A bacterial ABC transporter enables import of mammalian host glycosaminoglycans

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.

. 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 proteindependent 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.
Scientific RepoRts | 7: 1069 | DOI: 10.1038/s41598-017-00917-y polysaccharide-degrading enzymes are expressed under the control of cytoplasmic transcriptional regulators that sense substrates 21,22 . Inhibition of the import system results in reduced expression of these degrading enzymes, which may be a means to minimize the brunt of bacterial GAG-dependent infectious diseases. This study aimed to identify molecular components of the S. moniliformis GAG import system, structurally determine the GAG import proteins that interact and bind to sulfated and non-sulfated GAG disaccharides via X-ray crystallography, and functionally characterize the ABC transporter.

Results
Streptobacillus moniliformis GAG genetic cluster. Our previous reports 11, 12 indicated the presence of a genetic cluster responsible for depolymerization, degradation, and metabolism of GAGs in pathogenic bacteria, such as streptococci, enterococci, and clostridia. These bacterial GAG genetic clusters encode polysaccharide lyase and UGL. Hyaluronate and heparan sulfate lyases encoded by the cluster are classified in polysaccharide lyase (PL) families 8 and 12, respectively, as per the Carbohydrate Active enZyme (CAZy) database 23 , while all UGLs are solely members of the glycoside hydrolase (GH) family 88 according to the database. These depolymerizing and degrading enzymes are well conserved among bacterial GAG genetic clusters. In addition to such enzymes, genes coding for PTS are frequently observed in the cluster. There are, however, some variations in constitutive genes among GAG-related bacteria. For example, isomerases (streptococcal DhuI and clostridial KduI) and reductases (streptococcal DhuD and clostridial KduD) function as 4-deoxy-L-threo-5-hexosuloseuronate ketol-isomerase and 2-keto-3-deoxy-D-gluconate dehydrogenase, respectively, although little homology is observed between DhuI and KduI and between DhuD and KduD 11 . Thus, we decided to investigate GAG cluster organization in genome-sequenced bacteria.
Interestingly, the Gram-negative bacterium S. moniliformis 24 possessed a similar GAG genetic cluster containing genes which encode for a solute-binding protein-dependent ABC transporter (Fig. 2B). Based on the primary structure, the proteins encoded in the genetic cluster were annotated as follows:  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.
Smon0119, 4-deoxy-L-threo-5-hexosulose-uronate ketol-isomerase (KduI); Smon0120, multiple sugar transport system (ATP-binding protein); Smon0121, putative aldouronate transport system ( Some streptococci can assimilate hyaluronan as a sole carbon source 25,26 , and PTS encoded in the streptococcal GAG genetic cluster is suitable for import of unsaturated hyaluronan disaccharides containing no sulfate groups (Fig. 1, left). On the other hand, unsaturated disaccharides with a sulfate group at the C-6 position generated from sulfated GAGs, e.g., chondroitin sulfate C, are unsuitable for PTS substrates. The ABC transporter possibly enables bacteria to incorporate sulfated disaccharides. Thus, degradation and import systems of GAGs were analyzed in S. moniliformis.

