The digested products usually comprise oligosaccharides with some repeating units, which retain the immunogenicity of CPS. Many bacteriophages depolymerize the CPS of their host bacteria through their tailspike proteins (TSPs). Thus, alternate ways of accessing these glycans are important. The polysaccharide was enzymatically produced in glycoengineered Escherichia coli, but a low degree of pyruvylation was observed. Recently, the development of a K1 CPS-containing glycoconjugate vaccine against K1 K.
In an attempt to fragment K1 CPS into oligosaccharides via chemical methods, we failed to retain the pyruvylation and O-acetylation modifications in the products. The pyruvylation of the glucuronic acid and O-acetylation of the fucose are essential to the immune response induced by the K1 CPS. The chemical structure of the K1 CPS comprises repeating units of the trisaccharide (→ 3)-β-D-Glc p-(1 → 4)-β-D-Glc pA-(1 → 4)-α-L-Fuc p-(1 →). In contrast, glycoconjugate vaccines generated by conjugating CPS to a carrier protein can elicit a stronger and long-lasting immune response. On the basis of previous studies, however, whole CPS isolates from pathogenic bacteria could only induce relatively poor and T cell-independent immune responses. pneumoniae-induced liver abscess and lethality, suggesting that the K1 CPS is a promising target for vaccine development against K.
pneumoniae can enhance phagocytosis to combat invasion of the bacterium and protected mice from K. As an alternative to treatment with antibiotics, monoclonal antibodies against the K1 CPS of K. pneumoniae strains possess a hypermucovisous phenotype attributed to their unique capsular polysaccharides (CPSs), particularly the K1 CPS. Previous studies indicated that nearly all PLA K. Survivors of PLA-associated meningitis and endophthalmitis usually suffer from either severe neurological sequelae or blindness, respectively. Patients with PLA often have serious complications, such as endophthalmitis, meningitis, and necrotizing fasciitis, ultimately leading to a mortality of ~ 5%. pneumoniae, but also as a tool to prepare structurally-defined oligosaccharides for the generation of glycoconjugate vaccines against infections caused by this organism.įor more than two decades, pathogenic Klebsiella pneumoniae has been a community-acquired infection causing pyogenic liver abscesses (PLAs) in Asia. Our results provide insights into how the enzyme recognizes and depolymerizes the K1 CPS, and demonstrate the potential use of the protein not only as a therapeutic agent against K. pneumoniae infection, even against a high challenge dose. We also demonstrate that the enzyme is capable of protecting mice from K1 K. We demonstrate the tight interaction between the pyruvate moiety of K1 CPS and the enzyme in this second carbohydrate-binding site to be crucial to CPS depolymerization of the enzyme as well as phage absorption and infectivity. We also determined the 1.46-Å-resolution, product-bound crystal structure of the enzyme, revealing two distinct carbohydrate-binding sites in a trimeric β-helix architecture, which provide the first direct evidence for a second, non-catalytic, carbohydrate-binding site in bacteriophage-derived polysaccharide depolymerases. We found a bacteriophage-derived polysaccharide lyase that depolymerizes the K1 CPS into fragments of 1–3 repeating trisaccharide units with the retention of the pyruvylation and O-acetylation, and thus the important antigenic determinants of intact K1 CPS. pneumoniae-infected mice with the wild-type and mutant enzymes. We also performed site-directed mutagenesis, enzyme kinetic analysis, phage absorption and infectivity studies, and treatment of the K. The biochemical and biophysical properties of the enzyme were characterized and its crystal structures containing bound CPS products were determined. We analyzed the digested CPS products with NMR spectroscopy and mass spectrometry to reveal a bacteriophage-derived polysaccharide depolymerase specific to K1 CPS.
To date, however, obtaining the fragments of K1 CPS that contain the pyruvylation and O-acetylation for generating glycoconjugate vaccines still remains a challenge. The pyruvylation and O-acetylation modifications on the K1 CPS are essential to the immune response induced by the CPS. This K1 CPS has been reported as a promising target for development of glycoconjugate vaccines against K. Patients with PLA often have serious complications, ultimately leading to a mortality of ~ 5%. K1 capsular polysaccharide (CPS)-associated Klebsiella pneumoniae is the primary cause of pyogenic liver abscesses (PLA) in Asia.
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