A bacterial ABC transporter enables import of mammalian host glycosaminoglycans

Abstract

Glycosaminoglycans (GAGs), such as hyaluronan, chondroitin sulfate, and heparin, constitute mammalian extracellular matrices. The uronate and amino sugar residues in hyaluronan and chondroitin sulfate are linked by 1,3-glycoside bond, while heparin contains 1,4-glycoside bond. Some bacteria target GAGs as means of establishing colonization and/or infection, and bacterial degradation mechanisms of GAGs have been well characterized. However, little is known about the bacterial import of GAGs. Here, we show a GAG import system, comprised of a solute-binding protein (Smon0123)-dependent ATP-binding cassette (ABC) transporter, in the pathogenic Streptobacillus moniliformis. A genetic cluster responsible for depolymerization, degradation, and metabolism of GAGs as well as the ABC transporter system was found in the S. moniliformis genome. This bacterium degraded hyaluronan and chondroitin sulfate with an expression of the genetic cluster, while heparin repressed the bacterial growth. The purified recombinant Smon0123 exhibited an affinity with disaccharides generated from hyaluronan and chondroitin sulfate. X-ray crystallography indicated binding mode of Smon0123 to GAG disaccharides. The purified recombinant ABC transporter as a tetramer (Smon0121-Smon0122/Smon0120-Smon0120) reconstructed in liposomes enhanced its ATPase activity in the presence of Smon0123 and GAG disaccharides. This is the first report that has molecularly depicted a bacterial import system of both sulfated and non-sulfated GAGs.

Figure 1

Bacterial system for degradation and import of GAGs. GAGs such as hyaluronan and chondroitin sulfate are depolymerized to unsaturated disaccharides by extracellular or cell-surface polysaccharide lyases. The resultant unsaturated disaccharides are incorporated to cytoplasm by PTS or periplasmic binding protein-dependent ABC transporter. In the case of PTS, substrates (unsaturated disaccharides) are phosphorylated across the cytoplasmic membrane. Unsaturated disaccharides are degraded to constituent monosaccharides (unsaturated uronate and amino sugar) by UGL, and the resultant unsaturated uronate is metabolized to pyruvate and glyceraldehyde-3-phosphate (G-3-P) by subsequent reactions of isomerase, reductase, kinase and aldolase. The pathway and proteins/enzymes are detailed in the text. P enclosed in a yellow hexagon, phosphate group; S enclosed in a cyan hexagon, sulfate group.

Figure 2

Bacterial GAG genetic clusters. (A) Genetic cluster of Streptococcus agalactiae/Streptococcus pneumoniae. (B) Genetic cluster of Streptobacillus moniliformis. Genes for GAG depolymerization, degradation, and metabolism, except for import, are well conserved between the two bacterial clusters. Genetic clusters of Streptococcus and Streptobacillus encode PTS and the solute-binding protein-dependent ABC transporter, respectively, as an importer.

Figure 3

Degradation of GAGs. (A) S. moniliformis. (B) P. heparinus as a positive control. (C) E. coli as a negative control. Plates on the left and right represent before and after addition of acetic acid, respectively. Plates 1, 2, and 3 contained hyaluronan, chondroitin sulfate C, and heparin, respectively.

