title: Contribution of the mannan backbone of cryptococcal glucuronoxylomannan and a glycolytic enzyme of Staphylococcus aureus to contact-mediated killing of Cryptococcus neoformans.
abstract: The fungal pathogen Cryptococcus neoformans is killed by the bacterium Staphylococcus aureus, and the killing is inhibited by soluble capsular polysaccharides. To investigate the mechanism of killing, cells in coculture were examined by scanning and transmission electron microscopy. S. aureus attached to the capsule of C. neoformans, and the ultrastructure of the attached C. neoformans cells was characteristic of dead cells. To identify the molecules that contributed to the fungal-bacterial interaction, we treated each with NaIO(4) or protease. Treatment of C. neoformans with NaIO(4) promoted adherence. It was inferred that cleavage of xylose and glucuronic acid side chains of glucuronoxylomannan (GXM) allowed S. aureus to recognize mannose residues in the backbone, which resisted periodate oxidation. On the other hand, treatment of S. aureus with protease decreased adherence, suggesting that protein contributed to attachment in S. aureus. In confirmation, side chain-cleaved polysaccharide or defined alpha-(1-->3)-mannan inhibited the killing at lower concentrations than native GXM did. Also, these polysaccharides reduced the adherence of the two species and induced clumping of pure S. aureus cells. alpha-(1-->3)-Mannooligosaccharides with a degree of polymerization (DP) of >/=3 induced cluster formation of S. aureus in a dose-dependent manner. Surface plasmon resonance analyses showed interaction of GXM and surface protein from S. aureus; the interaction was inhibited by oligosaccharides with a DP of > or =3. Conformations of alpha-(1-->3) oligosaccharides were predicted. The three-dimensional structures of mannooligosaccharides larger than triose appeared curved and could be imagined to be recognized by a hypothetical staphylococcal lectin. Native polyacrylamide gel electrophoresis of staphylococcal protein followed by electroblotting, enzyme-linked immunolectin assay, protein staining, and N-terminal amino acid sequencing suggested that the candidate protein was triosephosphate isomerase (TPI). The enzymatic activities were confirmed by using whole cells of S. aureus. TPI point mutants of S. aureus decreased the ability to interact with C. neoformans. Thus, TPI on S. aureus adheres to the capsule of C. neoformans by recognizing the structure of mannotriose units in the backbone of GXM; we suggest that this contact is required for killing of C. neoformans.
authors.affiliation: Department of Microbiology, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan. ikeda@my-pharm.ac.jp
authors.forename: Reiko
authors.initials: R
authors.lastname: Ikeda
authors.forename: Fumito
authors.initials: F
authors.lastname: Saito
authors.forename: Miki
authors.initials: M
authors.lastname: Matsuo
authors.forename: Kenji
authors.initials: K
authors.lastname: Kurokawa
authors.forename: Kazuhisa
authors.initials: K
authors.lastname: Sekimizu
authors.forename: Masashi
authors.initials: M
authors.lastname: Yamaguchi
authors.forename: Susumu
authors.initials: S
authors.lastname: Kawamoto
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002236:Carbohydrate Conformation; D003455:Cryptococcus neoformans / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D004797:Enzyme-Linked Immunosorbent Assay; D020723:Glucuronic Acid / Q000378:metabolism; D008351:Mannans / Q000737:chemistry / Q000378:metabolism*; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism; D010504:Periodic Acid / Q000494:pharmacology; D017354:Point Mutation; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000201:enzymology / Q000235:genetics; D020349:Surface Plasmon Resonance; D014305:Triose-Phosphate Isomerase / Q000235:genetics / Q000378:metabolism*; D014994:Xylose / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008351:Mannans; D011134:Polysaccharides; D010504:Periodic Acid; C027478:glucuronoxylomannan; D020723:Glucuronic Acid; D014994:Xylose; C009288:metaperiodate; D010447:Peptide Hydrolases; D014305:Triose-Phosphate Isomerase