The GATA-type transcriptional activator Gat1 regulates nitrogen uptake and metabolism in the human pathogen Cryptococcus neoformans

Abstract

Nitrogen uptake and metabolism are essential to microbial growth. Gat1 belongs to a conserved family of zinc finger containing transcriptional regulators known as GATA-factors. These factors activate the transcription of Nitrogen Catabolite Repression (NCR) sensitive genes when preferred nitrogen sources are absent or limiting. Cryptococcus neoformans GAT1 is an ortholog to the Aspergillus nidulans AreA and Candida albicans GAT1 genes. In an attempt to define the function of this transcriptional regulator in C. neoformans, we generated null mutants (gat1Δ) of this gene. The gat1 mutant exhibited impaired growth on all amino acids tested as sole nitrogen sources, with the exception of arginine and proline. Furthermore, the gat1 mutant did not display resistance to rapamycin, an immunosuppressant drug that transiently mimics a low-quality nitrogen source. Gat1 is not required for C. neoformans survival during macrophage infection or for virulence in a mouse model of cryptococcosis. Microarray analysis allowed the identification of target genes that are regulated by Gat1 in the presence of proline, a poor and non-repressing nitrogen source. Genes involved in ergosterol biosynthesis, iron uptake, cell wall organization and capsule biosynthesis, in addition to NCR-sensitive genes, are Gat1-regulated in C. neoformans.

Nitrogen Metabolite Repression of Metabolism and Virulence in the Human Fungal Pathogen Cryptococcus neoformans

“…However, all but one of the C. neoformans GATA mutant strains exhibited growth equivalent to wild-type H99 on YNB defined medium supplemented with a wide panel of 38 usable nitrogen sources ( Figure 3 and Table S5). Consistent with previous studies conducted by Kmetzsch et al (2010), deletion of ORF CNAG00193.2 (GAT1) rendered C. neoformans incapable of utilizing a wide repertoire of nitrogen sources. The gat1D mutant, however, could still utilize a limited selection of amino acids, notably proline, strengthening our hypothesis that proline utilization is largely independent of nitrogen metabolite repression.…”

“…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. Kmetzsch et al 2010). Furthermore, sequence searches of these candidates in Pfam confirmed the presence of a predicted GATA zinc finger, supporting the hypothesis that all seven are bona fide GATA factors.…”

“…While the slightly quicker progression to morbidity in mice infected with the gat1/are1D mutant would not be considered great enough to classify this strain as "hypervirulent," it certainly highlights the complexity of the role of Gat1/Are1 in gene regulation of C. neoformans during infection. We note that Kmetzsch et al (2010) recently reported that the gat1/are1D mutant exhibited equivalent virulence to wild type when high doses (1 · 10 7 cells per strain) were used, which resulted in mice succumbing to infection as early as the third day postinfection. In contrast, we employed the more traditional inoculum of 1 · 10 5 cells to ensure the gradual progression of disease.…”

“…Cryptococcus assimilates a limited subset of nitrogen sources: Work on Cryptococcus has demonstrated the ability of this organism to assimilate a number of different nitrogen sources (Chaskes and Tyndall 1975;Staib et al 1976;Polacheck and Kwon-Chung 1980;Fiskin et al 1990;Kmetzsch et al 2010). However, these studies have not taken into consideration the significant advances that have been made in understanding the population structure of the species: the former variety Cryptococcus gattii has now been classified as a separate species, and eight distinct Cryptococcus haploid molecular types have been identified, each of which displays distinct physiological properties and virulence features (Kwon-Chung and Varma 2006;Litvintseva et al 2006).…”

The Ustilago maydis Nit2 Homolog Regulates Nitrogen Utilization and Is Required for Efficient Induction of Filamentous Growth

“…It is known that nitrate-metabolizing enzymes are transcriptionally repressed by ammonium in Hebeloma cylindrosporum and Ustilago maydis, but the mechanism behind this effect is unknown (4,38,39,51). Gat1, the Nit2 homolog of the basidiomycete human pathogen Cryptococcus neoformans, regulates nitrogen utilization and glucuronoxylomannan secretion, but the deletion of Gat1 has no effect on virulence (48).…”