Trehalose is a natural glucose disaccharide identified in the 19th century in fungi and insect cocoons, and later across the three domains of life. In members of the genus Mycobacterium, which includes the tuberculosis (TB) pathogen and over 160 species of nontuberculous mycobacteria (NTM), many of which are opportunistic pathogens, trehalose has been an important focus of research over the last 60 years. It is a crucial player in the assembly and architecture of the remarkable mycobacterial cell envelope as an element of unique highly antigenic glycolipids, namely trehalose dimycolate ('cord factor'). Free trehalose has been detected in the mycobacterial cytoplasm and occasionally in oligosaccharides with unknown function. TB and NTM infection statistics and death toll, the decline in immune responses in the aging population, human immunodeficiency virus/AIDS or other debilitating conditions, and the proliferation of strains with different levels of resistance to the dated drugs in use, all merge into a serious public-health threat urging more effective vaccines, efficient diagnostic tools and new drugs. This review deals with the latest findings on mycobacterial trehalose biosynthesis, catabolism, processing and recycling, as well with the ongoing quest for novel trehalose-related mechanisms to be targeted by novel TB therapeutics. In this context, the drug-discovery pipeline has recently included new lead compounds directed toward trehalose-related targets highlighting the potential of these pathways to stem the tide of rising drug resistance.

Strains of) was cloned from a gene library. The sequences of the three genes (including treS) were amplified by PCR and sequenced, revealing that the genes were structurally linked. To understand the role of trehalose during salt stress in T. thermophilus RQ-1, we constructed a mutant, designated RQ-1M6, in which TPS (otsA) and TPP (otsB) genes were disrupted by gene replacement. Mutant RQ-1M6 accumulated trehalose and mannosylglycerate in a medium containing yeast extract and NaCl. However, growth in a defined medium (without yeast extract, known to contain trehalose) containing NaCl led to the accumulation of mannosylglycerate but not trehalose. The deletion of otsA and otsB reduced the ability to grow in defined salt-containing medium, with the maximum salinity being 5% NaCl for RQ-1 and 3% NaCl for RQ-1M6. The lower salt tolerance observed in the mutant was relieved by the addition of trehalose to the growth media. In contrast to trehalose, the addition of glycine betaine, mannosylglycerate, maltose, and glucose to the growth medium did not allow the mutant to grow at higher salinities. The results presented here provide crucial evidence for the importance of the TPS/TPP pathway for the synthesis and accumulation of trehalose and the decisive contribution of this disaccharide to osmotic adaptation in T. thermophilus RQ-1.Thermophilic organisms, like the vast majority of other microorganisms, accumulate compatible solutes in response to water stress imposed by salt. However, the compatible solutes of thermophilic and hyperthermophilic prokaryotes are generally different from those of their mesophilic counterparts (34), and some compatible solutes, namely, di-myo-inositolphosphate, di-mannosyl-di-myo-inositol-phosphate, diglycerol phosphate, and mannosylglyceramide, are confined to organisms that grow at extremely high temperatures. Mannosylglycerate is also a common compatible solute of thermophiles and hyperthermophiles (21,28,35). Despite this association with organisms that grow at extremely high temperatures, mannosylglycerate was initially identified in red algae of the order Ceramiales (4, 15). Trehalose, a canonical compatible solute of mesophiles, also accumulates in a few thermophilic and hyperthermophilic organisms, where it appears to serve as a compatible solute (18,21,35). This nonreducing disaccharide has also been implicated in several stress responses in prokaryotes and eukaryotic microorganisms (10,32,36,37) and also serves as an intermediate in the synthesis of glycolipids, sulfolipids, and lipooligosaccharides in mycobacteria (3).The most common pathway for the synthesis of trehalose in bacteria involves trehalose-phosphate synthase (TPS), encoded by the gene otsA, which converts UDP-glucose and glucose-6-phosphate to trehalose-6-phosphate. This intermediate is subsequently dephosphorylated to yield trehalose via a specific trehalose-6-phosphate phosphatase (TPP), encoded by otsB (12). Another pathway converts maltose to trehalose via a trehalose synthase encoded by treS. The species of the...

