibody and secondary Alexa Fluor 488 conjugated anti-mouse IgG. The values are represented as the geometrical mean fluorescence intensity multiplied with the percent of gated events (GMFI × % gated events). B, dose-dependent binding of hTSP-1 to clinical isolates S. epidermidis 1457-M10 (pia-negative mutant of a 1457 catheter infection isolate) and S. epidermidis 1585 (liquor shunt infection isolate). The values are represented as the geometrical mean fluorescence intensity multiplied with percent gated events. C, dose-dependent binding of FITC-labeled hTSP-1 to different clinical isolates of S. aureus (H4862 and H9053, furuncle isolates, and caMRSA USA300). Bacterial suspensions of 5 × 10⁸ bacteria in 100 μl of PBS were incubated with increasing concentrations of FITC-labeled hTSP-1 (0–50 μg/ml) and analyzed for surface-bound hTSP-1 using flow cytometry. The values are represented as the geometrical mean fluorescence intensity multiplied with percent gated events.

FIGURE 2.

Proteolytic treatment diminishes TSP-1 binding activity of S. aureus. Binding of different concentrations of FITC-labeled hTSP-1 (50 and 100 μg/ml) to 2 × 10⁸ S. aureus SA113. Bacteria were pretreated with either Pronase E (ProE, 1 mg/ml) to digest potential proteinaceous binding partners on the bacterial surface or with sodium periodate (NalO₄, 0.05 mg/ml) to oxidize potential carbohydrate binding partners. Binding of FITC-labeled human TSP-1 was analyzed (n = 3) by flow cytometry in comparison with untreated bacteria (w/o). The values are represented as the geometrical mean fluorescence intensity multiplied with percent gated events (GMFI x % gated events). **, p < 0.01; ***, p < 0.001; ns, not significant.

FIGURE 3.

Binding of S. aureus surface proteins to hTSP-1 as analyzed by surface plasmon resonance. A and B, surface plasmon resonance sensorgrams demonstrating a dose-dependent binding of surface proteins enriched from S. epidermidis RP62A (A) and S. aureus SA113 (B) to hTSP-1 immobilized on a CM5 biosensor. C, binding of surface proteins of S. aureus SA113 (100 μg/ml) to immobilized hTSP-1 before (ctrl) and after incubation with Pronase E (ProE). The CM5 biosensor was coated with hTSP-1 (∼7500 RU), and enriched surface proteins were used as analytes at a flow rate of 10 μl/min. The affinity surface was regenerated between subsequent sample injections with 12.5 mm sodium hydroxide. The values of the control flow cell were subtracted from each sensorgram.

FIGURE 4.

Identification of the major staphylococcal autolysin as TSP-1 binding protein. A, representative silver-stained protein gel (12%) after two-dimensional separation of purified cell wall-associated and secreted proteins (40 μg) from S. aureus SA113. Proteins were subjected to isoelectric focusing with 7-cm IPG strips (pH 4–7), followed by SDS-PAGE. Single spots were excised after matching (circles) with a corresponding overlay blot with hTSP-1 (B). After digestion with trypsin, peptides of the spots were analyzed using mass spectrometry.

FIGURE 5.

Human TSP-1 binding to heterologously expressed AtlE derivates. A, silver-stained SDS gel (12%) of heterologously expressed part structures of AtlE and corresponding ligand overlay blot with hTSP-1. SDS-PAGE-separated proteins were blotted on a nitrocellulose membrane, incubated with hTSP-1 (50 μg/ml), and then binding was detected using a mouse polyclonal anti-hTSP-1 IgG followed by incubation with an alkaline phosphatase-coupled secondary anti-mouse antibody. M, marker PageRuler-prestained (Fermentas); lane 1, heterologously expressed N-acetyl-muramoyl-l-alanine amidase with repeats R1_ab-R2_ab (62.1 kDa); lane 2, amidase (24.3 kDa); lane 3, repeats R1_ab-R2_ab (35.98 kDa); lane 4, repeat R1_ab (16.3 kDa). B, domain arrangement and molecular weight of the heterologously expressed AtlE part structures used in this study. C, schematic of the crystal structure of repeat R2_ab of the AtlE amidase (PDB code 4EPC) generated with PyMOL. R2_ab consists of ∼170 amino acids folded into two SH3b domains, each containing a GW dipeptide motif. Blue, β strands; green, α helices.

