Microevolution of a standard strain of Cryptococcus neoformans resulting 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 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 phenotypic characteristics.

FIG. 1

Electrophoretic karyotypes of the nine ATCC 24067 isolates as obtained by CHEF analysis. The molecular weight marker (Saccharomyces cerevisiae chromosomal DNA; Bio-Rad) is shown in lane M, and the corresponding sizes in are indicated on the right. The asterisks in lanes D2 and G indicate the differences in the electrophoretic karyotypes of these isolates.

FIG. 2

Representative phenotypes of ATCC 24067 isolates distinguished by colony size (A), extracellular proteinase production (B), and melanin production (C). (A) From left to right, colonies of isolates F1, D2, and A after 7 days of growth in Sabouraud dextrose agar plates. (B) From left to right, colonies of isolates C, F1, and G in minimal medium agar plates supplemented with 0.1% azoalbumin after 12 days of growth. Note the difference in halo size for isolates C and F1, as well as the absence of a clearance halo for isolate G. Halo sizes are listed in Table 3. (C) From left to right, colonies of isolates E, C, and G after 15 days of growth in minimal medium agar plates containing l-dopa. Note the different levels of melanization among the isolates. All photographs were taken at the same magnification (×1.5 to ×2). Actual colony sizes are listed in Table 3. Note that the isolates shown and times of growth are different for rows A, B, and C and that comparisons between rows should not be made.

FIG. 3

Extracellular proteins of ATCC 24067 isolates on a sodium dodecyl sulfate–12% polyacrylamide gel. Each lane contained ∼400,000 total cpm from the indicated isolate. Molecular mass markers are indicated to the left in kilodaltons. A 6-day exposure is shown.

FIG. 4

Growth curves for the nine ATCC 24067 isolates in Sabouraud dextrose broth. This experiment was performed twice with similar results. For isolates A, B, D2, and F2 the results were confirmed by an additional experiment in which growth was measured by hemacytometer and CFU counts (not shown).

FIG. 5

¹H-NMR spectra of resolution-enhanced anomeric regions of the de-O-acetylated GXMs for the various isolates of ATCC 24067 used in this study. The ¹H-NMR data for the serotype D isolate 9375 is included as a reference (42).

FIG. 6

Repeating units of the reference serotype D GXM and for glucuronomannan with the observed chemical shifts for the mannosyl residues of the structure reporter group region at pD 10.

FIG. 7

Survival curves for adult A/J mice infected intravenously with 5 × 10⁵ C. neoformans organisms via the lateral tail vein. The average survival of mice infected with isolates D2, E, B, and A was 2.9 ± 0.9487, 8.2 ± 0.9487, 7.85 ± 1.0554, and 70 days, respectively (n = 10 per group). All mice infected with strain A were alive at the termination of the experiment at 70 days postinfection, and thus this data set contains censored values.