Development of a novel PCR assay for the identification of the black yeast, Exophiala (Wangiella) dermatitidis from adult patients with cystic fibrosis (CF)

Cystic fibrosis (CF) patients may suffer increased morbidity and mortality through colonisation, allergy and invasive infection from fungi. The black yeast, Exophiala dermatitidis (synonym Wangiella dermatitidis) has been found with increasing frequency in sputum specimens of CF patients, with reported isolation rates ranging from 1.1 to 15.7%. At present, no diagnostic PCR exists to aid with the clinical laboratory detection and identification of this organism. A novel species-specific PCR-based assay was developed for the detection of E. dermatitidis, based on employment of rDNA operons and interspacer (ITS) regions between these rDNA operons. Two novel primers, (designated ExdF & ExdR) were designed in silico with the aid of computer-aided alignment software and with the alignment of multiple species of Exophiala, as well as with other commonly described yeasts and filamentous fungi within CF sputum, including Candida. Aspergillus and Scedosporium. An amplicon of approximately 455 by was generated, spanning the partial ITS I region - the complete 5.8S rDNA region - partial ITS2 region, employing ExdF (forward primer [16-mer], 5'-CCG CCT ATT CAG GTC C-3' and ExdR (reverse primer [16-mer], 5'-TCT CTC CCA CTC CCG C-3', was employed and optimised on extracted genomic DNA from a well characterised culture of E. dermatitidis, as well as with high quality genomic DNA template from a further 16 unrelated fungi, including Candida albicans, C. dubliniensis, C. parapsilosis, C. glabrata, Scedosporium apiospermum, Penicillium sp., Aspergillus fumigatus, Aspergillus versicolor, Pichia guilliermondii, Rhodotorula sp., Trichosporon sp., Aureobasidium pullulans, Fusarium sp., Mucor hiemalis, Bionectria ochroleuca, Gibberella pulicaris. Results demonstrated that only DNA from E. dermatitidis gave an amplification product of the expected sire, whilst none of the other fungi were amplifiable. Subsequent employment of this primer pair detected this yeast from mycological cultures from 2/50 (4%) adult CF patients. These two patients were the only patients who were previously shown to have a cultural history of E. dermatitidis from their sputum. E. dermatitidis is a slow-growing fungus, which usually takes up to two weeks to culture in the microbiology laboratory and therefore is slow to detect conventionally, with the risk of bacterial overgrowth from common co-habiting pan- and multiresistant bacterial pathogens from sputum. namely Pseudomonas aeruginosa and Burkholderia cepacia complex organisms, hence this species-specific PCR assay may help detect this organism from CF sputum more specifically and rapidly. Overall, employment of this novel assay nay help in the understanding of the occurrence. aetiology and epidemiology of E. dermatitidis, as an emerging fungal agent in patients with CF. (C) 2008 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Total Synthesis of (±)-Phomactin G, a Platelet Activating Factor Antagonist from the Marine Fungus Phoma sp.

A total synthesis of phomactin G (3), which is a central intermediate in the biosynthesis of phomactin A (5) in Phoma sp. is described. The synthesis is based on a Cr(II)/Ni(II) macrocyclisation from the aldehyde vinyl iodide 9, leading to 16, followed by sequential conversion of 16 into the -epoxide 21 and the ketone 25 which, on deprotection, led to (±)-phomactin G. Phomactin G (3) shares an interesting structural homology with phomactin D (2), the most potent PAF-antagonist metabolite in Phoma sp. It is most likely converted into phomactin A (5), by initial allylic oxidation to the transient -alcohol phomactin structure 4, known as Sch 49028, followed by spontaneous pyran ring formation.

A comparative assessment of potential components of partial disease resistance to Fusarium head blight detected in a detached leaf assay of wheat, barley and oats.

