Trehalose-deficient Acinetobacter baumannii exhibits reduced virulence by losing capsular polysaccharide and altering membrane integrity.

Acinetobacter baumannii has become a leading cause of bacterial nosocomial infections, in part, due to its ability to resist desiccation, disinfection and antibiotics. Several factors contribute to the tenacity and virulence of this pathogen, including production of a broad range of surface glycoconjugates, secretory systems and efflux pumps. We became interested in examining the importance of trehalose in A. baumannii after comparing intact bacterial cells by high-resolution magic angle spinning nuclear magnetic resonance and by noting high levels of this disaccharide, obscuring all other resonances in the spectrum. Since this was observed under normal growth conditions, we speculated that trehalose must serve additional functions beyond osmolyte homeostasis. Using the virulent isolate A. baumannii AB5075 and mutants in the trehalose synthesis pathway, osmoregulatory trehalose synthesis proteins A and B (△otsA and △otsB), we found that the trehalose-deficient △otsA showed increased sensitivity to desiccation, colistin, serum complement and peripheral blood mononuclear cells, while trehalose-6-phosphate producing △otsB behaved similar to the wild-type. The △otsA mutant also demonstrated increased membrane permeability and loss of capsular polysaccharide. These findings demonstrate that trehalose deficiency leads to loss of virulence in A. baumannii AB5075.

Differential ear growth of two maize varieties to shading in the field environment: Effects on whole plant carbon allocation and sugar starvation response.

The interception of irradiation by smog pollution and cloud cover associated with extreme rainfall events has become an increasingly important limiting factor in crop production in China. Little is known about the adaptation of carbon (C) allocation to periodic low irradiance in field conditions. The trehalose signaling pathway plays a critical role in adapting C allocation to the environment in crops but its importance in adaptation to low light in field conditions is not known. To determine the effects of low irradiance on C economy and maize yield, two commonly grown hybrids (LY-16 and ZD-958) were subject to three levels of shading (15 %, 50 %, and 97 %) for one week from V13 stage in two successive seasons. Shading led to yield loss mainly due to decreased kernel number, which was greater in LY-16 than ZD-958. Effects of shading on leaf area and photosynthesis were similar in both varieties. Starch levels in leaves were maintained, whereas total soluble carbohydrates were reduced up to fivefold by shading in both varieties. Shading increased the proportion of photoassimilate retained in leaves relative to reproductive organs. Carbohydrates in ears and stem were decreased by shading similarly in both varieties. Amongst the parameters measured, the main difference between LY-16 and ZD-958 associated with yield penalty was the expression of class II trehalose phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) genes which were increased due to shading in leaves and ears, particularly in ears of LY-16. It is concluded that altered C fixation and allocation by low irradiance limited ear growth at pre-anthesis. Activation of TPSII and TPP genes indicates that the trehalose pathway likely plays a role in ear development under low light and could be a target for yield improvement under such conditions as with other stresses.

Time course analysis of Candida albicans metabolites during biofilm development.

Biofilm-associated infections are difficult to treat because of their decreased susceptibility to antimicrobial therapy. Candida albicans is the most common fungal pathogen associated with colonization and biofilm formation on the surfaces of indwelling medical devices which show intrinsic resistance to many commonly used antifungal agents. In this study, a metabonomic method using gas chromatography-mass spectrometry (GC/MS) was developed to characterize metabolic profiles during the whole biofilm developmental phases compared to the planktonic mode in C. albicans. Thirty-one differentially produced metabolites between the biofilm and planktonic specimens at each time point were identified, and they were mainly involved in the tricarboxylic acid (TCA) cycle, lipid synthesis, amino acid metabolism, glycolysis, and oxidative stress. Further experiments showed that lack of trehalose, one of the metabolites differentially produced between biofilm and planktonic cells, resulted in abnormal biofilm formation and increased sensitivity to amphotericin B and miconazole. This study provides a systemic view of the metabolic pattern during the development of C. albicans biofilms, indicating that multicomponent, phase-specific mechanisms are operative in the process of biofilm formation.

Glycoconjugate expression on the cell wall of tps1/tps1 trehalose-deficient Candida albicans strain and implications for its interaction with macrophages.

The yeast Candida albicans has developed a variety of strategies to resist macrophage killing. In yeasts, accumulation of trehalose is one of the principal defense mechanisms under stress conditions. The gene-encoding trehalose-6-phosphate synthase (TPS1), which is responsible for trehalose synthesis, is induced in response to oxidative stress, as in phagolysosomes. Mutants unable to synthesize trehalose are sensitive to oxidative stress in vitro. In mice, the TPS1-deficient strain, tps1/tps1, displays a lower infection rate than its parental strain (CAI4). We have previously demonstrated the reduced binding capacity of tps1/tps1 and its lower resistance to macrophages. At the same time, its outer cell wall layer was seen to be altered. In this study, we show that depending on the culture conditions, the tps1/tps1 strain regulates the carbohydrate metabolism in a different way to CAI4, as reflected by the enhanced ß-mannosylation of cell wall components, especially at the level of the 120 kDa glycoprotein species, accessible at the cell surface of tps1/tps1 when cultured in liquid medium, but not on solid medium. This leads to changes in its surface properties, as revealed by decreased hydrophobicity, and the lower levels of ERK1/2 phosphorylation and tumor necrosis factor-α (TNF-α) production in macrophages, thus increasing the resistance to these cells. In contrast, in solid medium, in which over-glycosylation was less evident, tps1/tps1 showed similar macrophage interaction properties to CAI4, but was less resistant to killing, confirming the protective role of trehalose. Thus, the lack of trehalose is compensated by an over-glycosylation of the cell wall components in the tps1/tps1 mutant, which reduces susceptibility to killing.

