Ikeda Reiko, Saito Fumito, Matsuo Miki, Kurokawa Kenji, Sekimizu Kazuhisa, Yamaguchi Masashi, Kawamoto Susumu
Department of Microbiology, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan.
J Bacteriol. 2007 Jul;189(13):4815-26. doi: 10.1128/JB.00412-07. Epub 2007 May 4.
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.
真菌病原体新型隐球菌可被金黄色葡萄球菌杀死，且可溶性荚膜多糖可抑制这种杀伤作用。为了研究杀伤机制，通过扫描电子显微镜和透射电子显微镜对共培养的细胞进行了检查。金黄色葡萄球菌附着在新型隐球菌的荚膜上，附着的新型隐球菌细胞的超微结构具有死细胞的特征。为了鉴定促成真菌 - 细菌相互作用的分子，我们分别用高碘酸钠（NaIO₄）或蛋白酶处理。用高碘酸钠处理新型隐球菌可促进其黏附。据推测，葡糖醛酸木聚糖（GXM）的木糖和葡萄糖醛酸侧链被切割后，金黄色葡萄球菌能够识别主链中的甘露糖残基，而这些甘露糖残基能抵抗高碘酸盐氧化。另一方面，用蛋白酶处理金黄色葡萄球菌会降低其黏附能力，这表明蛋白质在金黄色葡萄球菌的黏附中起作用。经证实，侧链被切割的多糖或特定的α-(1→3)-甘露聚糖在比天然GXM更低的浓度下就能抑制杀伤作用。此外，这些多糖降低了两种菌的黏附性，并诱导纯金黄色葡萄球菌细胞聚集。聚合度（DP）≥3的α-(1→3)-甘露寡糖以剂量依赖方式诱导金黄色葡萄球菌形成聚集体。表面等离子体共振分析显示GXM与金黄色葡萄球菌的表面蛋白相互作用；这种相互作用被DP≥3的寡糖抑制。预测了α-(1→3)寡糖的构象。大于三糖的甘露寡糖的三维结构呈弯曲状，可以想象它们会被一种假定的葡萄球菌凝集素识别。对金黄色葡萄球菌蛋白进行天然聚丙烯酰胺凝胶电泳，然后进行电转印、酶联免疫凝集素测定、蛋白质染色和N端氨基酸测序，结果表明候选蛋白是磷酸丙糖异构酶（TPI）。利用金黄色葡萄球菌的全细胞证实了其酶活性。金黄色葡萄球菌的TPI点突变体降低了与新型隐球菌相互作用的能力。因此，金黄色葡萄球菌上的TPI通过识别GXM主链中甘露三糖单元的结构附着在新型隐球菌的荚膜上；我们认为这种接触是杀死新型隐球菌所必需的。