S. moniliformis degrades GAGs.
To investigate degradation of GAGs by S. moniliformis, a plate method for halo detection 27 was adopted (Fig. 3). Bacterial cells were grown on nutrient medium plates containing bovine serum albumin (BSA) and each of the GAGs, and acetic acid was poured on the plates after colony formation. Degradation of GAGs caused clear zones, halos, although GAGs resulted in formation of white precipitates due to interaction with BSA in the presence of acetic acid. Hyaluronan, chondroitin sulfate C, and heparin were used as substrates. Chondroitin sulfate is classified as chondroitin sulfates A, B, and C based on position of the sulfate group 28 . Chondroitin sulfate C is sulfated at the C-6 position of GalNAc, while chondroitin sulfates A and B are sulfated at the C-4 position of GalNAc. The repeating units of chondroitin sulfates A, B, and C are GlcUA-GalNAc4S (GalNAc with a sulfate group at C-4 position), IdoUA-GalNAc4S, and GlcUA-GalNAc6S (GalNAc with a sulfate group at C-6 position), respectively 29 . Gram-negative Pedobacter heparinus is known to degrade hyaluronan, chondroitin sulfate, and heparin 30 and served as a positive control, while Escherichia coli served as a negative control.
P. heparinus cells formed halos on each GAG plate after addition of acetic acid ( Fig. 3B; plates 1, 2, and 3 on the right), while no halo formation was observed on E. coli plates ( Fig. 3C; plates 1, 2, and 3 on the right). In the case of S. moniliformis, halos were observed on plates containing hyaluronan or chondroitin sulfate C ( Fig. 3A; plates 1 and 2, right). Surprisingly, the bacterium did not grow in the presence of heparin ( Fig. 3A; plate 3, left). S. moniliformis in the presence of hyaluronan and chondroitin sulfate C in the nutrient medium grew better than when in the absence of GAG (Fig. S1).
Expression of the GAG genetic cluster in S. moniliformis. Because S. moniliformis degrades hyaluronan and chondroitin sulfate C, the expression of the bacterial GAG genetic cluster was examined by immunoblot analysis. The molecular masses of Smon0123 and Smon0127 were estimated to be 57 and 46 kDa, respectively, although Smon0123 was predicted to contain a signal peptide with a molecular mass of 2 kDa by a LipoP program 31 . As described later, anti-Smon0123 antibodies were prepared using recombinant Smon0123 as antigens. As Smon0127 exhibits a significant sequence identity (48%) with Streptococcus pyogenes UGL (SpyUGL),   32 were subjected to immunoblotting as the positive control ( Fig. 4A and B, lane 5). Both Smon0123 and Smon0127 were constitutively expressed in bacterial cells grown in the GAG-free medium ( Fig. 4A and B, lane 1). The intensity of both protein bands increased in the presence of GAG, particularly hyaluronan ( Fig. 4A and B, lane 2). These results indicated that the GAG genetic cluster was constitutively expressed in S. moniliformis cells and that its expression was enhanced in the presence of GAG.

Interaction between Smon0123 and unsaturated GAG disaccharides. Recombinant Smon0123,
the putative solute-binding protein, was expressed in E. coli cells, purified to homogeneity (Fig. 4D), and subjected to differential scanning fluorimetry (DSF) 33 to examine the interaction between Smon0123 and unsaturated GAG disaccharides (Fig. 5A). Our previous report evidenced that this method is feasible for analyzing the interaction between the solute-binding protein and the substrate 34 . In DSF, the reaction temperature was escalated in the mixture of a protein, ligand, and fluorescent chemical (SYPRO Orange). The relative fluorescent unit (RFU) was changed due to the formation of hydrophobic interaction between unfolded protein and SYPRO Orange. The inflection point of the increase on the RFU is defined as the melting temperature (T m ) 33 . As the ligand-bound protein is thermally more stable than the ligand-free form due to the conversion of the rigid form, T m in the presence of a ligand was higher than that in the absence of a ligand. Generally, the solute-binding protein adopts two conformations during substrate binding, substrate-free open form and substrate-bound closed form 35  were also calculated, these were estimated to basal levels in this assay. The binding ability of Smon0123 to GAG polysaccharides (hyaluronan, chondroitin sulfate C, and heparin) was also investigated and no specific interaction between Smon0123 and these GAG polysaccharides was observed.  Scientific RepoRts | 7: 1069 | DOI:10.1038/s41598-017-00917-y C-domains), and each domain has two small subdomains. N-domain is constituted by residues Pro27-Ile151 (N1 subdomain) and residues Gly328-Ala418 (N2 subdomain), and C-domain contains residues Lys152-Gly327 (C1 subdomain) and Lys419-Phe500 (C2 subdomain). Each domain included a parallel and an antiparallel β-sheet surrounded by many α-helices and formed the α/β sandwich structure. Both N-and C-domains were connected through three loops: residues Tyr146-Ser154 (N1-C1), residues Arg319-Thr324 (C1-N2), and residues Ala414-Ser415 (N2-C2). The ligand CΔ0S was bound to a cleft formed between the two N-and C-domains (Fig. 6B). A metal ion, possibly, a calcium ion based on its coordination, was located in the C-domain, far from the cleft. All the crystal structures of Smon0123 so far determined include metal ion in its molecule, suggesting that the metal ion contributes to protein folding. Because the metal ion is situated far from the substrate-biding site, the metal ion probably has no influence on the substrate-binding ability. Compared with the structure of CΔ0S-bound Smon0123 and that of ligand-free Smon0123, the N-and C-domains of ligand-bound Smon0123 were 47° more closed than those of ligand-free Smon0123 (Fig. 6C). There was no significant difference in the overall structure among Smon0123 proteins in complex with CΔ0S, CΔ4S, and CΔ6S.