Lack of trehalose accelerates H2O2-induced Candida albicans apoptosis through regulating Ca2+ signaling pathway and caspase activity.

Trehalose is a non-reducing disaccharide and can be accumulated in response to heat or oxidative stresses in Candida albicans. Here we showed that a C. albicans tps1Δ mutant, which is deficient in trehalose synthesis, exhibited increased apoptosis rate upon H(2)O(2) treatment together with an increase of intracellular Ca(2+) level and caspase activity. When the intracellular Ca(2+) level was stimulated by adding CaCl(2) or A23187, both the apoptosis rate and caspase activity were increased. In contrast, the presence of two calcium chelators, EGTA and BAPTA, could attenuate these effects. Moreover, we investigated the role of Ca(2+) pathway in C. albicans apoptosis and found that both calcineurin and the calcineurin-dependent transcription factor, Crz1p, mutants showed decreased apoptosis and caspase activity upon H(2)O(2) treatment compared to the wild-type cells. Expression of CaMCA1, the only gene found encoding a C. albicans metacaspase, in calcineurin-deleted or Crz1p-deleted cells restored the cell sensitivity to H(2)O(2). Our results suggest that Ca(2+) and its downstream calcineurin/Crz1p/CaMCA1 pathway are involved in H(2)O(2)-induced C. albicans apoptosis. Inhibition of this pathway might be the mechanism for the protective role of trehalose in C. albicans.

High yield of mannosylglycerate production by upshock fermentation and bacterial milking of trehalose-deficient mutant Thermus thermophilus RQ-1.

A production process, using upshock fermentation and osmotic downshock, for the effective production/excretion of mannosylglycerate (MG) by the trehalose-deficient mutant of the strain Thermus thermophilus RQ-1 has been developed. In the first phase of fed-batch fermentation, the knockout mutant was grown at 70 degrees C on a NaCl-free medium. After the culture reached the end of the exponential growth phase, upshift in temperature and NaCl concentration was applied. The temperature was increased to 77 degrees C, and NaCl was added up to 3.0% and kept constant during the second phase of fermentation. Although this shift in cultivation parameters caused a dramatic drop of cell density, a significant improvement in accumulation of MG up to 0.64 micromol/mg protein compared to batch fermentations (0.31 micromol/mg protein) was achieved. A total yield of 4.6 g MG/l of fermentation broth was obtained in the dialysis bioreactor with a productivity of 0.29 g MG l(-1) h(-1). The solute was released from the harvested biomass by osmotic downshock using demineralized water at 70 degrees C. More than 90% of the intracellularly accumulated solute was recovered from the water fraction. The process was very efficient, as hyperosmotic shock, release of the solute, and reiterative fed-batch fermentation could be repeated at least four times.

Anhydrobiosis without trehalose in bdelloid rotifers.

Eukaryotes able to withstand desiccation enter a state of suspended animation known as anhydrobiosis, which is thought to require accumulation of the non-reducing disaccharides trehalose (animals, fungi) and sucrose (plants), acting as water replacement molecules and vitrifying agents. We now show that clonal populations of bdelloid rotifers Philodina roseola and Adineta vaga exhibit excellent desiccation tolerance, but that trehalose and other disaccharides are absent from carbohydrate extracts of dried animals. Furthermore, trehalose synthase genes (tps) were not found in rotifer genomes. This first observation of animal anhydrobiosis without trehalose challenges our current understanding of the phenomenon and calls for a re-evaluation of existing models.

Role of antioxidant enzymatic defences against oxidative stress H(2)O(2) and the acquisition of oxidative tolerance in Candida albicans.

In Candida albicans, trehalose plays an essential role as a protector of cell integrity against oxidative challenge. A double homozygous mutant, tps1/tps1, deficient in trehalose synthesis, displayed severe cell mortality when exposed to high H(2)O(2) concentrations, compared with its congenic parental (CAI-4) strain (Alvarez-Peral et al., 2002). We have examined the putative role of a set of well-known antioxidant enzymes as components of the defence mechanism against oxidative challenges. When exposed to mild non-lethal oxidative treatment (0.5 mM H(2)O(2)), a significant induction of catalase, glutathione reductase (GR), and Cu,Zn-superoxide dismutase (SOD) was recorded in tps1/tps1 exponential cultures. However, in CAI-4 cells, subjected to the same conditions, there was only a clear activation of catalase, Mn-SOD and Cu,Zn-SOD activities. The degree of activation was always much more pronounced in the trehalose-deficient mutant than in its wild-type counterpart, except for Mn-SOD activity. After exposure to severe oxidative stress (50 mM H(2)O(2)) only GR and catalase activities increased in tps1/tps1 cultures, whereas in CAI-4 cells GR but not catalase was induced. In both cell strains, 50 mM H(2)O(2) caused inhibition of the Mn- and Cu,Zn-SOD isozymes, this inhibition being more pronounced in tps1/tps1 cells. C. albicans is able to acquire adaptive oxidative tolerance by pretreatment with a low non-stressing concentration of H(2)O(2) before exposure to a drastic oxidative challenge. When these antioxidant activities were measured during the adaptive response, a greater degree of enzymatic antioxidant induction was consistently observed in the tps1/tps1 mutant with respect to the CAI-4 strain. Together with a higher intrinsic sensitivity of tps1/tps1 cells, we suggest that this unexpected increase might be explained in terms of a compensatory mechanism to overcome the lack of endogenous trehalose upon drastic oxidative exposure, although this induction was not sufficient to improve the percentage of cell viability.