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. 2022 Apr 26;13(2):e0028322.
doi: 10.1128/mbio.00283-22. Epub 2022 Mar 8.

Lineages Derived from Cryptococcus neoformans Type Strain H99 Support a Link between the Capacity to Be Pleomorphic and Virulence

Affiliations

Lineages Derived from Cryptococcus neoformans Type Strain H99 Support a Link between the Capacity to Be Pleomorphic and Virulence

Kenya E Fernandes et al. mBio. .

Abstract

The pathogenic yeast Cryptococcus neoformans causes nearly 200,000 deaths annually in immunocompromised individuals. Cryptococcus cells can undergo substantial morphological change during mammalian infection, including increased capsule and cell size, the release of shed capsule, and the production of titan (>10 μm), micro (<2 μm)-, and irregular cells. We examined phenotypic variation under conditions designed to simulate in vivo stress in a collection of nine lineages derived from the C. neoformans type strain H99. These lineages are highly genetically similar but have a range of virulence levels. Strains from hypervirulent lineages had a larger average capsule size, greater variation in cell size, and an increased production of microcells and shed capsule. We tested whether disruption of SGF29, which encodes a component of the SAGA histone acetylation complex that has previously been implicated in the hypervirulence of some lineages, also has a role in the production of morphological variants. Deletion of SGF29 in a lineage with intermediate virulence substantially increased its production of microcells and released capsule, consistent with a switch to hypervirulence. We further examined SGF29 in a set of 52 clinical isolates and found loss-of-function mutations were significantly correlated with patient death. Expansion of a TA repeat in the second intron of SGF29 was positively correlated with cell and capsule size, suggesting it also affects Sgf29 function. This study extends the evidence for a link between pleomorphism and virulence in Cryptococcus, with a likely role for epigenetic mechanisms mediated by SAGA-induced histone acetylation. IMPORTANCE Cryptococcosis is a devastating cause of death and disease worldwide. During infection, Cryptococcus cells can undergo substantial changes to their size and shape. In this study, we used a collection of C. neoformans strains that are highly genetically similar but possess differing levels of virulence to investigate how morphological variation aligns with virulence. We found hypervirulent strains on average had larger capsules and greater variation in cell size and produced more microcells and shed capsule. These hypervirulent strains possessed a mutation in SGF29, which encodes a component of the SAGA complex involved in epigenetic regulation. Analysis of the SGF29 gene in a set of clinical isolates found strains with loss-of-function mutations were associated with higher patient death rates. The capacity to vary appears to be linked with virulence in Cryptococcus, and this can occur in the absence of genetic variation via epigenetic mechanisms.

Keywords: Cryptococcus neoformans; SAGA complex; giant cells; microcells; pleomorphism; virulence.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Origins of the C. neoformans H99 strains used in this study and their comparative virulence and growth dynamics. (A) The relationship between the H99 derivative strains used in this study. Mutations separating strains are shown by circles, and known or hypothesized directions of evolution are shown by arrows. Figure adapted from Arras et al. (9). CNS, central nervous system. (B) Diagram of the SGF29 and LMP1 genes showing disruptions found in some H99 derivative strains. Lighter-colored sections indicate areas of deletion, darker-colored sections indicate areas of insertion. Gray shading indicates introns. (C) Survival of G. mellonella larvae infected with an inoculum of 108 cells of each H99 strain or mock infected with PBS over 10 days; n = 10 for each treatment group. The left panel shows a representative survival plot (one of two independent replicates that gave similar results); the right panel shows photographs of representative larvae at days 0, 5, and 10 that had been mock infected (PBS) or infected with a less virulent (H99C), intermediate (H99O), or hypervirulent (H99S) strain. Treatment groups were compared by log rank test; H99C (P ≤ 0.017) and H99E (P ≤ 0.019) survived significantly longer than H99O, H99S, H99L, KN99a, KN99α, and H99F. (D) Growth curves of H99 strains incubated at 30°C with 180-rpm shaking averaged across three independent replicates, showing no substantial differences between strains.
FIG 2
FIG 2
Less virulent H99 strains produce significantly smaller capsules following growth under capsule-inducing conditions. Capsule thickness (A) and cell body diameter (B) of 100 individual cells of each strain grown in DMEM with 5% CO2 at 37°C for 5 days are shown. Strains were compared using two-tailed unpaired t tests with Welch’s correction (significance described in Results). Error bars show the means ±95% confidence intervals. Int., intermediate. Giant, micro-, and irregular cells were excluded from this analysis. (C) India ink preparations of each strain showing variation in capsule and cell size. Scale bar, 15 μm.
FIG 3
FIG 3
Hypervirulent H99 strains produce morphological variants at a much higher frequency than intermediate and less virulent strains following growth under capsule-inducing conditions. (A) India ink preparations of H99 strains showing morphological variants produced following growth in DMEM with 5% CO2 at 37°C for 5 days, including giant cells larger than 10 μm, microcells smaller than 2 μm, extracellular capsule clustered around cells (clustered capsule), and extracellular capsule released into the media (released capsule). Scoring on the left shows the approximate relative frequency of morphological variants seen in a typical field of view, as presented in panel B.
FIG 4
FIG 4
Deletion of SGF29 increases the production of morphological variants by H99O but not by H99S. Growth curves of wild-type and sgf29 knockout strains of H99O (A) and H99S (B) incubated at 30°C with 180-rpm shaking averaged across three independent replicates showing no substantial differences among strains. Capsule thickness (left) and cell body diameter (right) of wild-type and sgf29 knockout strains of H99O (C) and H99S (D) grown in DMEM with 5% CO2 at 37°C for 5 days. Strains were compared using two-tailed unpaired t tests with Welch’s correction. Error bars show the means ±95% confidence intervals, n = 100. The relative frequency of morphological variants in wild-type and sgf29 knockout strains of H99O (E) and H99S (F) are shown; see Fig. 3A for scoring process. H99O.2 and H99S.2 are independently sourced and presumed identical copies of H99O and H99S, respectively. H99OΔ = sgf29ΔH99O; H99OΔ+ = sgf29Δ + SGF29H99O; H99SΔ = sgf29ΔH99S; H99SΔ+ = sgf29Δ + SGF29H99S.
FIG 5
FIG 5
Mutations in SGF29 in clinical isolates correlate with clinical and phenotypic variables. (A) Strains with mutations occurring in the SGF29 gene resulting in truncated proteins. Lighter green sections indicate areas of deletion. (B) Strains with missense mutations occurring in the coding regions of the SGF29 gene (top) and the location of the mutations (bottom). Dark blue boxes indicate mutations predicted to be neutral, while red boxes indicate mutations predicted to be deleterious. (C) Tandem TA repeats ranging from 5 to 9 copies identified in the second intron of SGF29. (D) Significant correlations between clinical variables and strains possessing disruptions in SGF29 (including both mutations resulting in truncated proteins and deleterious missense mutations). (E) Significant correlations between clinical and phenotypic variables and the number of TA repeats in the second intron of SGF29.

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