Binding mode of Smon0123 to unsaturated GAG disaccharides. Interactions between Smon0123
and each unsaturated chondroitin disaccharide (CΔ0S, CΔ4S, or CΔ6S) are shown in Table 1   Three genes (smon0120, smon0121, and smon0122) coding for Smon0120, Smon0121, and Smon0122, respectively, locate sequentially in that order in the S. moniliformis genome and form the operon structure (Fig. 2B). Thus,    (Fig. 4F). Furthermore, the protein band corresponding to Smon0122 was detected by immunoblotting using anti-histidine tag antibodies (Fig. 4G). The ABC transporter seemed to have been eluted as a tetramer (theoretical molecular mass of approximately 150 kDa) in size on gel filtration (Fig. 4E, red arrow), although the eluted protein was thought to have molecular mass of slightly over 158 kDa based on the elution volume. This difference in molecular mass was possibly due to the presence of a detergent in the eluted buffer.  (Fig. 4H). The ATPase activity in the presence of non-GAG disaccharides as the negative control such as cellobiose and unsaturated alginate disaccharide (d2M) except for N,N′-diacetylchitobiose was comparable with that in the absence of disaccharides (PLS). The enhancement of the ATPase activity in the presence of N,N′-diacetylchitobiose was probably due to the similarity in component between GAG disaccharides and N,N′-diacetylchitobiose. Unsaturated hyaluronan and chondroitin disaccharides (ΔHA, CΔ0S, CΔ4S, and CΔ6S) significantly enhanced the ATPase activity of the proteoliposome, while the enzyme activity in the presence of unsaturated heparin disaccharides (HΔ0S and HΔ6S) was comparable with that in PLS.

