[Show abstract][Hide abstract] ABSTRACT: We identified in the methylotrophic yeast Hansenula polymorpha (syn. Pichia angusta) a novel hexose transporter homologue gene, HXS1 (hexose sensor), involved in transcriptional regulation in response to hexoses, and a regular hexose carrier gene, HXT1 (hexose transporter). The Hxs1 protein exhibits the highest degree of primary sequence similarity to the Saccharomyces cerevisiae transporter-like glucose sensors, Snf3 and Rgt2. When heterologously overexpressed in an S. cerevisiae hexose transporter-less mutant, Hxt1, but not Hxs1, restores growth on glucose or fructose, suggesting that Hxs1 is nonfunctional as a carrier. In its native host, HXS1 is expressed at moderately low level and is required for glucose induction of the H. polymorpha functional low-affinity glucose transporter Hxt1. Similarly to other yeast sensors, one conserved amino acid substitution in the Hxs1 sequence (R203K) converts the protein into a constitutively signaling form and the C-terminal region of Hxs1 is essential for its function in hexose sensing. Hxs1 is not required for glucose repression or catabolite inactivation that involves autophagic degradation of peroxisomes. However, HXS1 deficiency leads to significantly impaired transient transcriptional repression in response to fructose, probably due to the stronger defect in transport of this hexose in the hxs1Delta deletion strain. Our combined results suggest that in the Crabtree-negative yeast H. polymorpha, the single transporter-like sensor Hxs1 mediates signaling in the hexose induction pathway, whereas the rate of hexose uptake affects the strength of catabolite repression.
[Show abstract][Hide abstract] ABSTRACT: Inhibition of the biosynthesis of trehalose, a well-known stress protectant in pathogens, is an interesting approach for antifungal or antibacterial therapy. Deletion of TPS2, encoding trehalose-6-phosphate (T6P) phosphatase, results in strongly reduced virulence of Candida albicans due to accumulation of T6P instead of trehalose in response to stress. To further aggravate the deregulation in the pathogen, we have additionally deleted the GPR1 gene, encoding the nutrient receptor that activates the cyclic AMP-protein kinase A signaling pathway, which negatively regulates trehalose accumulation in yeasts. A gpr1 mutant is strongly affected in morphogenesis on solid media as well as in vivo in a mouse model but has only a slightly decreased virulence. The gpr1 tps2 double mutant, on the other hand, is completely avirulent in a mouse model for systemic infection. This strain accumulates very high T6P levels under stress conditions and has a growth defect at higher temperatures. We also show that a tps2 mutant is more sensitive to being killed by macrophages than the wild type or the gpr1 mutant. A double mutant has susceptibility similar to that of the single tps2 mutant. For morphogenesis on solid media, on the other hand, the gpr1 tps2 mutant shows a phenotype similar to that of the single gpr1 mutant. Taken together these results show that there is synergism between Gpr1 and Tps2 and that their combined inactivation results in complete avirulence. Combination therapy targeting both proteins may prove highly effective against pathogenic fungi with increased resistance to the currently used antifungal drugs.
Infection and immunity 05/2008; 76(4):1686-94. · 4.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigated the role in cell morphogenesis and pathogenicity of the Candida albicans GPR1 gene, encoding the G protein-coupled receptor Gpr1. Deletion of C. albicans GPR1 has only minor effects in liquid hypha-inducing media but results in strong defects in the yeast-to-hypha transition on solid hypha-inducing media. Addition of cAMP, expression of a constitutively active allele of the Galpha protein Gpa2 or of the catalytic protein kinase A subunit TPK1 restores the wild-type phenotype of the CaGPR1-deleted strain. Overexpression of HST7, encoding a component of the mitogen-activated protein kinase pathway, does not suppress the defect in filamentation. These results indicate that CaGpr1 functions upstream in the cAMP-protein kinase A (PKA) pathway. We also show that, in the presence of glucose, CaGpr1 is important for amino acid-induced transition from yeast to hyphal cells. Finally, as opposed to previous reports, we show that CaGpa2 acts downstream of CaGpr1 as activator of the cAMP-PKA pathway but that deletion of neither CaGpr1 nor CaGpa2 affects glucose-induced cAMP signaling. In contrast, the latter is abolished in strains lacking CaCdc25 or CaRas1, suggesting that the CaCdc25-CaRas1 rather than the CaGpr1-CaGpa2 module mediates glucose-induced cAMP signaling in C. albicans.
Molecular Biology of the Cell 05/2005; 16(4):1971-86. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Yeast-to-hypha transition in Candida albicans can be induced by a wide variety of factors, including specific nutrients. We have started to investigate the mechanism by which some of these nutrients may be sensed. The G-protein-coupled receptor Gpr1 is required for yeast-to-hypha transition on various solid hypha-inducing media. Recently we have shown induction of Gpr1 internalization by specific amino acids, e.g. methionine. This suggests a possible role for methionine as a ligand of CaGpr1. Here we show that there is a big variation in methionine-induced hypha formation depending on the type of carbon source present in the medium. In addition high glucose concentrations repress hypha formation whereas a concentration of 0.1%, which mimics the glucose concentration present in the bloodstream, results in maximal hypha formation. Hence, it remains unclear whether Gpr1 senses sugars, as in Saccharomyces cerevisiae, or specific amino acids like methionine.
