Nitrogen metabolite repression of metabolism and virulence in the human fungal pathogen Cryptococcus neoformans

Abstract

Proper regulation of metabolism is essential to maximizing fitness of organisms in their chosen environmental niche. Nitrogen metabolite repression is an example of a regulatory mechanism in fungi that enables preferential utilization of easily assimilated nitrogen sources, such as ammonium, to conserve resources. Here we provide genetic, transcriptional, and phenotypic evidence of nitrogen metabolite repression in the human pathogen Cryptococcus neoformans. In addition to loss of transcriptional activation of catabolic enzyme-encoding genes of the uric acid and proline assimilation pathways in the presence of ammonium, nitrogen metabolite repression also regulates the production of the virulence determinants capsule and melanin. Since GATA transcription factors are known to play a key role in nitrogen metabolite repression, bioinformatic analyses of the C. neoformans genome were undertaken and seven predicted GATA-type genes were identified. A screen of these deletion mutants revealed GAT1, encoding the only global transcription factor essential for utilization of a wide range of nitrogen sources, including uric acid, urea, and creatinine-three predominant nitrogen constituents found in the C. neoformans ecological niche. In addition to its evolutionarily conserved role in mediating nitrogen metabolite repression and controlling the expression of catabolic enzyme and permease-encoding genes, Gat1 also negatively regulates virulence traits, including infectious basidiospore production, melanin formation, and growth at high body temperature (39°-40°). Conversely, Gat1 positively regulates capsule production. A murine inhalation model of cryptococcosis revealed that the gat1Δ mutant is slightly more virulent than wild type, indicating that Gat1 plays a complex regulatory role during infection.

Figure 1.—

The predicted catabolic enzyme-encoding genes of uric acid, URO1 and DAL1, are sensitive to nitrogen metabolite repression. (A) Scheme representing the predicted (partial) uric acid degradation pathway of C. neoformans. (B) cDNA from wild-type H99 grown in YNB supplemented with ammonium, uric acid, or uric acid plus ammonium (10 mm each nitrogen source) were amplified via qRT-PCR using primers against URO1 (urate oxidase), DAL1 (allantoinase), URE1 (urease), and the control gene ACT1 (actin). In the presence of uric acid as the sole nitrogen source, the expression of URO1 and DAL1 was significantly increased while that of URE1 was slightly increased, but this upregulation was abolished when ammonium was also present. This nitrogen metabolite repression sensitivity of URO1 (* denotes P < 0.05) and DAL1 (** denotes P < 0.01) was statistically significant. Error bars represent standard errors across three biological replicates.

Figure 2.—

Nitrogen metabolite repression influences capsule and melanin formation. (A) India ink cell staining under light microscopy showed that wild-type H99 produces capsules that vary in size when grown on YNB supplemented with different nitrogen sources (10 mm each). Capsule size increased in the following order: (ammonium, glutamine, proline, alanine), asparagine, urea, uric acid, and creatinine. Upon coculture of each of these nitrogen sources with ammonium, capsule size was restored to that of the ammonium control. Scale bar, 10 µm. (B) Wild-type H99 produces varying amounts of melanin when grown on l-DOPA supplemented with different nitrogen sources (10 mm each). Melanization increased in the following order: (creatinine, uric acid), alanine, proline and (glutamine, asparagine, urea, ammonium). Upon coculture of each of these nitrogen sources with ammonium, melanin production was restored to that of the ammonium control, with the exception of the proline-grown cells, which melanized to the same extent with or without ammonium.

Figure 3.—

The gat1∆ mutant is unable to utilize a wide variety of nitrogen sources. Tenfold spot dilution assays of wild-type H99 and GATA-type deletion mutants for nitrogen utilization showed that the gat1∆ mutant is unable to grow on YNB supplemented with 10 mm ammonium, uric acid, urea, or creatinine but exhibits only a slight growth defect compared to wild type on 10 mm proline. Complementation of the gat1∆ mutant with the GAT1 gene restored wild-type nitrogen utilization phenotype (Figure S3).

