## 3 Results
### Glycerol-3-Phosphate Metabolic Flow Is Altered in Calcineurin and gpp2 Mutants
Most chitin deacetylases and chitinases were also induced or unaffected in all strains, except in _gpp_2\(\Delta\), where two of them were repressed (Figure 6A). All of the remaining DEGs were induced in the calcineurin-deficient and _gpp_2\(\Delta\) strains and repressed  
Figure 5: Gene expression of genes in the glycerol biosynthetic pathway (GO:0006072). (**A**) Genes, proteins, and metabolic intermediates involved in glycerol biosynthesis; (**B**) heat map of the DEGs involved in glycerol biosynthesis in the wild type and mutants; (**C**) glycerol biosynthesis gene codes in _C. neoformans_ and _S. cerevisiae_ and fold changes in expression in the wild type and mutants. or unaffected in the wild type, especially gluconeogenesis, glycogen mobilization, glycosyl hydrolases, pentose phosphate pathway, nucleotide sugar metabolism, and sphingolipid and sterol metabolism (Figure 6A). The induction of these genes suggests that the energetic mobilization of reserve carbohydrates is more pronounced in mutants than in the wild-type strain under cationic stress, which is in agreement with the metabolic flow channeling G3P and DHAP to gluconeogenesis and glycerolipid biosynthesis, as shown in Figure 5.  
Figure 6: _Cont._  
Translation (GO:006412) is a highly repressed category in the \(cnb1\Delta\), \(cna1\Delta\), and \(gpp2\Delta\) strains compared to the wild type. Sixty-four DEGs grouped in this GO category encode ribosomal proteins, which are highly repressed in the mutants (Figure 6B). Seventy genes encoding mostly large and small subunit ribosomal proteins were unaffected in the wild  
Figure 6: (**A**) A heat map of DEGs involved in the carbohydrate metabolic process (GO:0005975) in the wild type and mutants exposed to cationic stress; (**B**) a heat map of DEGs involved in translation (GO:0006412) in the wild type and mutants exposed to cationic stress; (**C**,**D**) a heat map of DEGs involved in transmembrane transport (GO:0055085) in the wild type and mutants exposed to cationic stress. type and highly repressed in the mutants, as shown. This result is in agreement with the low growth observed in the mutants compared to the wild type in 1 M NaCl (Figure 1A), suggesting that the inability to achieve osmobalance by glycerol accumulation is a very deleterious event that leads to slow growth or even growth arrest. At last, the GO biological process related to transmembrane transporters was underlined in the wild-type and calcineurin mutant strains; in the wild-type strain, more genes encoding transporters were repressed than induced, but in the \(cnb1\Delta\) and \(cna1\Delta\) mutants, 50 transporters were induced. The largest groups of DEGs are amino acids and sugar transporters (Figure 6C). Sugar transporters were mostly repressed or unaffected in the wild-type and \(gpp2\Delta\) strains but induced or unaffected in the calcineurin mutants. Also, the ATP-binding cassette category (ABC transporters) was mostly repressed in the wild type and induced in the mutants. These results are consistent with our previous observations that permease genes are induced in the \(gpp2\Delta\) mutant. The phenotypic data show that amino acids, such as proline, can rescue the sensitive phenotype of the \(gpp2\Delta\)[21]. Oxidative Stress Response and Glutathione Metabolism Are Induced in \(cna1\Delta\), \(cnb1\Delta\), and \(gpp2\Delta\) Mutant Strains  
Transcriptome analysis revealed that oxidative stress (GO:006979) and glutathione metabolism (GO:0006749) are induced in all three mutant strains (\(cna1\Delta\), \(cnb1\Delta\), and \(gpp2\Delta\)) compared to the wild type (Figure 7A) during cationic stress. This observation implies that the inability to cope with cationic stress must cause reactive oxygen species to accumulate, requiring a response, which leads to the induction of 11 genes related to oxidative stress: 4 catalase genes and 5 genes related to glutathione metabolism (Figure 7A). In agreement with this result, we asked whether glutathione could also affect the growth rate in the presence of cationic stress.