Discussion
In the present study, we found and characterized the bacterial solute-binding protein-dependent ABC transporter for the import system of GAGs for the first time, although a few ABC exporters for GAGs have already been identified in bacteria 39,40 and humans 41 . While PTS phosphorylates substrates at the C-6 position during transport, the ABC transporter imports substrates without any substrate modification. The S. moniliformis ABC transporter was demonstrated to be active on various unsaturated GAG disaccharides, particularly unsaturated sulfate group-free chondroitin and hyaluronan disaccharides and uniquely (relative to PTS GAG disaccharide import) unsaturated chondroitin disaccharide with a sulfate group at the C-6 position of GalNAc (C∆6S), which is an unsuitable substrate for PTS.
Based on the above-mentioned results and streptococcal GAG assimilation system 12 (Fig. 1, left), the corresponding Streptobacillus model for the degradation and import of GAG was postulated in Fig. 1 (Fig. 4H), indicating that the ABC transporter interacts with substrate-bound Smon0123 (closed form), but not the substrate-free form, and triggers ATP hydrolysis. Distinct from substrate-free Smon0123 in the open conformation, the binding protein in complex with the substrate adopts the closed conformation by accommodating the substrate at the cleft between the N-and C-domains (Fig. 6B). Therefore, the closed conformation of substrate-bound Smon0123 is considered to be important for association with the ABC transporter.
Structure determination of Smon0123 with CΔ6S is particularly important because this result directly demonstrates that CΔ6S, unsuitable for PTS, becomes a substrate for the S. moniliformis ABC transporter. The binding mode of Smon0123 to unsaturated chondroitin disaccharides is discussed as follows. The cleft of Smon0123 seems to have spatial allowance. In the interaction between Smon0123 and CΔ0S, numerous water molecules are located in the space where a sulfate group of CΔ4S or CΔ6S is accommodated (Fig. 7D). The sulfate group of each substrate is situated close to the binding site of Smon0123 and directly forms hydrogen bonds with several residues of Smon0123. The C-C contacts of Smon0123 with C∆6S also increase compared with those with CΔ0S. In addition, the pyranose ring of amino sugar in unsaturated GAG disaccharide with 1,4-glycoside bond would be in reverse to that in unsaturated chondroitin disaccharide, suggesting that the low affinity of Smon0123 with unsaturated GAG disaccharides with 1,4-glycoside bond is due to this structural difference in arrangement of the pyranose ring.
Judging from the substrate specificity of Smon0123 (binding protein) and Smon0127 (UGL), the degradation of hyaluronan and chondroitin sulfate by S. moniliformis is reasonable (Fig. 3A, 1 and 2, right). Heparin is lethal for this bacterium (Fig. 3A, 3, left), although putative heparinase (Smon0124) is encoded in the GAG genetic cluster (Fig. 2). There is a possibility that heparin oligosaccharide produced from heparin by putative heparinase inhibits bacterial growth. This result suggests that heparin is extremely scarce at the colonization site of S. moniliformis, contributing to the compositional analysis of GAGs in extracellular matrices of animal cells. There is a possibility that heparin is rich in the oral cavity in humans but scarce in rodents. Heparin is expected to become a potential anti-Streptobacillus agent.
S. moniliformis is normally indigenous to rodent oral cavity and belongs to a phylum of fusobacteria. Fusobacteria such as Fusobacterium and Leptotrichia usually inhabit the oral cavity and gastrointestinal tract of animals including humans 44 . Non-sulfated hyaluronan as well as sulfated GAGs such as chondroitin sulfate are abundant in animal oral cavities 45 . GAGs provide structures of tissues with a strong network due to the high water absorption. Gingiva in the oral mucosa is known to contain particularly rich sulfated GAGs due to the high pressure of mastication 46 . Besides, dermatan sulfate and chondroitin sulfate are rich in connective tissues of the oral mucosa and connective tissue papilla of the anterior and posterior palate in rodents 45 . Therefore, the degradation and import systems of sulfated GAGs in S. moniliformis are feasible for its colonization in rodent oral cavities, abundant in sulfated GAGs.
Homologous genes with S. moniliformis ABC transporter genes are found in the genome of some fusobacteria. Fusobacterium mortiferum (NCBI BioProject ID, 32421) and Leptotrichia goodfellowii (NCBI BioProject ID, 43669) form the GAG genetic cluster including ABC transporter genes in bacterial genomes, indicating that the GAG import system is common to these fusobacteria. Some fusobacteria are pathogenic and cause infection in some organs and tissues such as the lung, head neck, cranium, and meninx 47 . This fusobacterial system for the degradation and import of GAG may be involved in colonization and/or infection to host cells, and inhibitors for the bacterial system are expected to develop novel therapy agents.
In conclusion, for the first time, the bacterial import system of sulfated and non-sulfated GAGs was identified as the solute-binding protein-dependent ABC transporter and clarified to be functionally expressed in pathogenic S. moniliformis through molecular and structural biology.

