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Sci Rep
2019 Aug 15;91:11923. doi: 10.1038/s41598-019-48433-5.
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The regulation of the sulfur amino acid biosynthetic pathway in Cryptococcus neoformans: the relationship of Cys3, Calcineurin, and Gpp2 phosphatases.
de Melo AT
,
Martho KF
,
Roberto TN
,
Nishiduka ES
,
Machado J
,
Brustolini OJB
,
Tashima AK
,
Vasconcelos AT
,
Vallim MA
,
Pascon RC
.
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Cryptococcosis is a fungal disease caused by C. neoformans. To adapt and survive in diverse ecological niches, including the animal host, this opportunistic pathogen relies on its ability to uptake nutrients, such as carbon, nitrogen, iron, phosphate, sulfur, and amino acids. Genetic circuits play a role in the response to environmental changes, modulating gene expression and adjusting the microbial metabolism to the nutrients available for the best energy usage and survival. We studied the sulfur amino acid biosynthesis and its implications on C. neoformans biology and virulence. CNAG_04798 encodes a BZip protein and was annotated as CYS3, which has been considered an essential gene. However, we demonstrated that CYS3 is not essential, in fact, its knockout led to sulfur amino acids auxotroph. Western blots and fluorescence microscopy indicated that GFP-Cys3, which is expressed from a constitutive promoter, localizes to the nucleus in rich medium (YEPD); the addition of methionine and cysteine as sole nitrogen source (SD-N + Met/Cys) led to reduced nuclear localization and protein degradation. By proteomics, we identified and confirmed physical interaction among Gpp2, Cna1, Cnb1 and GFP-Cys3. Deletion of the calcineurin and GPP2 genes in a GFP-Cys3 background demonstrated that calcineurin is required to maintain Cys3 high protein levels in YEPD and that deletion of GPP2 causes GFP-Cys3 to persist in the presence of sulfur amino acids. Global transcriptional profile of mutant and wild type by RNAseq revealed that Cys3 controls all branches of the sulfur amino acid biosynthesis, and sulfur starvation leads to induction of several amino acid biosynthetic routes. In addition, we found that Cys3 is required for virulence in Galleria mellonella animal model.
Figure 1. Sulfur uptake and sulfur amino acid biosynthesis pathway proposed for C. neoformans. Based on S. cerevisiae and N. crassa pathways32.
Figure 2. (a) Growth pattern by spot dilution of wild type (H99), cys3Δ mutants (CNU123 and CNU124), and complemented strain (CNU136) in YEPD and SD (synthetic dextrose) without sulfur amino acids (SAA), with 20 mM of cysteine (Cys) and 20 mM of methionine (Met) in two different temperatures (30 and 37 °C). The last rows show the strains in YEPG (rich medium added with galactose) and 20 mM methionine and synthetic galactose (SG) with proline as nitrogen source; (b) growth pattern evaluated by optical density at 600 ηm (OD600) of wild type (H99) and cys3Δ mutant (CNU123) in YEPD supplemented with 25 mM homocysteine for 120 h at 30 °C. Asterisks represent values that are statistically significant between wild type and mutant at a given time points (*p < 0.05; **p < 0.01, and ***p < 0.001).
Figure 6. Yeast two-hybrid assay. Images depict representative colonies of S. cerevisiae expressing pairs of bait and prey fusion proteins, which are described at the left side of the panel. Each column represents a plate composition; DDO = double drop out (minus tryptophan and leucine), where no reporter gene is activated; QDO = quadruple drop out (minus tryptophan, leucine, histidine, and adenine), where two reporters are activated; and QDO/X/A = quadruple drop out with aurobasidin and X-α-Gal, where all four reporters are active. + represents the positive and − the negative control provided by Match Maker kit (Clontech).
Figure 8. Model of the sulfur amino acids biosynthesis regulation in C. neoformans.
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