Biochemical Society Transactions 03/2005; 33(Pt 1):291-3. · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An extended definition of the term metabolic engineering is given and its successful use in the construction of biorecognition elements of sensors is demonstrated. It is shown that genetic and chemical modifications of methylotrophic yeast cells provide directed changes in their physiological responses towards methanol, ethanol and formaldehyde resulting in enhanced selectivity and shorter time response of the corresponding potentiometric and amperometric biosensors.
FEMS Yeast Research 09/2002; 2(3):307-14. · 2.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cytochrome c from the methylotrophic yeast Hansenula polymorpha was isolated and purified to homogeneity for the first time. The final yield of the highly purified protein from 1.4 kg (wet weight) cells was about 20 mg. The hemoprotein has an apparent molecular mass of 12 kDa and isoelectric point (pI) of 9.3. The purified protein was characterized by electronic, EPR and NMR spectroscopies. The redox potential of the cytochrome, E degrees, measured by cyclic voltammetry measurements at neutral pH, is 0.302 V. Both NMR spectroscopy and electrochemical measurements confirm the presence in the solution of several acid-base equilibria, the most pronounced being characterized by a pK(a) of 8.3. The latter pK(a) was attributed to the detachment of the iron(III) ion-coordinated methionine and its replacement by a lysine residue. The electrochemically derived thermodynamic parameters for neutral and alkaline protein species (DeltaS degrees (rc) and DeltaH degrees (rc)) were obtained from the temperature dependence of the redox potential.
Biochimica et Biophysica Acta 12/2000; 1543(1):174-88. · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cytochrome c from the methylotrophic yeast Hansenula polymorpha was isolated and purified to homogeneity for the first time. The final yield of the highly purified protein from 1.4 kg (wet weight) cells was about 20 mg. The hemoprotein has an apparent molecular mass of 12 kDa and isoelectric point (pI) of 9.3. The purified protein was characterized by electronic, EPR and NMR spectroscopies. The redox potential of the cytochrome, E°, measured by cyclic voltammetry measurements at neutral pH, is 0.302 V. Both NMR spectroscopy and electrochemical measurements confirm the presence in the solution of several acid–base equilibria, the most pronounced being characterized by a pKa of 8.3. The latter pKa was attributed to the detachment of the iron(III) ion-coordinated methionine and its replacement by a lysine residue. The electrochemically derived thermodynamic parameters for neutral and alkaline protein species (ΔS°rc and ΔH°rc) were obtained from the temperature dependence of the redox potential.
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology 11/2000; 1543(1):174-188.
[Show abstract][Hide abstract] ABSTRACT: Two types of alcohol-specific microbial/electrochemical biosensors have been developed using specially constructed mutant cells of the methylotrophic yeast Hansenula polymorpha. The cells were immobilized in a calcium alginate gel, and placed between two membranes on the surface of oxygen or hydrogen peroxide-electrodes. The O2 electrode based biosensor contained mutant cells with strongly elevated alcohol oxidase activity. The peroxide electrode based biosensor consisted of catalase-defective mutant cells which produce hydrogen peroxide in the presence of alcohol. Both types of mutant cells were used in permeabilized form in order to release some components of the cellular respiration system, thus increasing the selectivity of the cellular respiration response to alcohol (cell/O2-biosensor) Permeabilization also increased sensitivity of the signal and shortened the response time (cell/H2O2-biosensor). Cell/O2 biosensors were linear up to 1.2 mM for ethanol and 0.35 mM for methanol, cell/H2O2 biosensors were linear up to 4.0 mM for ethanol, and 1.2 mM for methanol. Results were reproducible, sample pretreatment was not required, and the sensors exhibited good operational and storage stability. The use of sucrose, dulcitol or inositol during the preparation of the sensors resulted in increased stability of cells during their liophilization and storage in the dried state. Both biosensors had similar selectivity towards alcohols in the order of methanol (100%), ethanol (21%), and formaldehyde (12%). No signal was observed with glucose or glycerol as substrates.
[Show abstract][Hide abstract] ABSTRACT: The paper presents analytical parameters of an enzymatic method for alcohol assay in alcoholic beverages by the use of a new kit based on alcohol oxidase and peroxidase cata-lyzed reactions (AOP). Sensitivity, linearity, accuracy and reliability of the proposed method were tested. It has been shown that results of AOP-assays of alcohol in beers, wines, and strong drinks are in good correlation with those obtained by alcohol dehydrogenase (ADH) method (relations of ADH to AOP values are 0.9197±0.012; correla-tion coefficient R=0.9983; p<0.0001).