Cloning of a gene responsible for the formation of extracellular polysaccharide capsule in Cryptococcus neoformans, a major opportunistic fungal pathogen causing infection primarily in immunocompromised patients: The extracellular polysaccharide capsule of C.neoformans has been identified as the major virulence factor of the organism. A wild type genomic DNA library of C. neoformans B-3501 was used to complement an ura5 acapsular mutant (B-4131, cap 67). The library was constructed in a plasmid vector containing the URA5 gene as a selection marker. After electroporation, a plasmid containing a 4.5kb DNA insert was rescued in E.coli from one of the Cap+ transformants. By using overlapping subclones of the 4.5kb DNA fragment, the minimum region required for complementation of B-4131 Cap-phenotype was identified on a 2.9kb fragment. The transformants became acapsular when they lost their transforming plasmids during growth on non-selection media. In addition, the 2.9kb fragment isolated from the wild type strain was able to restore the Cap+phenotype in another acapsular mutant (cap 59). The CAP 59 was one of the five capsule-associated loci which were previously mapped. These results indicated that we cloned the gene CAP 67. A genomic probe for strain identification in C.neoformans was constructed for epidemiological study: A plasmid, UT-4, containing URA 5 gene with 3kb flanking sequences and telomeric sequence of C. neoformans was found to be a useful probe for DNA finger printing. The probe was specially useful for strain identification of the gattii variety of C. neoformans. Using the probe, we could identify that several strains of C.neoformans var.gattii.
NIH ExPorter
NIH ExPorter