Trehalose

Degradation-resistant trehalose analogues block utilization of trehalose by hypervirulent Clostridioides difficile.[Pubmed:30968891]

Chem Commun (Camb). 2019 Apr 10.

Trehalose is used as an additive in thousands of foods, cosmetics, and pharmaceutical products, and it is being investigated as a therapeutic for multiple human diseases. However, its ability to be used as a carbon source by microbes is a concern, as highlighted by the recent finding that Trehalose can be metabolized by and potentially enhance the virulence of epidemic Clostridioides difficile. Here, we show that Trehalose analogues designed to resist enzymatic degradation are incapable of being used as carbon sources by C. difficile. Furthermore, we demonstrate that Trehalose analogues, but not the known trehalase inhibitor validamycin A, inhibit native Trehalose utilization by hypervirulent C. difficile. Thus, degradation-resistant Trehalose analogues are valuable as trehalase inhibitors and as surrogates for or co-additives with Trehalose in applications where enzymatic breakdown is a concern.

Mechanism of Trehalose Induced Protein Stabilization from Neutron Scattering and Modeling.[Pubmed:30964287]

J Phys Chem B. 2019 Apr 9.

The sugar molecule Trehalose has been proven to be an excellent stabilizing co-solute for the preservation of biological materials. However, the stabilizing mechanism of Trehalose has been much debated during the previous decades and it is still not fully understood, partly because it has not been completely established how Trehalose molecules structure around proteins. Here we present a molecular model of a protein-water-Trehalose system, based on neutron scattering results obtained from neutron diffraction, quasielastic neutron scattering and different computer modeling techniques. The structural data clearly show how the proteins are preferentially hydrated, and analysis of the dynamical properties show that the protein residues are slowed down because of reduced dynamics of the protein hydration shell, rather than because of direct Trehalose-protein interactions. These findings are thereby giving strong support for previous models related to the preferential hydration model and contradicting other models based on water replacement at the protein surface. Furthermore, the results are important for understanding the specific role of Trehalose in biological stabilization, and more generally for providing a likely mechanism of how co-solutes affect the dynamics of proteins.

The trehalose-6-phosphate synthase TPS5 negatively regulates ABA signaling in Arabidopsis thaliana.[Pubmed:30963238]

Plant Cell Rep. 2019 Apr 8. pii: 10.1007/s00299-019-02408-y.

KEY MESSAGE: The TPS5 negatively regulates ABA signaling by mediating ROS level and NR activity during seed germination and stomatal closure in Arabidopsis thaliana. Trehalose metabolism is important in plant growth and development and in abiotic stress response. Eleven TPS genes were identified in Arabidopsis, divided into Class I (TPS1-TPS4) and Class II (TPS5-TPS11). Although Class I has been shown to have TPS activity, the function of most members of Class II remains enigmatic. Here, we characterized the biological function of the Trehalose-6-phosphate synthase TPS5 in ABA signaling in Arabidopsis. TPS5 expression was induced by ABA and abiotic stress, and expression in epidermal and guard cells was dramatically increased after ABA treatment. Loss-of-function analysis revealed that tps5 mutants (tps5-1 and tps5-cas9) are more sensitive to ABA during seed germination and ABA-mediated stomatal closure. Furthermore, the H2O2 level increased in the tps5-1 and tps5-cas9 mutants, which was consistent with the changes in the expression of RbohD and RbohF, key genes responsible for H2O2 production. Further, TPS5 knockout reduced the amounts of Trehalose and other soluble carbohydrates as well as nitrate reductase (NR) activity. In vitro, Trehalose and other soluble carbohydrates promoted NR activity, which was blocked by the tricarboxylic acid cycle inhibitor iodoacetic acid. Thus, this study identified that TPS5 functions as a negative regulator of ABA signaling and is involved in altering the Trehalose content and NR activity.

Hydrodynamic volume of trehalose and its water uptake mechanism.[Pubmed:30959240]

Biophys Chem. 2019 Apr 2;249:106145.

Trehalose ability to preserve water in biology has spawned research on this special disaccharide and its solutions. Trehalose unlike any other disaccharide, tend to mix with almost any amount of water. In water, Trehalose forms a hydrodynamic volume with bound waters (both coordination water and semicircular heterogeneities), capable of perturbing the very nature of normal bulk water. Switching of the two major conformational forms, defined by their helicities (i, i-H2O with lower helicity and ii, ii-H2O with higher helicity), were closely examined, using DFT/B3LYP- 6-311+G** level of theory, along with molecular dynamic (MD) calculations in aqueous media. Patterns in radial distribution functions (RDF) confirmed semicircular heterogeneities, including spines of water (rows of slow water molecules), in Trehalose hydration shell. Dynamics of Trehalose conformational switch and its coordination water are coupled to dynamics of these spines of water, which are themselves coupled to dynamics of the rest of Trehalose hydration shell waters. Like seamless cogwheels such energy cascade links the upstream slow dynamics of spines to the downstream collective bulk water dynamics. This lubricates Trehalose conformational switch through coordination water uptake, for which we proposed a mechanism here. We show how the coupling between Trehalose and bound waters in its hydrodynamic volume encompass both function and dynamic of the molecule and its hydration shell. Further simulations are needed to see how this ability is related to the evading and percolating nature of cryoprotectant water, also reported for the self-coordinating jelly behavior of biological water.