FIGURE 6.

Human TSP-1 binds preferentially to repeats R1_ab-R2_ab as shown by surface plasmon resonance studies. A, human TSP-1 (0.1 μg) was immobilized on 96-well plates (MaxiSorp) and incubated with various molecular ratios of AtlE R1_ab-R2_ab or AtlE R1_ab. The binding of repeats was detected using a polyclonal anti-AtlE-R1_ab-R2_ab IgG followed by incubation with a peroxidase-coupled secondary antibody. Results are expressed as means ± S.D. (n = 3). **, p < 0.01; ***, p < 0.001; ns, not significant. B, surface plasmon resonance sensorgrams of heterologously expressed AtlE R1_ab-R2_ab show a dose-dependent binding to immobilized hTSP-1. A CM5 biosensor was coated with hTSP-1 (∼4000 response units), and the heterologously expressed repeats R1_ab-R2_ab of AtlE were used as analytes. The values of the control flow cells were subtracted from each sensorgram. C, surface plasmon resonance sensorgrams of heterologously expressed AtlE R1_ab-2_ab show a dose-dependent binding to immobilized human vitronectin. Vn was immobilized on the CM5 biosensor (∼2500 response units), and the heterologously expressed repeats R1_ab-R2_ab of AtlE were used as analytes. The values of the control flow cells were subtracted from each sensorgram. D, low binding activity of heterologously expressed AtlE repeat R1_ab (25 μg/ml) to immobilized hTSP-1 as analyzed by an SPR study. Shown is an SPR sensorgram of a manual run.

FIGURE 7.

Reassociation of Atl repeats to S. aureus increases hTSP-1 binding. A, binding of hTSP-1 (6.25 and 12.5 μg/ml) to S. aureus SA113Δspa was analyzed using 5 × 10⁸ of untreated or 3 μg of AtlE repeats R1_ab-R2_ab (TSP 6.25 + R and TSP 12.5 + R) pretreated bacteria. Binding was analyzed by flow cytometry using a specific mouse anti-TSP-1 antibody and secondary Alexa Fluor 488-conjugated anti-mouse IgG. The values represent the geometrical mean fluorescence intensity multiplied with the percent of gated events (GMFI × % gated events) (n = 3). *, p < 0.05; ns, not significant. B, representative overlay histograms showing binding of hTSP to untreated S. aureus Sa113Δspa (black) and increased binding of hTSP-1 to S. aureus Δspa pretreated with 3 μg of AtlE repeats R1_ab-R2_ab (green).

FIGURE 8.

Human TSP-1 and vitronectin bind dose-dependently to the R1_ab-R2_ab repeats of Atl and compete for binding. A, binding of hTSP-1 to immobilized Atl repeats R1_ab-R2_ab. The heterologously expressed repeats R1_ab-R2_ab (0.5 μg) were immobilized on microtiter plates (MaxiSorp) and incubated with increasing amounts of hTSP-1. B, binding of human vitronectin to immobilized Atl repeats R1_ab-R2_ab. The heterologously expressed repeats R1_ab-R2_ab (0.5 μg) were immobilized on microtiter plates (MaxiSorp) and incubated with increasing amounts of Vn. C and D, human TSP-1 competes with human vitronectin for binding to the immobilized Atl repeats R1_ab-R2_ab. The heterologously expressed repeats R1_ab-R2_ab (0.5 μg) were immobilized on microtiter plates (MaxiSorp) and incubated with hTSP-1 (1000 ng/well) in the presence of increasing molecular ratios of Vn (C) or with Vn (125 ng/well) in the presence of increasing molecular ratios of hTSP-1 (D). Bound hTSP was detected using a polyclonal mouse anti-hTSP-1 IgG antibody followed by incubation with a peroxidase-coupled secondary anti-mouse antibody, and bound Vn was detected using a polyclonal rabbit anti-Vn IgG followed by incubation with a peroxidase-coupled secondary anti-rabbit antibody. Results are expressed as means ± S.D. (n = 3). *, p < 0.05; ***, p < 0.001; ns, not significant.