Abstract
Cryptococcus neoformans is an opportunist fungal pathogen that causes meningoencephalitis in immunocompromised patients. During infection, this basidiomycete yeast has to adapt to several adverse conditions, especially nutrient availability. The interruption on various amino acid biosynthetic pathways and on amino acid uptake causes reduced viability, inability to cope with various stresses, failure in virulence factors expression and avirulence in animal model of infection. The sulfur amino acid biosynthesis and uptake is an important feature for pathogen survival in vivo and in vitro. Our previous work demonstrates that C. neoformans Cys3 BZip transcription factor controls the gene expression in several steps of the sulfur assimilation and sulfur amino acid biosynthesis. Also, we have shown that Gpp2 phosphatase modulates Cys3 activity. In Saccharomyces cerevisiae Gpp2 is induced in response to hyper osmotic or oxidative stress and during diauxic shift. In this work, we will show that, in C. neoformans, Gpp2 is required to respond to stresses, mainly osmotic stress; also its transcription is induced during exposure to NaCl. Global transcriptional profile of gpp2Δ by RNAseq shows that CYS3 and other genes in the sulfur assimilation pathway are up regulated, which is consistent with our previous report, in which Gpp2 acts by avoiding Cys3 accumulation and nuclear localization. In addition, several transporters genes, especially amino acid permeases and oxidative stress genes are induced in the gpp2Δ strain; on the contrary, genes involved in glucose and tricarboxylic acid metabolism are down regulated. gpp2Δ strain fails to express virulence factors, as melanin, phospholipase, urease and has virulence attenuation in Galleria mellonella. Our data suggest that Gpp2 is an important factor for general pathogen adaptation to various stresses and also to the host, and perhaps it could be an interesting target for therapeutic use.
Highlights
Fungal pathogens can cause severe systemic mycoses which are difficult to treat (Garcia-Vidal et al, 2013)
Cryptococcus neoformans CNAG_01744 was identified in our earlier research by immune precipitation as part of a protein complex with the major sulfur amino acid biosynthesis transcription regulator Cys3 and the calcineurin catalytic and regulatory subunits Cna1 and Cnb1, respectively. We named this gene as GPP2 based on the amino acid sequence similarity to S. cerevisiae GPP2, a glycerol-3-phosphate phosphatase (Pahlman et al, 2001). This gene was found in C. neoformans by phosphoproteomics approach as substrate for the calcineurin complex (Park et al, 2016) and later it was named as HAD1 (Halo Acid Dehalogenase hydrolase) due to the predicted molecular function (Jung et al, 2018)
We have explored several phenotypes of C. neoformans survival and virulence associated to the deletion of the glycerol-3-phospate phosphatase, which is responsible to carry the last step in the biosynthesis of glycerol, a major cellular osmolyte that helps the cells to counter act the effects of osmotic and cold shock (Norbeck et al, 1996)
Summary
Fungal pathogens can cause severe systemic mycoses which are difficult to treat (Garcia-Vidal et al, 2013). C. neoformans GPP2 has been considered a target of the mitogen-activated protein kinase Hog; it is more than two fold induced in wild type in response to osmotic shock and is repressed in a hog strain, suggesting that this gene is a homolog of S. cerevisiae glycerol-3-phosphate phosphatase (Ko et al, 2009). These data indicate that GPP2 may be regulated by Hog and calcineurin. In connection with the virulence data this idea underlines the potential of the sulfur network for therapeutic use in C. neoformans
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