The relative resistance of 15 winter barley, three winter wheat and three winter oat cultivars on the UK recommended list 2003 and two spring wheat cultivars on the Irish 2003 recommended list were evaluated using Microdochium nivale in detached leaf assays to further understand components of partial disease resistance (PDR) and Fusarium head blight (FHB) resistance across cereal species. Barley cultivars showed incubation periods comparable to, and latent periods longer than the most FHB resistant Irish and UK wheat cultivars evaluated. In addition, lesions on barley differed from those on wheat as they were not visibly chlorotic when placed over a light box until sporulation occurred, in contrast to wheat cultivars where chlorosis of the infected area occurred when lesions first developed. The pattern of delayed chlorosis of the infected leaf tissue and longer latent periods indicate that resistances are expressed in barley after the incubation period is observed, and that these temporarily arrest the development of mycelium and sporulation. Incubation periods were longer for oats compared to barley or wheat cultivars. However, oat cultivars differed from both wheat and barley in that mycelial growth was observed before obvious tissue damage was detected under macroscopic examination, indicating tolerance of infection rather than inhibition of pathogen development, and morphology of sporodochia differed, appearing less well developed and being much less abundant. Longer latent periods have previously been related to greater FHB resistance in wheat. The present results suggest the longer latent periods of barley and oat cultivars, than wheat, are likely to play a role in overall FHB resistance if under the same genetic control as PDR components expressed in the head. However the limited range of incubation and latent periods observed within barley and oat cultivars evaluated was in contrast with wheat where incubation and latent periods were shorter and more variable among genotypes. The significance of the various combinations of PDR components detected in the detached leaf assay as components of FHB resistance in each crop requires further investigation, particularly with regard to the apparent tolerance of infection in oats and necrosis in barley, after the incubation period is observed, associated with retardation of mycelial growth and sporulation.

Components of resistance to Fusarium head blight in wheat detected in a seed germination assay with Microdochium majus and the relationship to FHB disease development and mycotoxin accumulation from Fusarium graminearum infection.

Components of partial disease resistance (PDR) to fusarium head blight (FHB), detected in a seed-germination assay, were compared with whole-plant FHB resistance of 30 USA soft red winter wheat entries in the 2002 Uniform Southern FHB Nursery. Highly significant (P <0·001) differences between cultivars in the in vitro seed-germination assay inoculated with Microdochium majus were correlated to FHB disease incidence (r = -0·41; P <0·05), severity (r = -0·47; P <0·01), FHB index (r = -0·46; P <0·01), damaged kernels (r = -0·52; P <0·01), grain deoxynivalenol (DON) concentration (r = -0·40; P <0·05) and incidence/severity/kernel-damage index (ISK) (r = -0·45; P <0·01) caused by Fusarium graminearum. Multiple linear regression analysis explained a greater percentage of variation in FHB resistance using the seed-germination assay and the previously reported detached-leaf assay PDR components as explanatory factors. Shorter incubation periods, longer latent periods, shorter lesion lengths in the detached-leaf assay and higher germination rates in the seed-germination assay were related to greater FHB resistance across all disease variables, collectively explaining 62% of variation for incidence, 49% for severity, 56% for F. graminearum-damaged kernels (FDK), 39% for DON and 59% for ISK index. Incubation period was most strongly related to disease incidence and the early stages of infection, while resistance detected in the seed germination assay and latent period were more strongly related to FHB disease severity. Resistance detected using the seed-germination assay was notable as it related to greater decline in the level of FDK and a smaller reduction in DON than would have been expected from the reduction in FHB disease assessed by visual symptoms.

The purification and characterization of phosphonopyruvate hydrolase, a novel carbon-phosphorus bond cleavage enzyme from Variovorax sp. Pal2.

Phosphonopyruvate hydrolase, a novel bacterial carbon-phosphorus bond cleavage enzyme, was purified to homogeneity by a series of chromatographic steps from cell extracts of a newly isolated environmental strain of Variovorax sp. Pal2. The enzyme was inducible in the presence of phosphonoalanine or phosphonopyruvate; unusually, its expression was independent of the phosphate status of the cell. The native enzyme had a molecular mass of 63 kDa with a subunit mass of 31.2 kDa. Activity of purified phosphonopyruvate hydrolase was Co2+-dependent and showed a pH optimum of 6.7–7.0. The enzyme had a Km of 0.53 mM for its sole substrate, phosphonopyruvate, and was inhibited by the analogues phosphonoformic acid, 3-phosphonopropionic acid, and hydroxymethylphosphonic acid. The nucleotide sequence of the phosphonopyruvate hydrolase structural gene indicated that it is a member of the phosphoenolpyruvate phosphomutase/isocitrate lyase superfamily with 41% identity at the amino acid level to the carbon-to-phosphorus bond-forming enzyme phosphoenolpyruvate phosphomutase from Tetrahymena pyriformis. Thus its apparently ancient evolutionary origins differ from those of each of the two carbon-phosphorus hydrolases that have been reported previously; phosphonoacetaldehyde hydrolase is a member of the haloacetate dehalogenase family, whereas phosphonoacetate hydrolase belongs to the alkaline phosphatase superfamily of zinc-dependent hydrolases. Phosphonopyruvate hydrolase is likely to be of considerable significance in global phosphorus cycling, because phosphonopyruvate is known to be a key intermediate in the formation of all naturally occurring compounds that contain the carbon-phosphorus bond.

