Microevolution of a Standard Strain ofCryptococcus neoformansResulting in Differences in Virulence and Other Phenotypes

Abstract

Cryptococcus neoformans is a major fungal pathogen for patients with debilitated 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) became attenuated after continuous in vitro culture prompted us to perform a comparative study of nine strain 24067 isolates obtained from six different research laboratories. Each isolate was characterized by DNA typing, virulence for mice, proteinase production, extracellular protein synthesis, melanin synthesis, carbon assimilation pattern, antifungal drug susceptibility, colony morphology, growth rate, agglutination titers, phagocytosis by murine macrophages, capsule size, and capsular polysaccharide structure. All isolates had similar DNA typing patterns consistent with their assignment to the same strain, although minor 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 rate, capsule size, proteinase production, and melanization. These findings imply that C. neoformans is able to undergo rapid changes in vitro, probably as a result of adaptation to laboratory conditions, and suggest the need for careful attention to storage and maintenance conditions. In summary, our results indicate thatC. 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 phenotypic characteristics.

capsule biosynthesis and regulation

“…For example, study of the serotype D laboratory strain ATCC 24067 conserved under different conditions in different laboratories showed that the GXM structure can vary over time [20]. Similar variations have been shown to occur in vivo during experimental infections in animals and clinical human infections [21,22].…”

Clinical isolates and laboratory referenceCandidaspecies and strains have varying abilities to form biofilms

“…Typically research on Candida biofilms uses laboratory-adapted strains that will have been subcultured multiple times and even for decades in some cases since their first isolation, and thus, they may not be a suitable representative for biofilm formation for current clinically relevant strains (Ramage et al, 2001;Andes et al, 2004). Fungal micro-evolution has been shown to occur under in vitro and in vivo growth conditions and affect major virulence factors (Franzot et al, 1998;Fries & Casadevall, 1998). This highlights the need for adequate attention to storage conditions of both clinical and laboratory strains and the need for careful selection of an appropriate laboratory strain for biofilm studies.…”

Characterization of a flocculation-like phenotype in Cryptococcus neoformans and its effects on pathogenesis

“…This effect was only seen at low infecting inocula that can be effectively cleared by the innate immune system. Franzot et al (1998) reported that strain ATCC 24067 could change in different laboratory environments over time. This microevolution was thought to explain inconsistencies in experimental results within and between laboratories.…”