Osmolyte Signatures for the Protection of Aspergillus sydowii Cells under Halophilic Conditions and Osmotic Shock

Abstract

Aspergillus sydowii is a moderate halophile fungus extensively studied for its biotechnological potential and halophile responses, which has also been reported as a coral reef pathogen. In a recent publication, the transcriptomic analysis of this fungus, when growing on wheat straw, showed that genes related to cell wall modification and cation transporters were upregulated under hypersaline conditions but not under 0.5 M NaCl, the optimal salinity for growth in this strain. This led us to study osmolyte accumulation as a mechanism to withstand moderate salinity. In this work, we show that A. sydowii accumulates trehalose, arabitol, mannitol, and glycerol with different temporal dynamics, which depend on whether the fungus is exposed to hypo- or hyperosmotic stress. The transcripts coding for enzymes responsible for polyalcohol synthesis were regulated in a stress-dependent manner. Interestingly, A. sydowii contains three homologs (Hog1, Hog2 and MpkC) of the Hog1 MAPK, the master regulator of hyperosmotic stress response in S. cerevisiae and other fungi. We show a differential regulation of these MAPKs under different salinity conditions, including sustained basal Hog1/Hog2 phosphorylation levels in the absence of NaCl or in the presence of 2.0 M NaCl, in contrast to what is observed in S. cerevisiae. These findings indicate that halophilic fungi such as A. sydowii utilize different osmoadaptation mechanisms to hypersaline conditions.

Keywords: Aspergillus; HOG; extremophile; halophile; osmolyte; osmotic shock.

Figure 1

Growth rate of A. sydowii (A) and osmolyte accumulation (B) in optimal, hypo- and hyper osmotic conditions. Data are means ± SD calculated from three independent experiments (n = 3). Statistical analyses are detailed in Supplementary Table S3.

Figure 2

Pathways for the synthesis of metabolites with possible osmolyte functions in response to salinity. The pathway has been modified from the Aspergillus nidulans KEGG pathway. Enzymes involved in the regulation of osmolyte concentration and depicted with the color grey: trehalose pathway; green: mannitol; blue: glycerol; purple: erythritol, and red: arabitol were evaluated by qPCR. The enzymes depicted in light orange were not evaluated. TPSA (Trehalose phosphate synthase), STPS (Heat shock trehalose phosphate synthase), CCG-9 (Trehalose phosphate synthase), TPP (Trehalose-6-P phosphatase), MTLD (Mannitol-1-phosphate 5-dehydrogenase), M2DH (Mannitol 2-dehydrogenase), MPP (Manitol/Hexitol phosphatase), HK (Hexokinase), GPI (Phospho-glucose isomerase), PFK (Phospho-fructokinase), ALD (Aldolase), GPD (Glycerol-3-P dehydrogenase), GPP (Glycerol-3-P phosphatase), GUT1 (Glycerol-kinase), HAD1(Halo-acid dehalogenase), DAK1 (Dihydroxyacetone kinase) GLD1 (Glycerol dehydrogenase). TKT (Transketolase), TAD (Trans-aldolase), LAR (L-arabinose), ARDH (L-arabinitol dehydrogenase), D-XK (D-Xylose Kinase), ER (Erythrose reductase).

Figure 3

Expression analysis of transcripts related to osmolytes synthesis of A. sydowii in different salinities after 7 d of culture. The values correspond to the average and standard deviations of three biological replicates (n = 3) and two technical qPCR replicates. Analyzed genes were grouped according to the pathway: (A) Trehalose synthesis, (B) Mannitol synthesis, (C) Pentose phosphate pathway (for arabitol and erythritol synthesis) and (D) Glycerol synthesis. Statistical significance (*) was assessed by a randomization test performed with the software Rest [46]. The dashed vertical lines correspond to a cutoff logFC = 2.

Figure 4

Accumulation of compatible solutes in A. sydowii after different osmotic shocks. The labels on the right indicate the culture conditions before and after the osmotic shock. The data represent the average and standard deviation of at least three replicates (n = 3). Statistical analyses of the data are summarized in Supplementary Table S5.

Figure 5

Transcriptional regulation of enzyme genes involved in the synthesis of osmolytes after hyperosmotic shock, hypoosmotic shock, and stress-inducing shock. The values correspond to the average and standard deviations of three biological replicates (n = 3) and two technical qPCR replicates. The statistical significance (*) was assessed by a randomization test performed with the software Rest [46]. The dashed horizontal lines correspond to a cutoff logFC = ±2.

Figure 6

Hog MAPK system in A. sydowii. (A) Reconstruction of MAPK phylogeny in selected Aspergilli, including Hog1, Hog2, and MpkC genes of A. sydowii. A more extensive phylogenetic tree can be observed in Supplementary Figure S3. (B) Hog gene homologs on A. sydowii showing protein size, molecular weight, conserved phosphorylation motifs, protein kinase (PK) domain, and the region corresponding to Y-215 and D3F9 recognized by the antibodies used to detect phosphorylated and total Hog, respectively, by Western blot. (C) Relative expression of Hog1 and Hog2 transcripts in salt-adapted A. sydowii growing without NaCl, 0.5 M or 2.0 M NaCl, representing the average and standard deviations of three biological replicates (n = 3) and two technical qPCR replicates. (D) Diagram of the shock conditions used to test the phosphorylation dynamics of Hog1 and Hog2 (E) Relative expression of Hog1 and Hog2 transcripts after osmotic shock in A. sydowii, representing the average and standard deviations of three biological replicates (n = 3) and two technical qPCR replicates. The statistical significance (*) was assessed by a randomization test performed with the software Rest [46]. The dashed horizontal lines correspond to a cutoff logFC = ±2. (F) Phosphorylation of Hog MAPK homologs after different osmotic shocks. Extracts from S. cerevisiae cultures shifted from a medium without NaCl to a medium with 1.0 M NaCl were used as positive controls for Hog1 phosphorylation.

Figure 7

Regulation of intracellular K⁺/Na⁺ ratio in response to salinity in A. sydowii cells. Dh: Debaryomyces hansenii was used as a positive control. Data are means ± SD. Statistical significance was determined using the Holm-Sidak method, with alpha = 0.05. Each row was analyzed individually, without assuming a consistent SD. (* p < 0.05, ** p < 0.01, **** p< 0.0001).

Figure 8

Oxidative stress and antioxidant markers evaluated in A. sydowii cultures under stable salinity conditions. Superoxide dismutase (SOD) and glutathione (GSH) (antioxidants markers; green in the heat maps), malondialdehyde (MDA) and protein advance oxidation products (PAOP) (oxidative damage markers; brown in the heat maps) were analyzed. Data are means ± SD. Bars with the asterisks (*) indicate the significant difference (** p < 0.01, * p < 0.05) between the control and its respective treated samples. Analyzed by a two-way ANOVA with Tukey’s multiple comparisons test.

Figure 9

Oxidative stress and antioxidant markers evaluated in A. sydowii after osmotic shock. Superoxide dismutase (SOD) and glutathione (GSH) (antioxidants markers, green in the heat maps), malondialdehyde (MDA) and protein advance oxidation products (PAOP) (oxidative damage markers, red in the heat maps) were analyzed. Columns represents the average of at least three replicates, and bars represent the standard deviation. Details of statistical differences are shown in Supplementary Table S7.