The capsule of Cryptococcus neoformans is known to be an important virulence factor. The major capsular polysaccharide is a straight-chain mannan with side chains of xylosyl, glucuronyl and O-acetyl groups, known as a glucuronoxylomannan (GXM). A method is proposed for obtaining mutants with genetically altered GXM in this yeast. First, GXM is chemically modified in one of 3 ways: 1) O-acetyl esters are removed by treatment with ammonia at pH 11; 2) all side-chains are removed by Smith degradation, leaving a """"""""straight-chain mannan;"""""""" 3) glucuronyl residues are removed with lithium in ethylenediamine, leaving desglucuronyl-GXM. Monoclonal antibodies are prepared against normal GXM and against the chemically modified carbohydrates. The monoclonals are covalently labeled with fluorescein. Mutants are induced by treatment of a population of normal, encapsulated cells with nitrosoguanidine or UV. Using fluorescent monoclonal antibody specific for modified GXM, mutants with modified capsular polysaccharides are specifically labeled. These cells are separated by use of a fluorescence-activated cell sorter. Putative mutants are characterized genetically by outcrossing, recombinational mapping and complementation testing. They are characterized biochemically by structural analysis of extracellular polysaccharide, enzymatic study of glucosyl transfer reactions and chromatographic analysis of lipid-linked oligosaccharides. Potential benefits to accrue from this study include development of monoclonal antibodies with many potential applications, the advancement of the genetics of C. neoformans in general and genetics of a fungal virulence factor in particular, the delineation of structure-function relationships in the GXM, and the identification of genetic loci which determine branching in the cryptococcal GXM.
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