Susceptibility of Candida glabrata biofilms to echinocandins: alterations in the matrix composition

Candidiases are the most recurrent fungal infections, especially among immunosuppressed patients. Although Candida albicans is still the most widespread isolated species, non-Candida albicans Candida species have been increasing. The goal of this work was to determine the susceptibility of C. glabrata biofilms to echinocandins and to evaluate their effect on the biofilm matrix composition, comparing the results with other Candida species. Drug susceptibilities were assessed through the determination of minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC) and minimum biofilm eradication concentration (MBEC) of caspofungin (Csf) and micafugin (Mcf). The β-1,3 glucans content of the matrices was assessed after contact with the drugs. The data suggest that, generally, after contact with echinocandins, the concentration of β-1,3 glucans increased. These adjustments in the matrix composition of C. glabrata biofilms and the chemical differences between Csf and Mcf, seem responsible and may determine the effectivity of the drug responses.     

Green synthesis of selenium nanoparticles and evaluate their effect on the expression of ERG3, ERG11 and FKS1 antifungal resistance genes in Candida albicans and Candida glabrata

Drug resistance in Candida species has been considerably increased in the last decades. Given the opposition to antifungal agents, toxicity and interactions of the antimicrobial drugs, identifying new antifungal agents seems essential. This study assessed the antifungal effects of nanoparticles (NPs) on the standard strains of Candida albicans and Candida glabrata and determined the expression genes, including ERG3, ERG11 and FKS1. Selenium nanoparticles (Se-NPs) were biosynthesized with a standard strain of C. albicans and approved by several methods including, ultraviolet-visible spectrophotometer, X-ray diffraction technique, Fourier-transform infrared analysis, field-emission scanning electron microscopy and EDX diagram. The antifungal susceptibility testing performed the minimum inhibitory concentrations (MICs) using the CLSI M27-A3 and M27-S4 broth microdilution method. The expression of the desired genes was examined by the real-time PCR assay between untreated and treated by antifungal drugs and Se-NPs. The MICs of itraconazole, amphotericin B and anidulafungin against C. albicans and C. glabrata were 64, 16 and 4 µg ml⁻¹. In comparison, reduced the MIC values for samples treated with Se-NPs to 1 and 0·5 µg ml⁻¹. The results obtained from real-time PCR and analysis of the ∆∆C_q values showed that the expression of ERG3, ERG11 and FKS1 genes was significantly down-regulated in Se-NPs concentrations (P < 0·05). This study's evidence implies biosafety Se-NPs have favourable effects on the reducing expression of ERG3, ERG11 and FKS1 antifungal resistance genes in C. albicans and C. glabrata.     

Atomic structure of the nuclear pore complex targeting domain of a Nup116 homologue from the yeast, Candida glabrata

The nuclear pore complex (NPC), embedded in the nuclear envelope, is a large, dynamic molecular assembly that facilitates exchange of macromolecules between the nucleus and the cytoplasm. The yeast NPC is an eightfold symmetric annular structure composed of ～456 polypeptide chains contributed by ～30 distinct proteins termed nucleoporins. Nup116, identified only in fungi, plays a central role in both protein import and mRNA export through the NPC. Nup116 is a modular protein with N‐terminal “FG” repeats containing a Gle2p‐binding sequence motif and a NPC targeting domain at its C‐terminus. We report the crystal structure of the NPC targeting domain of Candida glabrata Nup116, consisting of residues 882–1034 [CgNup116(882–1034)], at 1.94 Å resolution. The X‐ray structure of CgNup116(882–1034) is consistent with the molecular envelope determined in solution by small‐angle X‐ray scattering. Structural similarities of CgNup116(882–1034) with homologous domains from Saccharomyces cerevisiae Nup116, S. cerevisiae Nup145N, and human Nup98 are discussed. Proteins 2012; © 2012 Wiley Periodicals, Inc.     