Materials and Methods
Materials. Unsaturated chondroitin disaccharides (CΔ0S, CΔ4S, and CΔ6S) were purchased from Seikagaku Biobusiness. Unsaturated hyaluronan disaccharide (ΔHA) and unsaturated heparin disaccharides (HΔ6S and HΔNS6S) were purchased from Sigma-Aldrich. A sulfate group-free HΔ0S (heparin disaccharide IV-A) was purchased from Dextra. SYPRO Orange (Invitrogen), DDM (Nacalai tesque), and n-octyl-β-d-glucoside (n-OG) (Dojindo) were commercially available. Oligonucleotides were synthesized by Hokkaido System Science. Restriction endonucleases and DNA-modifying enzymes were purchased from Toyobo. All other analytical grade chemicals used in this study were commercially available. Plate method for detection of GAG degradation. Rapid plate method 27 was adopted to investigate the GAG-degrading ability of S. moniliformis. The bacterial cells were cultured in plates containing 1% BSA (Wako), 1% agar, 0.2% dialyzed GAG (hyaluronan, chondroitin sulfate C, or heparin), 0.8% nutrient broth, and 20% horse serum. After addition of 2 M acetic acid (1 ml) to the cultured plates, the remaining GAG was reacted with BSA as white precipitates and clear zones appeared as a halo in GAG-degraded regions.

Microorganisms
Immunoblotting. Antibodies  Enzyme assay. The Smon0127 UGL was assayed by monitoring the decrease of absorbance at 235 nm derived from the C=C double bond in unsaturated GAG disaccharides. The assay was conducted at 30 °C in reaction mixture of 20 mM Tris-HCl (pH 7.5), 0.2 mM substrate, and the enzyme. Unsaturated GAG disaccharides, ΔHA, CΔ0S, CΔ4S, CΔ6S, HΔ0S, and HΔ6S, were used as substrates. One unit of the enzyme activity was defined as the amount of enzyme required to degrade 1 µmol of substrate per minute as described previously 12,48 . The enzyme was also assayed using various concentrations (0 to 1 mM) of CΔ0S, and kinetic parameters (K m and k cat ) were determined according the Michaelis-Menten equation (Synergy Software). . Parameters under reaction conditions were determined as follows: excitation band width, 1 nm; emission band width, 10 nm; response, 2 s; sensitivity, high; excitation wavelength, 280 nm, start to end emission wavelength, 300 to 500 nm; data pitch, 1 nm; and scan speed, 100 nm/min. The reaction mixture contained 0.1 µM Smon0123, 50 mM Tris-HCl (pH 7.5), and 0 to 20 µM ligands. Various disaccharides such as CΔ0S, CΔ4S, CΔ6S, ΔHA, HΔ0S, HΔ6S, HΔNS6S, N,N′-diacetylchitobiose, and cellobiose were used as a ligand. The interaction between Smon0123 and GAG polysaccharides such as hyaluronan, chondroitin sulfate C, and heparin was also investigated using BSA as a negative control. The ratio of decreasing fluorescence intensity by addition of increasing ligands in comparison with the intensity in the absence of ligands was plotted and the dissociation constant (K d ) was determined using the least-squares method 49  . Each single crystal was picked up by a nylon loop, soaked in mother liquor containing 20% ethylene glycol as a cryoprotectant, and frozen in a cold nitrogen gas stream on the beamline BL-38B1 of SPring-8 (Harima, Japan). X-ray diffraction images were collected with a MAR225HE detector (Rayonix) with synchrotron radiation at wavelength 1.00 Å. The data were indexed, integrated, and scaled using the HKL-2000 program 50 . The structure was determined through molecular replacement method with the Molrep program 51 in the CCP4 program package. Structure refinement was conducted using the phenix refine program in the PHENIX program package 52 . After each refinement cycle, the model was adjusted manually with the win-Coot program 53  After stopping reaction at 80 min, a mixture of 6% ascorbic acid/1 M HCl and 1% ammonium molybdate (60 µl) was added to the reaction solution (60 µl) and incubated at room temperature for 5 min. Subsequently, a mixture of 2% sodium citrate, 2% sodium metaarsenite, and 2% acetic acid (90 µl) was added to the solution and incubated at 37 °C for 10 min 38 . This reaction mixture was measured in absorbance at 850 nm to determine phosphate ion concentration generated through ATP hydrolysis. ATPase activity was represented as phosphate ion (nmol) produced by 1 mg Smon0121-Smon0122(10xHis)/Smon0120-Smon0120 per 1 min. The value measured using proteoliposome and disaccharides was subtracted from that using liposomes without any proteins and ligands.