The regulation of the sulfur amino acid biosynthetic pathway in Cryptococcus neoformans: the relationship of Cys3, Calcineurin, and Gpp2 phosphatases

Abstract

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.

The Glycerol Phosphatase Gpp2: A Link to Osmotic Stress, Sulfur Assimilation and Virulence in Cryptococcus neoformans

“…Calcineurin has been linked to osmotic stress and cell wall integrity response in C. neoformans (Kraus and Heitman, 2003; Fan et al, 2007). Also, the relationship between calcineurin complex and GPP2 identified in our previous work and others reinforces the idea that this phosphatase complex plays an important role on osmobalance (Park et al, 2016; Jung et al, 2018; de Melo et al, 2019).…”

“…Interruptions on amino acid biosynthesis or failure to uptake amino acids by permeases lead to growth arrest, defects in virulence factor and decreased virulence in mouse and Galleria mellonella animal model of infection (Yang et al, 2002; Pascon et al, 2004; Fernandes et al, 2015; Martho et al, 2016; Calvete et al, 2019). Recently, we showed the regulation of sulfur amino acid biosynthesis is essential for C. neoformans survival in vivo and in vitro (de Melo et al, 2019). Cys3 transcription factor, the major regulator of the sulfur uptake network, is part of a protein complex and physically interacts with calcineurin and Gpp2 phosphatase.…”

“…Calcineurin regulatory and catalytic subunits are important in order to maintain high levels of Cys3 in the nucleus and Gpp2 seems to be important to send Cys3 to degradation. Deletion of GPP2 gene causes abnormal accumulation of Cys3 protein (de Melo et al, 2019).…”

BoaγPLI: Structural and functional characterization of the gamma phospholipase A2 plasma inhibitor from the non-venomous Brazilian snake Boa constrictor

“…Peptides were eluted on a capillary analytical column (C18 nanoAcquity BEH 75 μm x 150 mm, 1.7 μm column) using a gradient of 93% A (0.1% formic acid) and 7% B (99.9% ACN 0.1% formic acid) to 35% B over 30 min in a flow of 275 nl / min. Data were acquired in MS E mode [32,33] in duplicate. Protein identification, PTM and homology searches were performed in PEAKS Studio 7.5 software (Bioinformatics Solutions Inc.) by MS/MS search against the Serpentes databases obtained from Uniprot (2844 sequences, downloaded in October 25 th , 2018).…”

Involvement of Sulfur in the Biosynthesis of Essential Metabolites in Pathogenic Fungi of Animals, Particularly Aspergillus spp.: Molecular and Therapeutic Implications

“…Similarly to MET3 and MET6, MET2 is also necessary for Met biosynthesis in C. neoformans and disruption of this gene leads to Met auxotrophy and avirulence (Nazi et al, 2007). Further, it has been revealed that CYS3 encodes a transcriptional activator that upon deletion causes a Met/Cys auxotrophy and that Cys3 is the major regulator of inorganic sulfur uptake in C. neoformans (de Melo et al, 2019).…”