Response to oxidative stress caused by H(2)O(2) in Saccharomyces cerevisiae mutants deficient in trehalase genes

The role of trehalose as cell protector against oxidative stress induced by H(2)O(2) has been studied in Saccharomyces cerevisiae mutants in which the two trehalase genes ATH1 and NTH1 are deleted. The addition of low H(2)O(2) concentrations to proliferating cultures of either strain did not harm cell viability and induced a marked activity to Nth1p, but with no significant level of trehalose accumulation. This pattern was reversed after a more severe H(2)O(2) treatment that caused drastic cell killing. The most severe phenotype corresponded to the Delta nth1 mutant. Under these conditions, the increase in Nth1p was abolished and a three-fold rise in trehalose content was recorded concomitant with activation of the trehalose synthase complex. The behavior of the double-disruptant Delta ath1Delta nth1 mutant was identical to that of wild-type cells, although in exponential cultures Ath1p activity was virtually undetectable upon exposure to H(2)O(2). Furthermore, these strains displayed an adaptive response to oxidative stress that was independent of intracellular trehalose synthesis. Our data strongly suggest that trehalose storage in budding yeasts is not an essential protectant in cell defense against oxidative challenge.     

Human trehalase is a stress responsive protein in Saccharomyces cerevisiae

Three trehalases ATH1, NTH1, and NTH2 have been identified in Saccharomyces cerevisiae. ATH1, and NTH1 hydrolyze trehalose to glucose to provide energy and assist in recovery from stress. Human trehalase (TREH) is expressed in the intestine and kidney and probably hydrolyzes ingested trehalose in the intestine and acts as marker of renal tubular damage in kidney. Since trehalose is not present in circulation or kidney tubules, its renal effect suggests it has other yet unidentified actions. Here we examined the function of human trehalase in budding yeast. We constructed three yeast trehalase mutants (NTH1Δ, NTH2Δ, and ATH1Δ) and then transformed TREH into these mutants. NTH1Δ did not grow on media containing trehalose as the carbon source, and TREH did not rectify NTH1Δ dysfunction and also did not grow on trehalose medium, suggesting that TREH is not responsible for utilization of exogenous trehalose in yeast. In experiments involving exposure to heat, osmotic and oxidative stresses, NTH1Δ showed no recovery. Interestingly, ATH1Δ-TREH showed high sensitivity to all three stressors. ATH1Δ and NTH2Δ showed very low neutral trehalase activity and NTH1Δ did not show any neutral trehalase activity, and trehalose concentrations were higher. Increased neutral trehalase activity (equivalent to the wild type), reduction of trehalose content and brisk sensitivity to stressors were noted in TREH-ATH1Δ strain, but not in TREH-NTH1Δ or -NTH2Δ. Our results suggest that TREH acts as a stress-response protein in the kidney rather than involved in utilization of exogenous trehalose.     

Characterization of Beauveria bassiana neutral trehalase (BbNTH1) and recognition of crucial stress-responsive elements to control its expression in response to multiple stresses

A neutral trehalase (NTH1) of fungal entomopathogen Beauveria bassiana was characterized for the first time as a 743-aa enzyme (84.4 kDa). To identify crucial stress-responsive elements (STREs) to control the expression of the NTH-coding gene (BbNTH1) in response to different stresses, the full-length promoter (−2713 bp) upstream of its open reading frame and three upstream-truncated fragments (−1912, −1060 and −560 bp) were fused to the reporter gene eGFP and then transformed into B. bassiana, respectively. Consequently, eGFP was well expressed as intensive fluorescence in mycelia, conidiogenic cells and forming conidia controlled by the full-length promoter with five STREs. Surprisingly, transformants controlled by the shortest fragment with last two STREs at −315 and −274 bp exhibited consistently brightest fluorescence in mycelia under 3-h oxidative adaption of 0.3-1.2 mM menadione, and in colonies under 6-day osmotic stress of 0.5-1 M NaCl and thermal stress of 15-540 min at 40 °C after 3-day growth at 25 °C. Single or dual site-directed mutations of the two STREs from CCCCT to CATCT significantly altered the gene response to the multiple stresses. Thus, the two STREs in the downstream 560-bp region of the promoter are crucial to regulating not only constitutive but stress-inducible expression of the target gene.