Catabolism of 4-hydroxybenzoate in Candida parapsilosis proceeds through initial oxidative decarboxylation by a FAD-dependent 4-hydroxybenzoate 1-hydroxylase

Abstract The first two steps in the catabolism of 4-hydroxybenzoate by the ascomycetous yeast Candida parapsilosis CBS604 were investigated. In contrast to the well-known bacterial pathways and to what was previously assumed, metabolism of 4-hydroxybenzoate in C. parapsilosis proceeds through initial oxidative decarboxylation to give 1,4-dihydroxybenzene. This reaction is catalyzed by a NAD(P)H and FAD-dependent 4-hydroxybenzoate 1-hydroxylase. Further metabolism of 1,4-dihydroxybenzene to the ring-fission substrate 1,2,4-trihydroxybenzene is catalyzed by a NADPH-specific FAD-dependent aromatic hydroxylase acting on phenolic compounds. ¹⁹F-NMR experiments with cell extracts and 2-fluoro-4-hydroxybenzoate as the model compound confirm this metabolic pathway and exclude the alternative pathway proceeding through initial 3-hydroxylation followed by oxidative decarboxylation in the second step.     

Characterisation of a partially purified uracil phosphoribosyltransferase from the opportunistic pathogenCandida albicans

This paper describes for the first time the partial purification and properties of uracil phosphoribosyltransferase (UPRTase) from the yeastCandida albicans. UPRTase was purified 38 fold by acid precipitation, DEAE-Sephacel chromatography and ultrafiltration. Further purification of UPRTase was unsuccessful due to the labile nature of the enzyme and the failure in obtaining satisfactory stabilizing conditions. SDS-PAGE suggested that the enzyme exists as a dimer of two dissimilar subunits with molecular masses of 47 and 38 kDa. The pH optimum for phosphoribosylation was about 7.5 and the optimal Mg⁺⁺ concentration was 2 mM. The kinetics of the enzymes for its substrates, uracil and 5-phosphoribosyl-1-pyrophosphate (PRPP) were determined by measuring initial enzyme velocities over a wide range of concentrations of either substrate at different fixed concentrations of the second substrate. Graphic analysis of the data by Hanes-Woolf plots indicated that the reaction is indistinguishable from a double displacement reaction. Ping pong mechanism has been previously reported for other phosphoribosyltransferases. The enzyme has a low affinity for its substrates (K _m=70.5 and 186 µM for uracil and PRPP, respectively) as compared with those ofE. coli and baker's yeast. Inhibition studies indicate that 5-fluorouracil acts as an alternative substrate for UPRTase with 1.6 times higher specific activity.Abbreviations UPRTase Uracil phosphoribosyltranferase - PRTases phosphoribosyltransferases - PRPP 5-phosphoribosyl-1-pyrophosphate - 5-FC 5-fluorocytosine - 5-FU 5-fluorouracil - PEI polyethyleneimine - DTT dithiothreitol - DMSO dimethyl sulphoxide - PMSF phenylmethylsulphonyl fluoride - UMP uridine mono-phosphate     

A comparative study on cell wall antigens and cell surface hydrophobicity in clinical isolates ofCandida albicans

Characterization of common cell surface-bound antigens inCandida albicans strains, particularly those expressed in the walls of mycelial cells might be useful in the diagnosis of systemic candidiasis. Hence, antigenic similarities among wall proteins and mannoproteins fromC. albicans clinical serotype A and B isolates, were studied using polyclonal (mPAbs) and monoclonal (MAb 4C12) antibodies raised against wall antigens from the mycelial form of a commonC. albicans serotype A laboratory strain (ATCC 26555). Zymolyase digestion of walls isolated from cells of the different strains studied grown at 37°C (germination conditions), released, in all cases, numerous protein and mannoprotein components larger than 100 kDa, along with a 33–34 kDa species. The pattern of major antigens exhibiting reactivity towards the mPAbs preparation was basically similar for all the serotype A and B isolates, though minor strain-specific bands were also observed. The immunodeterminant recognized by MAb 4C12 was found to be absent or present in very low amounts inC. albicans isolates other than the ATCC 26555 strain, yet high molecular weight species similar in size (e.g., 260 kDa) to the wall antigen against which MAb 4C12 was raised, were observed, particularly in wall digests from serotype A strains. Cell surface hydrophobicity, an apparently important virulence factor inC. albicans, of the cell population of each serotype B strain was lower than that of the corresponding serotype A counterparts, which is possibly due to the fact that the former strains exhibited a reduced ability to form mycelial filaments under the experimental conditions used.Abbreviations CSH cell surface hydrophobicity - IIF indirect immunofluorescence     

Enzymatic Resolutions of Heterocyclic Alcohols

The butyrates and acetates of heterocyclic alcohols like 3 - hydroxy tetrahydrofuran and - pyran, 3- and 4 - chromanol as well as the corresponding sulfur heterocycles were hydrolyzed using lipase from Candida rugosa (CRL) and from Pseudomonas cepacia, (PCL). Poor to excellent enantioselectivities were obtained depending on the structure of the substrates. An electrostatic amendment to the steric substrate model for PGL is proposed.     

