A trade-off between proliferation and defense in the fungal pathogen Cryptococcus at alkaline pH is controlled by the transcription factor GAT201

Cryptococcus is a fungal pathogen whose virulence relies on proliferation in and dissemination to host sites, and on synthesis of a defensive yet metabolically costly polysaccharide capsule. Regulatory pathways required for Cryptococcus virulence include a GATA-like transcription factor, Gat201, that regulates Cryptococcal virulence in both capsule-dependent and capsule-independent ways. Here we show that Gat201 is part of a negative regulatory pathway that limits fungal survival. RNA-seq analysis found strong induction of GAT201 expression within minutes of transfer to host-like media at alkaline pH. Microscopy, growth curves, and colony forming units to test viability show that in host-like media at alkaline pH wild-type Cryptococcus neoformans yeast cells produce capsule but do not bud or maintain viability, while gat201Δ cells make buds and maintain viability, yet fail to produce capsule. GAT201 is required for transcriptional upregulation of a specific set of genes in host-like media, the majority of which are direct Gat201 targets. Evolutionary analysis shows that Gat201 is conserved within pathogenic fungi but lost in model yeasts. This work identifies the Gat201 pathway as controlling a trade-off between proliferation, which we showed is repressed by GAT201, and production of defensive capsule. The assays established here will allow characterisation of the mechanisms of action of the Gat201 pathway. Together, our findings urge improved understanding of the regulation of proliferation as a driver of fungal pathogenesis.

. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 14, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 Proliferation-defense trade-off in Cryptococcus 49 Figure S3, related to figure 2: GAT201 promotes capsule biosynthesis and represses budding in RPMI medium (without serum) at 37°C 2 hours after inoculation. There is no obvious difference in phenotype in rich YPD medium. Micrographs show GAT201 (H99), gat201∆m, and complemented GAT201-C1 strains, stained with India Ink. Scale bar is 10µm.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 14, 2023. ; https://doi.org/10.1101/2023.06.14.543486 doi: bioRxiv preprint Proliferation-defense trade-off in Cryptococcus 50 Figure S4, related to Figure 2: Partial complementation of Gat201 mRNA expression shown by RT-qPCR of GAT201, GAT204, and LIV3 genes compared to 3 reference genes (ACT1, GPD1, SRP14). In complemented strains, GAT201 mRNA abundance is roughly 10x lower (log2 fold-change~3.3) than in wild-type GAT201 (KN99alpha). Cultures were inoculated from overnight growth in YPD and grown in RPMI at 37°C for 7 hours. The figure shows log2 fold-change (∆∆Cq) values from 3 biological replicates (median of 3 technical replicates), and a mean value across the biological replicates. The low value of GAT201 detected in gat201∆m represents background, and was only detected at all in 2 out of 3 biological replicates.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 14, 2023. ; https://doi.org/10.1101/2023.06.14.543486 doi: bioRxiv preprint Proliferation-defense trade-off in Cryptococcus 51 Figure S5: GAT201 has a minor effect on growth in media buffered to near-neutral pH with mono and dibasic sodium phosphate and β-glycerophosphate. Cells were grown as in Figure   2C and their OD595 measured, but in Gibco™ CO 2 -independent media.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 14, 2023. ; https://doi.org/10.1101/2023.06.14.543486 doi: bioRxiv preprint Proliferation-defense trade-off in Cryptococcus 52 Figure S6, related to Figure 3A: GAT201 promotes capsule biosynthesis and represses budding in RPMI medium both with and without serum at 37°C, 2 hours after inoculation. Strains are GAT201 (KN99alpha) and gat201∆m, here a wider field of view is shown than in Figure 3A.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 14, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 Proliferation-defense trade-off in Cryptococcus 53 Figure S7, related to Figure 3B: Principal Component Analysis of RNA-seq dataset 2, comparing GAT201 (wild-type) to gat201∆ strains. Note PC 1 vs 2 panel is a repeat of 3B.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 14, 2023. ; https://doi.org/10.1101/2023.06.14.543486 doi: bioRxiv preprint Proliferation-defense trade-off in Cryptococcus 54 Figure S8, related to Figure 3D: Thirty representative genes show distinct expression patterns and GAT201 dependence, again in log2 fold-change per gene. See Figure 3 legend for details.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 14, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 Proliferation-defense trade-off in Cryptococcus 55 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 14, 2023. ; https://doi.org/10.1101/2023.06.14.543486 doi: bioRxiv preprint Proliferation-defense trade-off in Cryptococcus 56 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 14, 2023. ; https://doi.org/10.1101/2023.06.14.543486 doi: bioRxiv preprint