The GATA-like transcription factor Gat201 determines alkaline-restricted growth in Cryptococcus neoformans

Abstract

The fungus Cryptococcus neoformans is an opportunistic human pathogen that causes fatal meningitis through uncontrolled proliferation in host tissues. Evasion of host defenses relies on a protective polysaccharide capsule, regulated, in part, by the GATA-like transcription factor Gat201. Gat201 additionally contributes to virulence through capsule-independent mechanisms. Here, we show that Gat201 affects the proliferation of C. neoformans: in RPMI-1640 cell culture media at an alkaline pH that restricts wild-type cell growth, gat201∆ strains show increased budding, growth, and viability. RNA-seq analysis shows that Gat201 pathway genes, including co-factors GAT204 and LIV3, are rapidly activated within minutes of inoculating C. neoformans in RPMI media, and strains mutated for GAT204 and, to a lesser extent, LIV3 also show improved growth. The effect of Gat201 on growth is pH-dependent: gat201∆ cells grow better than wild-type cells at high pH but worse than wild-type cells at neutral pH, in otherwise identical media. Together, this identifies the Gat201 pathway as an alkaline-responsive regulator of proliferation: Gat201 appears to govern an environment-dependent trade-off between proliferation and production of the defensive capsule. Furthermore, evolutionary analysis shows that Gat201 is in a subfamily of GATA-like transcription factors that is conserved within diverse fungi but absent in model yeasts. Together, our findings urge improved understanding of proliferation in diverse environmental niches in order to understand the mechanistic basis of fungal pathogenesis.IMPORTANCEInfectious microorganisms must adapt to differences between external and host environments in order to colonize and cause disease. Cryptococcus neoformans is an encapsulated fungal pathogen that can infect human airways and travel to the brain to cause life-threatening meningitis. The airway is a dynamic environment characterized by nutrient limitation, high temperature (37°C), CO₂, and transiently high pH (>8.5). In both the lung and brain, fungal proliferation through budding is a major driver of pathogenesis; however, the regulators of Cryptococcus proliferation are poorly understood and distinct from other model yeasts. In this work, we explore how Cryptococcus adapts to shifting environments and identify that the transcription factor Gat201, known to regulate capsule production, negatively regulates proliferation under alkaline conditions. Our findings highlight the need for improved understanding of proliferation/adaptation and its regulation in non-model systems.

Keywords: Cryptococcus neoformans; pH; transcriptional regulation.

Fig 1

GAT201 represses the proliferation and viability of C. neoformans during reactivation in RPMI medium. (A) GAT201 promotes capsule biosynthesis and represses budding in RPMI-1640 medium (without serum) at 37°C 2 h after inoculation. Micrographs show GAT201 (H99 wild-type), gat201∆m, and complemented GAT201-C1 strains, stained with India Ink, capsule highlighted with red arrow, and buds highlighted with green arrows. GAT201-C1 complements the budding phenotype but does not clearly complement the capsule phenotype. (B) Quantification of budding index at 2 h (% budded cells) shows that gat201∆m cells reactivate to produce buds in RPMI (n = >100 cells per replicate, with three biological replicates per condition). Panels A and B are taken from the same experiment, and larger sets of representative cells are shown in Fig. S4. (C) GAT201 (H99) cell populations reactivating in RPMI show a fall in density after 10 h of growth, which is absent in gat201∆ strains and absent during growth in rich YPD media. Growth curves of optical density at 595 nm (OD₅₉₅) were collected via plate reader from seven biological replicates, three technical replicates each, at 37°C. Note the different y-axis limits in the subpanels, reflecting higher final OD in rich media. (D) GAT201 (H99) cells reactivating in RPMI or RPMI+ serum show a decline in viability after 12–24 h, which is absent in gat201∆ and partially restored by complementing GAT201. The decline in viability is more severe in RPMI without serum than it is in RPMI with serum. Colony-forming units per milliliter of culture were measured by serial dilution on plates, in three biological replicates; individual replicates are plotted as dots with a dashed line connecting the medians.

Fig 2

GAT201 co-factors GAT204 and LIV3 also restrict growth. Growth curves of optical density at 595 nm (OD₅₉₅) were collected via plate reader from four biological replicates, three technical replicates each, at 37°C. Fig. S13 shows individual replicates.

Fig 3

The effect of GAT201 on growth depends on sodium bicarbonate (NaHCO₃). Starting with an RPMI formulation lacking NaHCO₃, we added either 0 mM, 1.5 mM, 6 mM, or 24 mM NaHCO₃ and grew Cryptococcus for 24 h. Wild-type GAT201 cells grow in 0 mM NaHCO₃ but do not grow in 24 mM NaHCO₃, whereas gat201∆ cells have opposite phenotypes of no growth in 0 mM and growth at 24 mM. These cells have intermediate phenotypes at intermediate concentrations of NaHCO₃, whereas complemented strains have growth phenotypes resembling wild-type. This figure shows the median of three technical replicates from a single biological replicate, and two further biological replicates are shown in Fig. S16.

Fig 4

C. neoformans Gat201 is homologous to other GATA-family zinc finger proteins that regulate fungal growth and environmental responses. (A) Domain structure of Gat201 and 4 close homologs, with GATA-like zinc finger domain shown in red (Interpro IPR013088) and predicted unstructured regions in blue (MobiDB Lite consensus disorder), taken from Interpro (35). (B) Multiple sequence alignment of the GATA-like zinc finger domains of homologs made with MUSCLE (36). Conserved cysteine residues typical of GATA-like zinc fingers are indicated with asterisks. An extended phylogeny and homology analysis are shown in Fig. S18.

Fig 5

The Gat201 pathway promotes Cryptococcus virulence and represses proliferation. Gat201 acts in parallel to the serum-responsive cAMP/Pka pathway and the major pH-responsive Rim101 pathway. Gat201 requires mutual activators, Gat204 and Liv3, to suppress proliferation.