Isolation of yeast candidates for efficient sophorolipids production: their production potentials associate to their lineage

Eleven biosurfactant-producing strains were newly isolated from environmental samples using a drop-collapse assay and thin-layer chromatography (TLC). According to the TLC analysis, the separation patterns of the glycolipid spots of nine dominant strains corresponded to that of the sophorolipids produced by a Starmerella bombicola type strain. The retention factor values of the spot patterns of two strains were less than those of the others. Two representative major products were purified, and their molecular structures were determined. The major products were identified as diacetylated lactonic and acidic sophorolipids. The fatty acid moieties of both compounds were estimated to be 17-hydroxymethyl hexadecenoic acid. The amounts of glycolipids ranged from 5.0 to 22.9 g/L after 4 d of cultivation. According to a phylogenetic analysis, the strains were identified as Starmerella bombicola and Candida floricola.     

Implementing CRISPR-Cas technologies in conventional and non-conventional yeasts: Current state and future prospects

Within five years, the CRISPR-Cas system has emerged as the dominating tool for genome engineering, while also changing the speed and efficiency of metabolic engineering in conventional (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and non-conventional (Yarrowia lipolytica, Pichia pastoris syn. Komagataella phaffii, Kluyveromyces lactis, Candida albicans and C. glabrata) yeasts.Especially in S. cerevisiae, an extensive toolbox of advanced CRISPR-related applications has been established, including crisprTFs and gene drives. The comparison of innovative CRISPR-Cas expression strategies in yeasts presented here may also serve as guideline to implement and refine CRISPR-Cas systems for highly efficient genome editing in other eukaryotic organisms.     

Antibiotic tetaine — a selective inhibitor of chitin and mannoprotein biosynthesis in Candida albicans

The antibiotic tetaine inhibits in Candida albicans the biosynthesis of two important cell wall constituents, chitin and mannoprotein. This effect is a consequence of inactivation of the enzyme glucosamine-6-phosphate synthetase. Due to the lack of glucosamine-6-phosphate the effective secretion of mannoprotein enzymes, acid phosphatase and invertase, by Candida albicans spheroplasts is inhibited. In the presence of tetaine, probably a modified mannoprotein, lacking a branched polymannan, is synthesized. The antibiotic action decreases the viability of Candida albicans cells, especially that of mycelial forms of this fungus.Abbreviations GlcNAc N-acetyl-d-glucosamine - GlcN-6-P d-glucosamine-6-phosphate - ManNAc N-acetyl-d-mannosamine - -MM -methylmannoside - EGTA 1,2 di/2-aminoethoxy/ethane - N,N,N,N tetra-acetic acid     

Experimental rat vaginal infection with Candida parapsilosis

The experimental vaginopathic potential of Candida parapsilosis was determined in ovariectomized rats maintained under pseudoestrus by estrogen administrations. Of the 3 strains of C. parapsilosis tested, that isolated from the vagina of a woman affected by vulvovaginal candidosis gave a prolonged and sustained experimental vaginitis, not different in extent and duration from that caused by a vaginal isolate of C. albicans from a vaginitis patient. The other two isolates of C. parapsilosis (one from the vagina of an asymptomatic subject and another from soil) were unable to infect rat vagina. Microscopic observations of PAS-stained vaginal smears from rats infected with the vaginopathic isolate of C. parapsilosis showed pronounced adherence of yeasts to exfoliated cells. In addition, this isolate of C. parapsilosis produced an elevated quantity of acid proteinase in vitro.     

Cultivation ofCandida blankii in simulated bagasse hemicellulose hydrolysate

Summary All fourCandida blankii isolates evaluated for growth in simulated bagasse hemicellulose hydrolysate utilized the sugars and acetic acid completely. The utilization ofd-xylose,l-arabinose and acetic acid were delayed by the presence ofd-glucose, but after glucose depletion the other carbon sources were utilized simultaneously. The maximum specific growth rate of 0.36 h^–1 and cell yield of 0.47 g cells/g carbon source assimilate compared with published results obtained withC. utilis. C. blankii appeared superior toC. utilis for biomass production from hemicellulose hydrolysate in that it utilizedl-arabinose and was capable of growth at higher temperatures.