Kluyveromyces marxianus developing ethanol tolerance during adaptive evolution with significant improvements of multiple pathways.[Pubmed:30949239]

Biotechnol Biofuels. 2019 Mar 22;12:63.

Background: Kluyveromyces marxianus, the known fastest-growing eukaryote on the earth, has remarkable thermotolerance and capacity to utilize various agricultural residues to produce low-cost bioethanol, and hence is industrially important to resolve the imminent energy shortage crisis. Currently, the poor ethanol tolerance hinders its operable application in the industry, and it is necessary to improve K. marxianus' ethanol resistance and unravel the underlying systematical mechanisms. However, this has been seldom reported to date. Results: We carried out a wild-type haploid K. marxianus FIM1 in adaptive evolution in 6% (v/v) ethanol. After 100-day evolution, the KM-100d population was obtained; its ethanol tolerance increased up to 10% (v/v). Interestingly, DNA analysis and RNA-seq analysis showed that KM-100d yeasts' ethanol tolerance improvement was not due to ploidy change or meaningful mutations, but founded on transcriptional reprogramming in a genome-wide range. Even growth in an ethanol-free medium, many genes in KM-100d maintained their up-regulation. Especially, pathways of ethanol consumption, membrane lipid biosynthesis, anti-osmotic pressure, anti-oxidative stress, and protein folding were generally up-regulated in KM-100d to resist ethanol. Notably, enhancement of the secretory pathway may be the new strategy KM-100d developed to anti-osmotic pressure, instead of the traditional glycerol production way in S. cerevisiae. Inferred from the transcriptome data, besides ethanol tolerance, KM-100d may also develop the ability to resist osmotic, oxidative, and thermic stresses, and this was further confirmed by the cell viability test. Furthermore, under such environmental stresses, KM-100d greatly improved ethanol production than the original strain. In addition, we found that K. marxianus may adopt distinct routes to resist different ethanol concentrations. Trehalose biosynthesis was required for low ethanol, while sterol biosynthesis and the whole secretory pathway were activated for high ethanol. Conclusions: This study reveals that ethanol-driven laboratory evolution could improve K. marxianus' ethanol tolerance via significant up-regulation of multiple pathways including anti-osmotic, anti-oxidative, and anti-thermic processes, and indeed consequently raised ethanol yield in industrial high-temperature and high-ethanol circumstance. Our findings give genetic clues for further rational optimization of K. marxianus' ethanol production, and also partly confirm the positively correlated relationship between yeast's ethanol tolerance and production.

Ethanol stimulates trehalose production through a SpoT-DksA-AlgU dependent pathway in Pseudomonas aeruginosa.[Pubmed:30936375]

J Bacteriol. 2019 Apr 1. pii: JB.00794-18.

Pseudomonas aeruginosa frequently resides among ethanol-producing microbes, making its response to these microbially-produced concentrations of ethanol relevant to understanding its biology. Our transcriptome analysis found that genes involved in Trehalose metabolism were induced by low concentrations of ethanol, and biochemical assays showed levels of intracellular Trehalose increased significantly upon growth with ethanol. The increase in Trehalose was dependent on the TreYZ pathway, but not other Trehalose metabolic enzymes TreS or TreA. The sigma factor AlgU (AlgT), a homolog of RpoE in other species, was required for increased expression of the treZ gene and Trehalose levels, but induction was not controlled by the well-characterized proteolysis of its antisigma factor MucA. Growth with ethanol led to increased SpoT-dependent (p)ppGpp accumulation, which stimulates AlgU-dependent transcription of treZ and other AlgU-regulated genes through DksA, a (p)ppGpp and RNA polymerase binding protein. Ethanol stimulation of Trehalose also required acylhomoserine lactone (AHL)-mediated quorum sensing, as induction was not observed in a DeltalasRDeltarhlR strain. A network analysis using a model, eADAGE, built from publicly available P. aeruginosa transcriptome datasets (1) provided strong support for our model that treZ and co-regulated genes are controlled by both AlgU and AHL-mediated QS (QS). Consistent with (p)ppGpp and AHL-mediated quorum sensing regulation, ethanol, even when added at the time of culture inoculation, stimulated treZ transcript levels and Trehalose production in cells from post-exponential phase cultures but not from exponential phase cultures. These data highlight the integration of growth and cell density cues in the P. aeruginosa transcriptional response to ethanol.Importance Pseudomonas aeruginosa is often found with bacteria and fungi that produce fermentation products including ethanol. At concentrations similar to those produced by environmental microbes, we found that ethanol stimulated expression of Trehalose biosynthetic genes and cellular levels of Trehalose, a disaccharide that protects against environmental stresses. The induction of Trehalose by ethanol required the alternative sigma factor AlgU through DksA and SpoT-dependent (p)ppGpp. Trehalose accumulation also required AHL quorum sensing and only occurred in post-exponential phase cultures. This work highlights how cells integrate cell-density and growth cues in their responses to products made by other microbes and reveals a new role for (p)ppGpp in the regulation of AlgU activity.