MICROEVOLUTION OF A STANDARD STRAIN OF CRYPTOCOCCUS-NEOFORMANS RESULTING IN DIFFERENCES IN VIRULENCE AND OTHER PHENOTYPES

Cryptococcus neoformans is a major fungal pathogen for patients with d ebilitated immune systems. However, no information is available on the stability of virulence or of phenotypes associated with virulence for C. neoformans laboratory strains. A serendipitous observation in our laboratory that one isolate of C. neoformans ATCC 24067 (strain 52D) b ecame attenuated after continuous in vitro culture prompted us to perf orm a comparative study of nine strain 24067 isolates obtained from si x different research laboratories. Each isolate was characterized by D NA typing, virulence for mice, proteinase production, extracellular pr otein synthesis, melanin synthesis, carbon assimilation pattern, antif ungal drug susceptibility, colony morphology, growth rate, agglutinati on titers, phagocytosis by murine macrophages, capsule size, and capsu lar polysaccharide structure. All isolates had similar DNA typing patt erns consistent with their assignment to the same strain, although min or chromosome size polymorphisms were observed in the electrophoretic karyotypes of two isolates. Several isolates had major differences in phenotypes that may be associated with virulence, including growth rat e, capsule size, proteinase production, and melanization. These findin gs imply that C. neoformans is able to undergo rapid changes in vitro, probably as a result of adaptation to laboratory conditions, and sugg est the need for careful attention to storage and maintenance conditio ns. In summary, our results indicate that C. neoformans (i) can become attenuated by in vitro culture and (ii) is capable of microevolution in vitro with the emergence of variants exhibiting new genotypic and p henotypic characteristics.