Establishment of a transgenic yeast screening system for estrogenicity and identification of the anti-estrogenic activity of malachite green

Endocrine disruptors refer to chemical compounds in the environment which interfere with the endocrine systems of organisms. Among them, environmental estrogens pose serious problems to aquatic organisms, in particular fish. It is therefore important and necessary to have a fast and low-cost system to screen the large number of different chemical compounds in the aquatic environment for their potential endocrine disrupting actions. In this study, a screening platform was developed to detect xenoestrogens in the aquatic environment using the fission yeast Schizosaccharomyces pombe, and applied for compound screening. The aim was to demonstrate any significant potential differences between the fish screening system and the human screening system. To this end, a yeast expression vector harboring a fish estrogen receptor alpha and a reporter vector containing the estrogen responsive element fused with the Escherichia coli LacZ gene were constructed. After transformation with these two vectors, the transformed yeast clones were confirmed by Western blotting and selected on the basis of the beta-galactosidase activity. In this transgenic yeast system, the natural estrogen (estradiol) and other known xenoestrogens such as diethylstilbestrol, bisphenol A, genistein and dichloro-diphenyl-trichloroethane exhibited dose-dependent activities. Using this system, more than 40 putative endocrine disruptors including phytoestrogens, pesticides, herbicides, industrial dyes and other industrial chemicals were screened. Ten of them were demonstrated to exhibit estrogenic actions. Industrial dyes such as malachite green (MG) that disrupt thyroid hormone synthesis are extensively used and are widely distributed in the aquatic environment. Using this system, MG did not show any estrogenic action, but was demonstrated to exhibit anti-estrogenic activity.     

DNA probes for the rapid identification of medically important Candida species using a multianalyte profiling system

Recently, a new flow cytometric technology to detect multiple DNA target sequences in a single microtiter well plate was developed [multianalyte profiling (MAP) System, Luminex Corp., Austin, TX]. DNA probes, directed to the internal transcribed spacer 2 region of ribosomal DNA, were therefore designed to detect and differentiate PCR amplicons from six medically important Candida species using this system. Each probe was covalently linked to one of 100 available microsphere (bead) sets. Biotinylated PCR amplicons were then hybridized to the complementary probe on each bead set. Bound amplicons were detected fluorometrically using a streptavidin-linked reporter dye, R-phycoerythrin. Specific hybridization was noted for all six Candida species probes (mean sample-to-background ratio+/-standard error: Candida albicans, 58.7+/-1.2; Candida tropicalis, 53.2+/-3.8; Candida glabrata, 46.9+/-2.1; Candida parapsilosis, 59.9+/-1.6; Candida krusei, 54.7+/-3.7 vs. 0.9+/-0.03 for all heterologous Candida species DNA targets and vs. 1.0+/-0.1 for samples containing water instead of DNA; P < 0.001). The limit of test sensitivity was 0.5 pg of DNA. A sample could be processed and analyzed within 1 h post-PCR amplification. Therefore, the multianalyte profiling system was rapid, sensitive and specific for the detection and differentiation of the most medically important species of Candida.     

Development of a set of plasmid vectors for genetic manipulations of the pathogenic yeast Candida parapsilosis

A system for genetic transformation of the yeast Candida parapsilosis, recently developed in our laboratory, opened a venue for investigation of this pathogenic species at the molecular level. In this study we extend the range of available experimental tools by construction of a genomic DNA library suitable for screening and isolation of genes by functional complementation of yeast mutants and a set of replicative plasmid vectors for genetic manipulation of C. parapsilosis cells. The plasmids are based on auxotrophic (CpGAL1, CpURA3, CpMET2, CpLYS4) and dominant (CaIMH3) selection markers. In addition, we constructed plasmid derivatives containing reporter genes yEGFP3 and KlLAC4 coding for enhanced version of the green fluorescent protein and Kluyveromyces lactis beta-galactosidase, respectively. The vectors facilitate propagation and expression of cloned genes in C. parapsilosis cells and allow intracellular localization of gene products and/or monitoring the activity of promoter sequences.     

