Accumulation Of Intracellular Trehalose Research Articles

Antimicrobial property of (+)-lyoniresinol-3α-O-β-d-Glucopyranoside isolated from the root bark ofLycium chinense Miller against human pathogenic microorganisms

(+)-Lyoniresinol-3alpha-O-beta-D-glucopyranoside (1) was isolated from an ethyl acetate extract of the root bark from Lycium chinense Miller, and its structure was determined using 1D and 2D NMR spectroscopy including DEPT, HMQC, and HMBC. (+)-Lyoniresinol-3alpha-O-beta-D-glucopyranoside exhibited potent antimicrobial activity against antibiotic-resistant bacterial strains, methicillin-resistant Staphylococcus aureus (MRSA) isolated from patients, and human pathogenic fungi without having any hemolytic effect on human erythrocytes. In particular, compound 1 induced the accumulation of intracellular trehalose on C. albicans as stress response to the drug, and disrupted the dimorphic transition that forms pseudo-hyphae caused by the pathogenesis. This indicates that (+)-lyoniresinol-3alpha-O-beta-D-glucopyranoside has excellent potential as a lead compound for the development of antibiotic agents.

Metabolite profiling for analysis of yeast stress response during very high gravity ethanol fermentations

A laboratory strain and an industrial strain of Saccharomyces cerevisiae were grown at high substrate concentration, so-called very high gravity (VHG) fermentation. Simultaneous saccharification and fermentation (SSF) was applied in a batch process using 280 g/L maltodextrin as carbon source. It was shown that known ethanol and osmotic stress responses such as decreased growth rate, lower viability, higher energy consumption, and intracellular trehalose accumulation occur in VHG SSF for both strains when compared with standard laboratory medium (20 g/L glucose). The laboratory strain was the most affected. GC-MS metabolite profiling was applied for assessing the yeast stress response influence on cellular metabolism. It was found that metabolite profiles originating from different strains and/or fermentation conditions were unique and could be distinguished with the help of multivariate data analysis. Several differences in the metabolic responses to stressing conditions were revealed, particularly the increased energy consumption of stressed cells was also reflected in increased intracellular concentrations of pyruvate and related metabolites.

Trehalose accumulation induced during the oxidative stress response is independent of TPS1 mRNA levels in Candida albicans.

Growing cells of the Candida albicans trehalose-deficient mutant tps1/tps1 were extremely sensitive to severe oxidative stress exposure (H2O2). However, their viability was not affected after saline stress or heat-shock treatments, being roughly equivalent to that of the parental strain. In wild-type cells, these adverse conditions induced the intracellular accumulation of trehalose together with activation of trehalose-6P synthase, whereas the endogenous trehalose content and the corresponding biosynthetic activity were barely detectable in the tps1/tps1 mutant. The addition of cycloheximide did not prevent the marked induction of trehalose-6P synthase activity. Furthermore, the presence of H2O2 decreased the level of TPS1 mRNA expression. Hence, the conspicuous trehalose accumulation in response to oxidative stress is not induced by increased transcription of TPS1. Our results are consistent with a specific requirement of trehalose in order to withstand a severe oxidative stress in C. albicans, and suggest that trehalose accumulation observed under these conditions is a complex process that most probably involves post-translational modifications of the trehalose synthase complex.

Trehalose accumulation, invertase activity and physiological characteristics of yeasts isolated from 24 h fermentative cycles during the production of artisanal Brazilian cachaça

Trehalose accumulation, invertase activity and physiological characteristics of 86 yeast isolates from short fermentative cycles during the production of cachaca in three artisanal distilleries of the State of Minas Gerais were studied. Among these isolates, 70% were able to grow at temperatures between 40 and 42oC. Only Saccharomyces cerevisiae isolates were able to grow over 40oC. Lower temperatures (<40oC) favoured the growth of yeasts such as Candida parapsilosis-like, C. maltosa-like, Kloeckera japonica, S. exiguus and C. bombicola-like. The isolates from all three distilleries were ethanol tolerant, produced invertase, and accumulate trehalose in the presence of glucose. The strains isolated from distillery A presented more resistance to ethanol (around 84.2% of the strains were able to grow in the presence of 12% ethanol) when compared to the ones from distilleries C and B (9.5% and no strain, respectively). The strains of S. cerevisiae isolated from the three distilleries presented a higher capacity to produce invertase and accumulate trehalose in the presence of glucose. Based on the results of thermal and ethanol stress experiments, it was possible to identify strong relationship between intracellular trehalose accumulation and cell viability. The increase in cell viability was even more pronounced when the strains were subjected to a pre-treatment at sublethal temperatures.

Role of trehalose biosynthesis in environmental survival and virulence of Salmonella enterica serovar typhimurium

The otsA and otsB genes, encoding trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase respectively, have been isolated from Salmonella enterica serovar typhimurium and nucleotide-sequenced. Induction of trehalose biosynthesis by exposure of bacteria to high osmotic strength resulted in the intracellular accumulation of trehalose. An otsA mutant of S. enterica serovar typhimurium was more susceptible to killing by heat, and grew poorly under conditions of high osmolarity. The wild-type and otsA mutant strains showed similar abilities to colonise spleen tissues after oral dosing of mice. These findings suggest that the otsBA gene products play a role in environmental survival, but not in virulence, of S. enterica serovar typhimurium.

