Abstract
Certain bacterial species target the polysaccharide glycosaminoglycans (GAGs) of animal extracellular matrices for colonization and/or infection. GAGs such as hyaluronan and chondroitin sulfate consist of repeating disaccharide units of uronate and amino sugar residues, and are depolymerized to unsaturated disaccharides by bacterial extracellular or cell-surface polysaccharide lyase. The disaccharides are degraded and metabolized by cytoplasmic enzymes such as unsaturated glucuronyl hydrolase, isomerase, and reductase. The genes encoding these enzymes are assembled to form a GAG genetic cluster. Here, we demonstrate the Streptococcus agalactiae phosphotransferase system (PTS) for import of unsaturated hyaluronan disaccharide. S. agalactiae NEM316 was found to depolymerize and assimilate hyaluronan, whereas its mutant with a disruption in the PTS genes included in the GAG cluster was unable to grow on hyaluronan, while retaining the ability to depolymerize hyaluronan. Using toluene-treated wild-type cells, the PTS activity for import of unsaturated hyaluronan disaccharide was significantly higher than that observed in the absence of the substrate. In contrast, the PTS mutant was unable to import unsaturated hyaluronan disaccharide, indicating that the corresponding PTS is the only importer of fragmented hyaluronan, which is suitable for PTS to phosphorylate the substrate at the C-6 position. This is distinct from Streptobacillus moniliformis ATP-binding cassette transporter for import of sulfated and non-sulfated fragmented GAGs without substrate modification. The three-dimensional structure of streptococcal EIIA, one of the PTS components, was found to contain a Rossman-fold motif by X-ray crystallization. Docking of EIIA with another component EIIB by modeling provided structural insights into the phosphate transfer mechanism. This study is the first to identify the substrate (unsaturated hyaluronan disaccharide) recognized and imported by the streptococcal PTS. The PTS and ABC transporter for import of GAGs shed light on bacterial clever colonization/infection system targeting various animal polysaccharides.
Highlights
Extracellular matrices of all animal tissues and organs serve as physical scaffolds for cellular constituents, cell differentiation and proliferation, homeostasis, and tissue formation [1]
The ability of S. agalactiae to degrade and assimilate hyaluronan allows the measurement of GAG-phosphotransferase system (PTS) import activity using an unsaturated disaccharide derived from hyaluronan degradation
The GAG-PTS import of unsaturated hyaluronan disaccharide in bacterial wild-type cells grown in the absence of hyaluronan was significantly higher than in controls using no substrate or GlcN6P; this indicates that S. agalactiae incorporates unsaturated hyaluronan disaccharide via a PTS
Summary
Extracellular matrices of all animal tissues and organs serve as physical scaffolds for cellular constituents, cell differentiation and proliferation, homeostasis, and tissue formation [1]. Glycosaminoglycans (GAGs), constituents of the matrices [2], are acidic polysaccharides consisting of repeating disaccharide units of uronate and amino sugar residues. Hyaluronan, chondroitin sulfate, heparin, and heparan sulfate are classified as GAGs based on their constituent monosaccharides, glycoside linkages, and sulfation patterns [3, 4]. Hyaluronan consists of D-glucuronate (GlcUA) and N-acetyl-D-glucosamine (GlcNAc), chondroitin sulfates of GlcUA and N-acetyl-D-galactosamine (GalNAc), and heparin and heparan sulfate of GlcUA or L-iduronate (IdoUA), and D-glucosamine (GlcN) or GlcNAc [5] (S1 Fig). The uronate and amino sugar residues in hyaluronan and chondroitin sulfate are linked by 1,3-glycoside bonds, whereas the residues in heparin and heparan sulfate are connected by 1,4-glycoside bonds. With the exception of hyaluronan, these GAGs frequently contain sulfate groups in the uronate and/or amino sugar residues, and function as protein-binding proteoglycans in extracellular matrices
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