Structure and kinetics of phosphonopyruvate hydrolase from Variovorax sp. Pal2: New insight into the divergence of catalysis within the PEP mutase/isocitrate lyase superfamily .

Phosphonopyruvate (P-pyr) hydrolase (PPH), a member of the phosphoenolpyruvate (PEP) mutase/isocitrate lyase (PEPM/ICL) superfamily, hydrolyzes P-pyr and shares the highest sequence identity and functional similarity with PEPM. Recombinant PPH from Variovorax sp. Pal2 was expressed in Escherichia coli and purified to homogeneity. Analytical gel filtration indicated that the protein exists in solution predominantly as a tetramer. The PPH pH rate profile indicates maximal activity over a broad pH range.The steady-state kinetic constants determined for a rapid equilibrium ordered kinetic mechanism with Mg+2 binding first (Kd =140 ± 40 M), are kcat = 105 ± 2 s-1 and P-pyr Km = 5 ± 1 M. PEP (slow substrate kcat = 2 × 10-4 s-1), oxalate, and sulfopyruvate are competitive inhibitors with Ki values of 2.0 ± 0.1 mM, 17 ± 1 M, and 210 ± 10 M, respectively. Three PPH crystal structures have been determined, that of a ligand-free enzyme, the enzyme bound to Mg2+ and oxalate (inhibitor), and the enzyme bound to Mg2+ and P-pyr (substrate). The complex with the inhibitor was obtained by cocrystallization, whereas that with the substrate was obtained by briefly soaking crystals of the ligand-free enzyme with P-pyr prior to flash cooling. The PPH structure resembles that of the other members of the PEPM/ICL superfamily and is most similar to the functionally related enzyme, PEPM. Each monomer of the dimer of dimers exhibits an (/)8 barrel fold with the eighth helix swapped between two molecules of the dimer. Both P-pyr and oxalate are anchored to the active site by Mg2+. The loop capping the active site is disordered in all three structures, in contrast to PEPM, where the equivalent loop adopts an open or disordered conformation in the unbound state but sequesters the inhibitor from solvent in the bound state. Crystal packing may have favored the open conformation of PPH even when the enzyme was cocrystallized with the oxalate inhibitor. Structure alignment of PPH with other superfamily members revealed two pairs of invariant or conservatively replaced residues that anchor the flexible gating loop. The proposed PPH catalytic mechanism is analogous to that of PEPM but includes activation of a water nucleophile with the loop Thr118 residue.

Crystallization and preliminary X-ray diffraction analysis of naphthalene dioxygenase from Rhodococcus sp strain NCIMB 12038

The three-component naphthalene dioxygenase (NDO) enzyme system carries out the first step in the aerobic degradation of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene by Rhodococcus sp. strain NCIMB 12038. The terminal oxygenase component (naphthalene 1,2-dioxygenase) that catalyzes this reaction belongs to the aromatic ring hydroxylating dioxygenase family and has been crystallized. These enzymes utilize a mononuclear nonheme iron centre to catalyze the addition of dioxygen to their respective substrates. In this reaction, two electrons, two protons and a dioxygen molecule are consumed. The Rhodococcus enzyme has only 33 and 29% sequence identity to the corresponding alpha- and beta-subunits of the NDO system of Pseudomonas putida NCIMB 9816-4, for which the tertiary structure has been reported. In order to determine the three-dimensional structure of the Rhodococcus NDO, diffraction-quality crystals have been prepared by the hanging-drop method. The crystals belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 87.5, b = 144, c = 185.6 Angstrom, alpha = beta = gamma = 90degrees, and diffract to 2.3 Angstrom resolution.