Overexpression of Trehalose Synthase and Accumulation of Intracellular Trehalose in 293H and 293FTetR:Hyg Cells

A humanized clone containing the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase (otsA/B) has been constructed. Using the Gateway Cloning System (Invitrogen, Inc.), the otsA/B genes have been placed under the control of the CMV promoter (pEXPcmv-otsA/B) or the CMV promoter and the tet operator (pEXP cmv TetO-otsA/B). The pEXPcmv-otsA/B clone has been introduced into 293H cells using LIPOFECTAMINE 2000 and the intracellular concentration of trehalose has been evaluated. The 293H cells accumulate 4–5 μg trehalose/mg dry weight and this concentration increases to 7–10 μg trehalose/mg dry weight if trehalose is included in the growth medium. The pEXPcmv TetO-otsA/B clone has been transfected into 293FTetR:Hyg cells which contain the tet repressor integrated into the genome. When these transfected cells are grown in the absence of tetracycline, no intracellular trehalose is detected. Inclusion of 0.3 μg/ml tetracycline in the growth medium results in the accumulation of 11–14 μg trehalose/mg dry weight, a value which increases to 19–20 μg trehalose/mg dry weight if trehalose is included in the growth medium. The data for the 293FTetR:Hyg cells indicate that intracellular trehalose accumulates in response to the addition of tetracycline. This system will allow us to manipulate the intracellular concentration of trehalose and to evaluate the desiccation tolerance of these cells as a function of intracellular trehalose concentration.

Changes in the yeast metabolism at very high-gravity wort fermentation.

The rate of ethanol production increased with increasing wort gravity up to the initial wort concentration of 24%, reaching the maximum ethanol concentration of 6.2%, but its attenuation reached only 49%. The intracellular trehalose accumulation was proportional to the initial wort gravity, at 24 or 30% wort fermentation increased 3 or 4.5 times, respectively, compared to 12% wort fermentation. Trehalose accumulation began after exhaustion of glucose, ceased after uptake of approximately 65% reducing saccharides, despite of increasing ethanol or remaining saccharide concentration in the environment.

Analysis of the Heat-Shock Response Displayed by Two Chaetomium Species Originating from Different Thermal Environments

Three features of the heat shock response, reorganization of protein expression, intracellular accumulation of trehalose, and alteration in unsaturation degree of fatty acids were investigated in the thermophilic fungus Chaetomium thermophile and compared to the response displayed by a closely related mesophilic species, C. brasiliense. Thermophilic heat shock response paralleled the mesophilic response in many respects like (i) the temperature difference observed between normothermia and the upper limit of translational activity, (ii) the transient nature of the heat shock response at the level of protein expression including both the induction of heat shock proteins (HSPs) as well as the repression of housekeeping proteins, (iii) the presence of representatives of high-molecular-weight HSPs families, (iv) intracellular accumulation of trehalose, and finally (v) modifications in fatty acid composition. On the other hand, a great variability between the two organisms was observed for the proteins expressed during stress, in particular a protein of the HSP60 family that was only observed in C. thermophile. This peptide was also present constitutively at normal temperature and may thus fulfil thermophilic functions. It is shown that accumulation of trehalose does not play a part in thermophily but is only a stress response. C. thermophile contains less polyunsaturated fatty acids at normal temperature than C. brasiliense, a fact that can be directly related to thermophily. When subjected to heat stress, both organisms tended to accumulate shorter and less unsaturated fatty acids.

Trehalose metabolism and leavening ability of bakers' yeast grown in the presence of sodium chloride

We examined the relationship between trehalose metabolism and the leavening ability of bakers' yeast grown in the presence of NaCl. The yeast cells were cultured at 30°C in media (5% glucose, 1% peptone, 0.5% yeast extract, 0.1% KH 2PO 4, and 0.1% MgSO 4) containing 0–3% NaCl. The cells were grown to the stationary phase for 24 h irrespective of the NaCl concentration in the medium, but the cell yield was decreased by addition of NaCl. The presence of 1% NaCl in the medium transiently enhanced the accumulation of intracellular trehalose after 24 h, however, the accumulated trehalose was hydrolyzed during further growth, and 2 or 3% NaCl decreased the intracellular trehalose content. The neutral trehalase activity in yeast cells grown for 24 h increased with increasing NaCl concentration, while the acid trehalase activity decreased. Trehalose-6-phosphate synthase (TPS) activity in cells grown for 24 h increased significantly in the presence of 1 and 2% NaCl, but decreased in the presence of 3% NaCl. When we determined the leavening ability of bakers' yeast cells grown for 24 h in the presence of 0–3% NaCl using dough without addition of NaCl, the leavening ability increased with increasing NaCl concentration in the culture medium. The cells having the highest leavening ability were those that had the lowest amounts of trehalose and the lowest TPS activity among the conditions tested. The leavening ability of bakers' yeast grown in the presence of NaCl appears not to correlate with intracellular trehalose content.

Intracellular accumulation of trehalose during streptomycin formation by Streptomyces griseus.

When washed mycelium of Streptomyces griseus HUT 6037 was incubated in 0.5% NaCl solution containing 14C-gl cosamine with shaking at 28°C, the activity of the mycelium to incorporate radioactivity into the cell wall decreased rapidly, while that into streptomycin increased. During this physiological change, UDP-Af-acetylmuramyl-pentapeptide, UDP-N-acetylglucosamine, glutamic acid and trehalose accumulated in the mycelium. The latter two substances accumulated much more than the former two. When the time courses of the activities of incorporation of 14C-glucosamine into the four substances, mucopeptide and streptomycin were examined, a decrease in the activity into peptidoglycan led to an increase into streptomycin, glutamic acid and trehalose. In a pH-stat batch culture with a defined medium, trehalose was accumulated in the cell before glucose was consumed. However, after glucose was consumed, the consumption of trehalose began. Streptomycin production continued until intracellular trehalose completely disappeared in spite of the lack of glucose in the culture medium.