Functionality and prevalence of trehalose-based oligosaccharides as novel compatible solutes in ascospores of Neosartorya fischeri (Aspergillus fischeri) and other fungi

Abstract

Ascospores of Neosartorya, Byssochlamys and Talaromyces can be regarded as the most stress-resistant eukaryotic cells. They can survive exposure at temperatures as high as 85°C for 100 min or more. Neosartorya fischeri ascospores are more viscous and more resistant to the combined stress of heat and desiccation than the ascospores of Talaromyces macrosporus which contain predominantly trehalose. These ascospores contain trehalose-based oligosaccharides (TOS) that are novel compatible solutes, which are accumulated to high levels. These compounds are also found in other members of the genus Neosartorya and in some other genera within the order Eurotiales that also include Byssochlamys and Talaromyces. The presence of oligosaccharides was observed in species that had a relatively high growth temperature. TOS glasses have a higher glass transition temperature (Tg ) than trehalose, and they form a stable glass with crystallizing molecules, such as mannitol. Our data indicate that TOS are important for prolonged stabilization of cells against stress. The possible unique role of these solutes in protection against dry heat conditions is discussed.

Figure 1

Germination (% of total) of N. fischeri and T. macrosporus ascospores after heating for 0–30 min at 85°C in ACES buffer (A) or after drying and storage for 1 week at 22–25°C at a RH of 45–85% (humid) or 0.5–2% (dry), a 1 h exposure at 25°C, 60°C, 70°C or 80°C in the absence of water, and a heat activation for 2 min at 85°C in ACES buffer (B).

Figure 2

The intensity of the ESR spectra obtained from ascospores of N. fischeri (A, B) and T. macrosporus (C, D) that were labelled with the spin probe TEMPONE. The spectra are composed of a signal originating from the cell wall and the medium (A, C) and an intracellular signal. The latter is calculated by subtracting the signal of the cell wall and the medium from the total signal (B, D). Supernatant is the extracellular solution in which the spores are suspended and composed of demi water, TEMPONE and FC (ferricyanide).

Figure 3

HPLC profiles of cell-free extracts of N. fischeri (A) and T. macrosporus (B) ascospores that had been isolated from 40-day-cultures. Besides trehalose and mannitol, the N. fischeri extract has peaks with shorter retention times than trehalose (C) TLC of N. fischeri ascospore extract (NF) and a sugar standard (ST) consisting of glucose (1), trehalose (2), raffinose (3), stachyose (4) and verbascose (5). Neosartorya fischeri spores contain oligosaccharides of different size: a disaccharide, trisaccharide, tetrasaccharide and pentasaccharide.

Figure 4

Compatible solutes in N. fischeri (non-shaded bars) and T. macrosporus (grey-shaded bars) ascospores expressed as pg spore⁻¹ (A) and mM (B).

Figure 5

Linear regression of the wavenumber of the OH stretching band as function of the temperature of the samples consisting of mannitol (A), trehalose (B), sucrose (C), isobemisiose (D), neosartose (E), fischerose (F), trehalose/mannitol (G), mannitol/trehalose/isobemisiose/neosartose/fischerose (H) and mannitol/sucrose/raffinose/stachyose/verbascose (I). No regression line could be determined in the mannitol sample due to crystallization (A). Mannitol crystallization was also observed in the trehalose/mannitol mixture (G). The intersection of the regression lines represents the glass transition temperature with T_g1 and T_g2 determined from the 1st and 2nd scan respectively. The steepness of the regression line corresponds with the WTC value (cm⁻¹ °C⁻¹).

Figure 6

Absorbance spectrum of mannitol (A), fischerose (B) and trehalose + mannitol (C, D) as measured by FTIR. The spectra of A, B and D are from the 2nd scan (after heating and re-cooling), while C is of the 1st scan. The change of peak shape of the trehalose + mannitol sample (C, D) is due to crystallization. The mannitol sample (A), but not fischerose (B), also shows crystallization.

Figure 7

Phylogenetic tree of the family Trichocomaceae indicating the presence or absence of trehalose and TOS in the ascospores. The presence of oligosaccharides is indicated in the boxes (labelled with di-, tri-, tetra- and penta- for corresponding oligosaccharides) at the right side of the Figure. (Oligo)saccharides were measured by TLC and the grey scale indicates the intensity of staining of the TLC band.