Trehalose is the primary organic solute inRubrobacter xylanophilus under all conditions tested, including those for optimal growth. We detected genes of four different pathways for trehalose synthesis in the genome of this organism, namely, the trehalose-6-phosphate synthase (Tps)/trehalose-6-phosphate phosphatase (Tpp), TreS, TreY/TreZ, and TreT pathways. Moreover, R. xylanophilus is the only known member of the phylum Actinobacteria to harbor TreT. The Tps sequence is typically bacterial, but the Tpp sequence is closely related to eukaryotic counterparts. Both the Tps/Tpp and the TreT pathways were active in vivo, while the TreS and the TreY/TreZ pathways were not active under the growth conditions tested and appear not to contribute to the levels of trehalose observed. The genes from the active pathways were functionally expressed in Escherichia coli, and Tps was found to be highly specific for GDP-glucose, a rare feature among these enzymes. The trehalose-6-phosphate formed was specifically dephosphorylated to trehalose by Tpp. The recombinant TreT synthesized trehalose from different nucleoside diphosphate-glucose donors and glucose, but the activity in R. xylanophilus cell extracts was specific for ADP-glucose. The TreT could also catalyze trehalose hydrolysis in the presence of ADP, but with a very high K m . Here, we functionally characterize two systems for the synthesis of trehalose in R. xylanophilus, a representative of an ancient lineage of the actinobacteria, and discuss a possible scenario for the exceptional occurrence of treT in this extremophilic bacterium.

A mannosylglycerate synthase (MgS) gene detected in the genome of Selaginella moellendorffii was expressed in E. coli and the recombinant enzyme was purified and characterized. A remarkable and unprecedented feature of this enzyme was the ability to efficiently synthesize mannosylglycerate (MG) and glucosylglycerate (GG) alike, with maximal activity at 50 °C, pH 8.0 and with Mg(2+) as reaction enhancer. We have also identified a novel glycoside hydrolase gene in this plant's genome, which was functionally confirmed to be highly specific for the hydrolysis of MG and GG and named MG hydrolase (MgH), due to its homology with bacterial MgHs. The recombinant enzyme was maximally active at 40 °C and at pH 6.0-6.5. The activity was independent of cations, but Mn(2+) was a strong stimulator. Regardless of these efficient enzymatic resources we could not detect MG or GG in S. moellendorffii or in the extracts of five additional Selaginella species. Herein, we describe the properties of the first eukaryotic enzymes for the synthesis and hydrolysis of the compatible solutes, MG and GG.

Gene expression alterations occurring with aging have been described for a multitude of species, organs, and cell types. However, most of the underlying studies rely on static comparisons of mean gene expression levels between age groups and do not account for the dynamics of gene expression throughout the lifespan. These studies also tend to disregard the pairwise relationships between gene expression profiles, which may underlie commonly altered pathways and regulatory mechanisms with age. To overcome these limitations, we have combined segmented regression analysis with weighted gene correlation network analysis (WGCNA) to identify high-confidence signatures of aging in the brain, heart, liver, skeletal muscle, and pancreas of C57BL/6 mice in a publicly available RNA-Seq dataset (GSE132040). Functional enrichment analysis of the overlap of genes identified in both approaches showed that immune- and inflammation-related responses are prominently altered in the brain and the liver, while in the heart and the muscle, aging affects amino and fatty acid metabolism, and tissue regeneration, respectively, which reflects an age-related global loss of tissue function. We also explored sexual dimorphism in the aging mouse transcriptome and found the liver and the muscle to have the most pronounced gender differences in gene expression throughout the lifespan, particularly in proteostasis-related pathways. While the data showed little overlap among the age-dysregulated genes between tissues, aging triggered common biological processes in distinct tissues, which we highlight as important features of murine tissue physiological aging.