Concomitant osmotic and chaotropicity-induced stresses in Aspergillus wentii: compatible solutes determine the biotic window

Abstract

Whereas osmotic stress response induced by solutes has been well-characterized in fungi, less is known about the other activities of environmentally ubiquitous substances. The latest methodologies to define, identify and quantify chaotropicity, i.e. substance-induced destabilization of macromolecular systems, now enable new insights into microbial stress biology (Cray et al. in Curr Opin Biotechnol 33:228-259, 2015a, doi: 10.1016/j.copbio.2015.02.010 ; Ball and Hallsworth in Phys Chem Chem Phys 17:8297-8305, 2015, doi: 10.1039/C4CP04564E ; Cray et al. in Environ Microbiol 15:287-296, 2013a, doi: 10.1111/1462-2920.12018 ). We used Aspergillus wentii, a paradigm for extreme solute-tolerant fungal xerophiles, alongside yeast cell and enzyme models (Saccharomyces cerevisiae and glucose-6-phosphate dehydrogenase) and an agar-gelation assay, to determine growth-rate inhibition, intracellular compatible solutes, cell turgor, inhibition of enzyme activity, substrate water activity, and stressor chaotropicity for 12 chemically diverse solutes. These stressors were found to be: (i) osmotically active (and typically macromolecule-stabilizing kosmotropes), including NaCl and sorbitol; (ii) weakly to moderately chaotropic and non-osmotic, these were ethanol, urea, ethylene glycol; (iii) highly chaotropic and osmotically active, i.e. NH4NO3, MgCl2, guanidine hydrochloride, and CaCl2; or (iv) inhibitory due primarily to low water activity, i.e. glycerol. At ≤0.974 water activity, Aspergillus cultured on osmotically active stressors accumulated low-M r polyols to ≥100 mg g dry weight(-1). Lower-M r polyols (i.e. glycerol, erythritol and arabitol) were shown to be more effective for osmotic adjustment; for higher-M r polyols such as mannitol, and the disaccharide trehalose, water-activity values for saturated solutions are too high to be effective; i.e. 0.978 and 0.970 (25 ºC). The highly chaotropic, osmotically active substances exhibited a stressful level of chaotropicity at physiologically relevant concentrations (20.0-85.7 kJ kg(-1)). We hypothesized that the kosmotropicity of compatible solutes can neutralize chaotropicity, and tested this via in-vitro agar-gelation assays for the model chaotropes urea, NH4NO3, phenol and MgCl2. Of the kosmotropic compatible solutes, the most-effective protectants were trimethylamine oxide and betaine; but proline, dimethyl sulfoxide, sorbitol, and trehalose were also effective, depending on the chaotrope. Glycerol, by contrast (a chaotropic compatible solute used as a negative control) was relatively ineffective. The kosmotropic activity of compatible solutes is discussed as one mechanism by which these substances can mitigate the activities of chaotropic stressors in vivo. Collectively, these data demonstrate that some substances concomitantly induce chaotropicity-mediated and osmotic stresses, and that compatible solutes ultimately define the biotic window for fungal growth and metabolism. The findings have implications for the validity of ecophysiological classifications such as 'halophi...

Fungal stress biology: a preface to the Fungal Stress Responses special edition

“…However, this resistance and the associated robust phenotype come at a price of low conidial yield. Rangel et al (2015) give insights into the physicochemical properties and ecophysiological roles of erythritol, trehalose, and mannitol in relation to fungal cell biology (see also Alves et al 2015;Rangel et al 2006aRangel et al , b, 2008a. The authors have also shown that three levels of phenotypic plasticity occur for stress tolerance exhibited by M. robertsii, and this has profound consequences for its stress biology, virulence, and ecology.…”

“…Their paper reports a systems-inspired approach based on quantification of fungal growth rates, intracellular compatible solutes, mycotoxin concentrations, gene expression for key genes involved in sugar transport and mycotoxin production, and creation of a model that can be used to predict conditions under which production of mycotoxins occurs. The solute stressors studied were NaCl, which is an ionic osmotic stressor, and glycerol, which reduces water activity (and is chaotropic at high concentrations) and yet does not impose osmotic stress (see Alves et al 2015 in this Special Issue). Medina et al focus on interactions between temperature and stressor concentration as determinants for aspects of fungal growth phenotype and physiology in Aspergillus, Penicillium, and Fusarium.…”

“…Osmoregulation is critical for cell shape and turgor as well as intracellular dynamics. Glycerol at high concentrations can act as a stressor Williams and Hallsworth 2009;Alves et al 2015) and is used as a compatible solute, playing diverse roles in yeast physiology. The author summarizes different aspects of the HOG MAPK signaling pathway, including the Sln1 branch and the Sho1 branch that appear to function independently and have different roles.…”

Modulating Nucleation by Kosmotropes and Chaotropes: Testing the Waters

“…The constant pH level is required to quantitatively compare the effects of the chao-/kosmotropic additives during the early stages of calcium carbonate formation because it ensures a constant carbonate/bicarbonate ratio and removes corresponding, otherwise disturbing effects on supersaturation and ionic speciation. As examples of kosmotropes and chaotropes in the field of cellular stress biology [65], the effects of trehalose and urea are compared to reference experiments at pH 9.75. These additives do not influence the time required for mineral nucleation but do significantly affect the equilibrium between free and carbonate-associated Ca 2+ ions, as evident from the slope in the prenucleation regime before the sudden Ca 2+ concentration drop upon nucleation.…”

“…In view of the observed equilibrium shifts between free and bound ion species, the distinct bicarbonate and carbonate ratio appear to impart a pH-dependent physicochemistry to the PNCs, thus consequently determining additive-ion and -ion cluster interactions. As examples of kosmotropes and chaotropes in the field of cellular stress biology [65], the effects of trehalose and urea are compared to reference experiments at pH 9.75. These additives do not influence the time required for mineral nucleation but do significantly affect the equilibrium between free and carbonate-associated Ca 2+ ions, as evident from the slope in the prenucleation regime before the sudden Ca 2+ concentration drop upon nucleation.…”