The contribution of the mannan backbone of cryptococcal glucuronoxylomannan and a glycolytic enzyme of Staphylococcus aureus to contact mediated killing of Cryptococcus neoformans

Department of Microbiology, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan; Laboratory of Microbiology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 3-1, 7-Chome, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8673, Japan

^* To whom correspondence should be addressed. Email: ikeda{at}my-pharm.ac.jp.

	Abstract

The fungal pathogen Cryptococcus neoformans is killed by the bacterium Staphylococcus aureus and this death is inhibited by soluble capsular polysaccharides. To investigate the mechanism of killing, cells in co-culture were examined under 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 NaIO₄ or protease. Treatment of C. neoformans with NaIO₄ 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 -(13)-mannan inhibited the killing at lower concentrations than native GXM. Also, these polysaccharides reduced the adherence of the two species and induced clumping of pure S. aureus cells. -(13)-mannooligosaccharides of degree of polymerization (DP) 3 induced cluster formation of S. aureus in a dose dependent manner. Surface-plasmon-resonance (SPR) analyses showed interaction of GXM and surface protein from S. aureus; the interaction was inhibited by oligosaccharides of DP 3. Conformations of -(13) oligosaccharides were predicted. The three dimensional structures of mannooligosaccharides sized more than triose appeared curved and could be imagined to be recognized by a hypothetical staphylococcal lectin. Native PAGE 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.