The secreted acid trehalase encoded by the CgATH1 gene is involved in Candida glabrata virulence

BACKGROUND Candida glabrata yeast is the second cause of candidiasis worldwide. Differs from other yeasts since assimilates only glucose and trehalose (a characteristic used in rapid identification tests for this pathogen) by secreting into the medium a highly active acid trehalase encoded by the CgATH1 gene. OBJECTIVE This study aimed to characterise the function of the acid trehalase in the physiopathology of C. glabrata. METHODS Gene deletion was performed to obtain a mutant ath1Δ strain, and the ability of the ath1Δ strain to grow in trehalase, or the presence of trehalase activity in the ath1Δ yeast cells, was verified. We also tested the virulence of the ath1Δ strain in a murine model of infection. FINDINGS The ath1Δ mutant strain grows normally in the presence of glucose, but loses its ability to grow in trehalose. Due to the high acid trehalase activity present in wild-type cells, the cytoplasmic neutral trehalase activity is only detected in the ath1Δ strain. We also observed a significantly lower virulence of the ath1Δ strain in a murine model of infection with either normal or immunocompromised mice. MAIN CONCLUSIONS The acid trehalase is involved in the hydrolysis of external trehalose by C. glabrata, and the enzyme also plays a major virulence role during infectivity.

Shared control of maltose and trehalose utilization in Candida utilis

Trehalose biosynthesis and its hydrolysis have been extensively studied in yeast, but few reports have addressed the catabolism of exogenously supplied trehalose. Here we report the catabolism of exogenous trehalose by Candida utilis. In contrast to the biphasic growth in glucose, the growth of C. utilis in a mineral medium with trehalose as the sole carbon and energy source is aerobic and exhibits the Kluyver effect. Trehalose is transported into the cell by an inducible trehalose transporter (K M of 8 mM and V MAX of 1.8 æmol trehalose min-1 mg cell (dry weight)-1. The activity of the trehalose transporter is high in cells growing in media containing trehalose or maltose and very low or absent during the growth in glucose or glycerol. Similarly, total trehalase activity was increased from about 1.0 mU/mg protein in cells growing in glucose to 39.0 and 56.2 mU/mg protein in cells growing in maltose and trehalose, respectively. Acidic and neutral trehalase activities increased during the growth in trehalose, with neutral trehalase contributing to about 70 percent of the total activity. In addition to the increased activities of the trehalose transporter and trehalases, growth in trehalose promoted the increase in the activity of alpha-glucosidase and the maltose transporter. These results clearly indicate that maltose and trehalose promote the increase of the enzymatic activities necessary to their catabolism but are also able to stimulate each other's catabolism, as reported to occur in Escherichia coli. We show here for the first time that trehalose induces the catabolism of maltose in yeast

Evidence for a modulation of neutral trehalase activity by Ca2+ and cAMP signaling pathways in Saccharomyces cerevisiae

Saccharomyces cerevisiae neutral trehalase (encoded by NTH1) is regulated by cAMP-dependent protein kinase (PKA) and by an endogenous modulator protein. A yeast strain with knockouts of CMK1 and CMK2 genes (cmk1cmk2) and its isogenic control (CMK1CMK2) were used to investigate the role of CaM kinase II in the in vitro activation of neutral trehalase during growth on glucose. In the exponential growth phase, cmk1cmk2 cells exhibited basal trehalase activity and an activation ratio by PKA very similar to that found in CMK1CMK2 cells. At diauxie, even though both cells presented comparable basal trehalase activities, cmk1cmk2 cells showed reduced activation by PKA and lower total trehalase activity when compared to CMK1CMK2 cells. To determine if CaM kinase II regulates NTH1 expression or is involved in post-translational modulation of neutral trehalase activity, NTH1 promoter activity was evaluated using an NTH1-lacZ reporter gene. Similar ß-galactosidase activities were found for CMK1CMK2 and cmk1cmk2 cells, ruling out the role of CaM kinase II in NTH1 expression. Thus, CaM kinase II should act in concert with PKA on the activation of the cryptic form of neutral trehalase. A model for trehalase regulation by CaM kinase II is proposed whereby the target protein for Ca2+/CaM-dependent kinase II phosphorylation is not the neutral trehalase itself. The possible identity of this target protein with the recently identified trehalase-associated protein YLR270Wp is discussed

Periplasmic trehalase from Escherichia coli: characterization and immobilization on spherisorb

1. Trehalase was partially purified from Escherichia coli and characterized. The Km for trehalose was 0.78 mM, the pH optimum 5.5 and the temperature optimum 30 degrees C. 2. Trehalase represented approximately 50 per cent of the total protein released by osmotic shock. The preparation was free of nonspecific carbohydrate hydrolases, which act on sucrose, galactose and maltose, permitting trehalose determination in biological samples, such as insect hemolymph and free cell extracts among others. 3. The enzyme was stable in 50 mM maleate buffer, pH 6.2, at -8 degrees C for at least 6 months and could be used to determine trehalose in the range of 6 to 30 nmol. 4. Immobilization of the enzyme was achieved by covalent linkage to spherisorb-5NH2 (spherical silica gel). Retention of total catalytic activity averaged 32 per cent . 5. The reactor, stored for one month at -5 degrees C, retained 98 per cent of its initial immobilized activity. 6. This immobilized form of the enzyme could be used routinely for specific determinations of trehalose

Activation of yeast trehalase by heat shock

1. Activation of Saccharomyces cerevisiae trehalase by heat shock was shown in all strains tested, including mutants in which the reponse to a glucose signal was absent. A low concentration of cAMP favored the response as seen in 2nd log cells or in ras2 and cyr1ts mutant strains. The heat shock effect upon trehalse activity was not observed under conditions of catabolite repession. 2 Neither hexokinase PII nor the heat shock protein hsp26 seemed to be involve in the axtivation of trehalase by heat shock. However, mutant strains deleted in the polyubiquitin gene showed only a 2-fold activation of the enzyme while in control strains a 5-to 7-fold irreversible activation was observed. 3. An alternative mechanism of trehalase activation by removal of an inhibitor through ligation with ubiquitin is discussed. Activation by cAMP-independent phosphorylation is also considered

Efecto de la ingestión de pan integral sobre la actividad disacaridasica intestinal en ratas / Effects if ingestion of whole-wheat bread on intestinal disacharidase activity in rats

Se realizó un experimento para evaluar el efecto de la ingestión de pan integral sobre la actividad disacaridásica intestinal. Se utilizó un total de 21 ratas macho, las cuales se agruparon según: a) dieta control con caseína más metionina, b) dieta con blanco, y c) dieta con pan integral. Despúes del periodo experimental de 10 días, se determinó la actividad específica de lactasa, mitasa, sacarasa y trealasa en distintos niveles de localización en la microvellosidad. Todas las enzimas presentaron una disminución significativa (p<0,01) de su actividad en la fracción luminal en las ratas alimentadas con pan integral. Sólo la lactasa y la maltasa mostraron una disminución de su actividad (p<0.01) en la fracción de membrana para dicha dieta. La fracción enterocitaria no mostró diferencia cuando se comparó con la dieta de pan blanco. En todos los nivles de localización la actividad disacaridásica fue mayor en la dieta control (p<0.01). Los resultados obtenidos sugieren que el efecto por "arrastre mecánico" de la fibra dietética contenida en el pan integral es el fundamental en la interacciòn fibra-actividad disacaridásica y que, por tanto, su presencia en el intestino no afecta sensiblemente la biosíntesis de dichas enzimas en el enterocito