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Front Microbiol
2019 Jan 01;10:2728. doi: 10.3389/fmicb.2019.02728.
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The Glycerol Phosphatase Gpp2: A Link to Osmotic Stress, Sulfur Assimilation and Virulence in Cryptococcus neoformans.
Martho KF
,
Brustolini OJB
,
Vasconcelos AT
,
Vallim MA
,
Pascon RC
.
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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.
FIGURE 1. Growth pattern of the gpp2Δ mutant (CNU125 and CNU 126) compared to wild type (H99) and reconstituted strain (CNU135) in (A) YEPD and three different temperatures (22, 30, and 37°C); (B) the same strains on Synthetic dextrose (SD) supplemented with ammonium sulfate and amino acids (tryptophan, histidine, and methionine), SD with ammonium sulfate only and SD supplemented with 10 mM of proline as the sole nitrogen source at 30°C. Spot dilutions are 1 × 105 to 1 cell at the last spot.
FIGURE 2. Growth rate of gpp2Δ mutant (CNU125 and CNU 126) compared to wild type (H99) and reconstituted strain (CNU135) in (A) YEPD, YEPD supplemented with 0.03% of SDS and YEPD supplemented with 0.5% of Congo red. (B) Growth rate of the same strains in different pHs (6.4, 7.0, and 8.0) at 30 and 37°C and (C) growth rate of the same strains in the presence of osmotic stress (0.75 and 1 M of NaCl and KCl) in YEPD and SD supplemented with the preferred nitrogen source; (D) growth rate of gpp2Δ mutants, wild type and reconstituted strain in SD, SD supplemented with 0.5 M NaCl and SD plus 0.5 M NaCl and 20 mM of proline (left panel), the same strains in YEPD, YEPD supplemented with 0 M NaCl and YEPD plus 0.5 M NaCl and glycerol (right panel); (E) effect of the temperature (22°C) on wild type, reconstituted and mutant strains in YEPD with and without glycerol. Spot dilutions are 1 × 105 to 1 cell at the last spot. (F) qPCR showing the induction of GPP2 transcript in the presence of 1 M NaCl relative to the control YEPD in wild type strain H99. Statistical significant differences were tested by Two-way ANOVA, ∗∗∗p < 0.001.
FIGURE 3. Virulence factor evaluation of the gpp2 mutant compared to wild type and reconstituted strains. (A) urease, (B) phospholipase, (C) melanin, (D) capsule, and (E) invertebrate animal model in G. mellonella.
FIGURE 4. Graphs represent the DEGs found to be up (A,B) and down (C,D) regulated according to GO categories based on biological processes (A up regulated and C down regulated) and molecular function (B up regulated and D down regulated). The cutoff used was 1.0 Log2. (E) Graph shows CYS3 gene quantification by Real time qPCR in gpp2Δ strain relative to wild type. RNA was extracted from cultures growing in YEPD for 2 h at 30°C.
FIGURE 5. Expression of permease genes in the gpp2Δ mutant relative to wild type in (A) YEPD and (B) SD during nitrogen starvation by qPCR. Dotted line represents the expression level of the reference strain (wild type). Statistical significant differences were tested by Two-way ANOVA, ∗∗∗∗p < 0.0001 and ∗∗∗p < 0.001. (C) Growth of wild type, reconstituted (CNU135) and mutant strains (CNU125 and CNU 126) in YEPD without and with 0.1 g/L of eugenol (left panel) and the same strains in SD plus proline without and with 0.1 g/L eugenol (right panel).
FIGURE 6. Expression pattern of CYS3
(A) and SUL1
(B) in wild type strain under osmotic stress by qPCR. YEPD was supplemented with 1 M of NaCl. Statistical significant differences were tested by Two-way ANOVA, ∗∗∗∗p < 0.0001 and ∗p < 0.05.
FIGURE 7. Model representing the main findings in this work. AA = amino acids; transmembrane drawing = amino acid permeases; Cna1 and Cnb1 = calcineurin catalytic and regulatory subunits respectively; GSH = glutathione.
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