Figure 4

Expression and characterization of binding protein-dependent ABC transporter. SDS-PAGE followed by immunoblotting using anti-Smon0123 antibodies (A) and anti-SpyUGL antibodies (B). S. moniliformis cells grown in liquid nutrient medium (100 μl) in the presence or absence of GAG (hyaluronan or chondroitin sulfate (C) were collected at OD₆₀₀ = 0.37 by centrifugation. The cells were lysed with SDS, and the resultant cell lysates were subjected to immunoblotting for detection of Smon0123 (solute-binding protein) and Smon0127 (UGL) encoded in the GAG genetic cluster. Lane M, protein standards with molecular masses of 120, 100, 80, 60, 50, 40, 30, and 20 kDa used for immunoblotting; lane 1, S. moniliformis cells in the absence of GAG; lane 2, S. moniliformis cells in the presence of hyaluronan; lane 3, S. moniliformis cells in the presence of chondroitin sulfate C; lane 4, E. coli cells in the absence of GAG; lane 5, positive control [purified Smon0123 (A) and SagUGL (B)]. (C) and (D) SDS-PAGE followed by staining with Coomassie Brilliant Blue (CBB). Lane M, protein standards with molecular masses of 250, 150, 100, 75, 50, 37, 25, and 20 kDa for CBB staining; lane 1, purified Smon0127 (C) and Smon0123 (D). (E) Elution profile of Smon0121-Smon0122(10xHis)/Smon0120-Smon0120 via gel filtration chromatography. Left and right-sided peaks show an aggregate and a tetramer, respectively. Volumes required for elution of the standard ferritin (440 kDa) and aldolase (158 kDa) are indicated by black arrows. (F) SDS-PAGE followed by CBB staining. Lane M, protein standards with molecular masses of 250, 150, 100, 75, 50, 37, 25, and 20 kDa; lane 1, purified Smon0121-Smon0122(10xHis)/Smon0120-Smon0120. (G) Immunoblotting using anti-histidine tag antibodies. Lane M, protein standards with molecular masses of 120, 100, 80, 60, 50, 40, 30, and 20 kDa; lane 1, purified Smon0121-Smon0122(10xHis)/Smon0120-Smon0120. (H) ATPase activity of the Smon0121-Smon0122(10xHis)/Smon0120-Smon0120 in liposomes in the presence or absence of various disaccharides. PLS, proteoliposome without disaccharides; chitobiose, N,N′-diacetylchitobiose; d2M, unsaturated alginate disaccharide. The ATPase activity in PLS was considered 100%. Each data represents the average of triplicate individual experiments (means ± standard errors of the means).

Figure 5

Affinity of Smon0123 with unsaturated GAG disaccharides. (A) DSF analysis. Upper, fluorescence profile of Smon0123 with C∆0S (green: 0 mM and cyan: 1 mM) or 1 mM N,N′-diacetylchitobiose (red). Middle, negative derivative curve plot derived from the fluorescence profile. Lower, the values of T _open and T _closed in the presence or absence of various unsaturated GAG disaccharides. (B) Fluorescence spectrum analysis. Upper, wavelength-scanned fluorescence intensity of Smon0123 with C∆0S (blue: 0 μM; red: 0.25 μM; green: 0.50 μM; purple: 0.75 μM; cyan: 1.0 μM; orange: 2.0 μM; lilac: 5.0 μM; pink: 10 μM; and olive: 20 μM). Middle, the relative fluorescence intensity by addition of increasing ligand concentrations was plotted after modification based on volume change in the cuvette. K _d was determined using the least-squares method. Lower, dissociation constants of Smon0123 with various unsaturated GAG disaccharides. Chitobiose, N,N′-diacetylchitobiose.

Figure 6

Overall structures of Smon0123. Structures (stereo-diagram with wall-eyed viewing) of ligand-free Smon0123 (N-28/C-5) (A) and CΔ0S-bound Smon0123 (N-18/C-5) (B). Cyan, α-helices; purple, β-strands; pink, loops and coils. CΔ0S bound to Smon0123 is shown in ball model (green, carbon atom; red, oxygen atom; and blue, nitrogen atom). Orange ball shows the metal ion. Structural comparison with ligand-free Smon0123 (N-28/C-5) (olive) and CΔ0S-bound Smon0123 (N-18/C-5) (blue) (C). Both N-domains of ligand-free and -bound Smon0123 proteins were superimposed. There is a structural difference (47° in angle) in the N- and C-domains between C∆0S-free and -bound Smon0123.

Figure 7

Binding mode of Smon0123 to unsaturated GAG disaccharides. Interactions between Smon0123 and CΔ0S (A), CΔ4S (B), or CΔ6S (C). CΔ0S, CΔ4S, and CΔ6S are shown in sticks (green, carbon atom; red, oxygen atom; blue, nitrogen atom; and yellow, sulfur atom) (stereo-diagram with wall-eyed viewing). Carbon atoms of Smon0123 residues interacting with the substrates are shown in gray lines. Direct and water-mediated hydrogen bonds between Smon0123 and substrates are shown in dashed lines colored with magenta and cyan, respectively. Water molecules (oxygen atoms) are shown as cyan balls. (D) Large space for sulfate groups of substrates. In the interaction between Smon0123 and CΔ0S (left), a number of water molecules are located in the space where a sulfate group of CΔ6S is accommodated (right).