Figure 4.—

Gat1 regulates nitrogen metabolite repression. (A) Scheme representing the predicted proline degradation pathway of C. neoformans. (B) cDNA from wild-type H99 and gat1∆ mutant grown in YNB supplemented with proline or proline plus ammonium (10 mm each nitrogen source) were amplified via qRT-PCR using primers against PUT1 (proline oxidase), PUT5 (proline oxidase), PUT2 (pyrroline-5-carboxylate dehydrogenase), and the control gene ACT1 (actin). One of the predicted catabolic enzyme-encoding genes of proline, PUT1, was sensitive to nitrogen metabolite repression in wild type (*** denotes P < 0.0001) but not in the gat1∆ mutant. The remaining two catabolic genes, PUT5 and PUT2, were nitrogen metabolite repression insensitive in both strains. Error bars represent standard errors across three biological replicates.

Figure 5.—

Gat1 regulates the expression of the ammonium assimilation enzyme and permease-encoding genes. (A) Scheme representing the predicted pathway of central nitrogen metabolism in C. neoformans. (B) cDNA from wild-type H99 and gat1∆ mutant grown in YPD were amplified via qRT-PCR using primers against GDH1 (NADPH-GDH), GLN1 (GS), GLT1 (GOGAT), GDH2 (NAD-GDH), AMT1 (ammonium transporter 1), AMT2 (ammonium transporter 2), and the control gene ACT1 (actin). The expression of GDH1, AMT1, and AMT2 was significantly lower in the gat1∆ mutant compared to wild type, while that of GLN1, GLT1, and GDH2 was slightly lower. This difference in level of expression for GDH1 (*** denotes P < 0.0001), AMT1 (** denotes P < 0.01), and AMT2 (* denotes P < 0.05) was statistically significant. Error bars represent standard errors across three biological replicates.

Figure 6.—

Gat1 inhibits filament formation during mating. Filamentation assays on V8 and MS mating media (pH 5.0) showed that filament formation was enhanced in unilateral gat1Δ crosses in comparison to the wild-type crosses. Filamentation was enhanced even further in a bilateral gat1Δ cross. Complementation of the H99α gat1Δ and KN99a gat1Δ mutants with the GAT1 gene restored wild-type mating in all tested crosses (data not shown).

Figure 7.—

Gat1 is required for capsule synthesis, but negatively regulates melanization and growth at 39° or 40°. (A) India ink cell staining under light microscopy revealed that the wild-type H99 and gat1Δ + GAT1 strains produce enlarged capsules while the gat1∆ mutant produces residual amount of capsule when strains were cultured under serum-induced growth conditions. Scale bar, 10 µm. (B) The gat1∆ mutant produces more melanin compared to both the wild-type and gat1∆ + GAT1 strains when grown on l-DOPA medium supplemented with 10 mm proline at 37°. In contrast, all three strains melanized to the same extent when grown at 30°. (C) Unlike the negative control ure1∆ mutant, the wild-type, gat1∆ mutant and gat1∆ + GAT1 strains all had the ability to produce urease when grown on Christensen’s urea agar as reflected by the bright pink clearing surrounding the aliquot of spotted cells. (D) Tenfold spot dilution assays on YPD medium at human body temperature showed that the gat1∆ mutant exhibits enhanced growth compared to both the wild-type and gat1∆ + GAT1 strains at 39° and 40°.

Figure 8.—

The gat1Δ mutant kills C. elegans as efficiently as wild-type H99, but exhibits modestly enhanced virulence in a murine host. (A) C. elegans infection: ∼50 nematode worms were transferred to a lawn of wild-type, gat1Δ, or gat1Δ + GAT1 cells as the sole food source on both BHI and 2.5% pigeon guano media, and survival was monitored at 24-hr intervals. There was no observable difference in C. elegans killing by all three strains on both BHI (wild type vs. gat1Δ, P = 0.1066; gat1Δ vs. gat1Δ + GAT1, P = 0.9230) and pigeon guano media (wild type vs. gat1Δ, P = 0.0250; gat1Δ vs.gat1Δ + GAT1, P = 0.3614). (B) Mus musculus infection: 10 mice were infected intranasally with either 1 × 10⁵ cells of wild type, gat1Δ, or gat1Δ + GAT1 strains, and survival was monitored daily. Mice infected with the wild-type and gat1Δ + GAT1 strains progress to morbidity at the same rate (wild type vs. gat1Δ + GAT1, P = 0.6691), whereas mice infected with the gat1Δ strain progress to morbidity more rapidly (wild type vs. gat1Δ, P = 0.0151).