Cryptococcus neoformans capsule structure evolution in vitro and during murine infection

Abstract

Cryptococcus neoformans capsule structure modifications after prolonged in vitro growth or in vivo passaging have been reported previously. However, nothing is known about the dynamics of these modifications or about their environmental specificities. In this study, capsule structure modifications after mouse passaging and prolonged in vitro culturing were analyzed by flow cytometry using the glucuronoxylomannan-specific monoclonal antibody E1. The capsule structures of strains recovered after 0, 1, 8, and 35 days were compared by using the level of E1-specific epitope expression and its cell-to-cell heterogeneity within a given cell population. In vitro, according to these parameters, the diversity of the strains was higher on day 35 than it was initially, suggesting the absence of selection during in vitro culturing. In contrast, the diversity of the strains recovered from the brain tended to decrease over time, suggesting that selection of more adapted strains had occurred. The strains recovered on day 35 from the spleen and the lungs had different phenotypes than the strains isolated from the brain of the same mouse on the same day, thus strongly suggesting that there is organ specificity for C. neoformans strain selection. Fingerprinting of the strains recovered in vitro and in vivo over time confirmed that genotypes evolved very differently in vitro and in vivo, depending on the environment. Overall, our results suggest that organ-specific selection can occur during cryptococcosis.

FIG. 1.

Clinical isolate virulence assays. The survival of mice infected with 10⁴ viable yeast cells from one of seven clinical isolates recovered from the same patient are shown.

FIG. 2.

Heterogeneity of the capsule structure within a clonal cell population. (A) Cells from strain C45a were grown on induction medium and labeled with the anti-GXM MAb E1. The arrows indicate strongly labeled cells (white arrows) and negative cells (black arrows). (B) FACS analysis of this cell population.

FIG. 3.

(A) Evolution of CENTEL hybridization patterns of strains isolated from the brain over time. Stars indicate profile changes compared to the original strain C45a (profile I). Roman numerals refer to the different profiles. (B) Schematic representation of the evolution of CENTEL hybridization patterns of strains isolated from continuous culture (▴) or from different organs (▪). B, brain; S, spleen; L, lungs. Roman numerals refer to the different profiles. Only two strains isolated from the lungs on day 8 gave interpretable profiles, and they are not included in this figure.

FIG. 4.

Capsule structure evolution as assessed by median fluorescence intensity (A) and heterogeneity of the cell population (CVF; see Materials and Methods) (B) after in vitro or in vivo passaging. Two in vivo independent experiments were done, one with a large inoculum (10⁵ cells per mouse) and one with a small inoculum (10⁴ cells per mouse).