OsCD1 encodes a putative member of the cellulose synthase-like D sub-family and is essential for rice plant architecture and growth

The cell wall plays important roles in plant architecture and morphogenesis. The cellulose synthase-like super-families were reported to contain glycosyltransferases motif and are required for the biosynthesis of cell wall polysaccharides. Here, we describe a curled leaf and dwarf mutant, cd1, in rice, which exhibits multiple phenotypic traits such as the reduction of plant height and leaf width, curled leaf morphology and a decrease in the number of grains and in the panicle length. Map-based cloning indicates that a member of the cellulose synthase-like D (CSLD) group is a candidate for OsCD1. RNAi transgenic plants with the candidate CSLD gene display a similar phenotype to the cd1 mutant, suggesting that OsCD1 is a member of the CSLD sub-family. Furthermore, sequence analysis indicates that OsCD1 contains the common D,D,D,QXXRW motif, which is a feature of the cellulose synthase-like super-family. Analysis of OsCD1 promoter with GUS fusion expression shows that OsCD1 exhibits higher expression in young meristem tissues such as fresh roots, young panicle and stem apical meristem. Cell wall composition analysis reveals that cellulose content and the level of xylose are significantly reduced in mature culm owing to loss of OsCD1 function. Take together, the work presented here is useful for expanding the understanding of cell wall biosynthesis.     

Analysis of PEAM4, the pea AP1 functional homologue, supports a model for AP1-like genes controlling both floral meristem and floral organ identity in different plant species

APETALA1 (AP1) and its homologue SQUAMOSA (SQUA) are key regulatory genes specifying floral meristem identity in the model plants Arabidopsis and Antirrhinum. Despite many similarities in their sequence, expression and functions, only AP1 appears to have the additional role of specifying sepal and petal identity. No true AP1/SQUA-functional homologues from any other plant species have been functionally studied in detail, therefore the question of how the different functions of AP1-like genes are conserved between species has not been addressed. We have isolated and characterized PEAM4, the AP1/SQUA-functional homologue from pea, a plant with a different floral morphology and inflorescence architecture to that of Arabidopsis or Antirrhinum. PEAM4 encodes for a polypeptide 76% identical to AP1, but lacks the C-terminal prenylation motif, common to AP1 and SQUA, that has been suggested to control the activity of AP1. Nevertheless, constitutive expression of PEAM4 caused early flowering in tobacco and Arabidopsis. In Arabidopsis, PEAM4 also caused inflorescence-to-flower transformations similar to constitutive AP1 expression, and was able to rescue the floral organ defects of the strong ap1-1 mutant. Our results suggest that the control of both floral meristem and floral organ identity by AP1 is not restricted to Arabidopsis, but is extended to species with diverse floral morphologies, such as pea.     

PlantRGS: a web server for the identification of most suitable candidate reference genes for quantitative gene expression studies in plants

Normalization of quantitative gene expression data with a suitable reference gene is essential for accurate and reliable results. However, the availability and choice of most suitable reference gene(s) showing uniform expression across all the experimental conditions remain a drawback. We have developed a web server, PlantRGS (http://www.nipgr.res.in/PlantRGS), for the identification of most suitable candidate reference gene(s) at the whole-genome level using microarray data for quantitative gene expression studies in plants. Microarray data from more than 11 000 tissue samples for nine plant species have been included in the PlantRGS for meta-analysis. The web server provides a user-friendly graphical user interface-based analysis tool for the identification of most suitable reference genes in the selected plant species under user-defined experimental conditions. Various parameter options and output formats will help users to investigate desired number of most suitable reference genes with wide range of expression levels. Validation of results revealed that novel reference genes identified by the PlantRGS outperforms the traditionally used reference genes in terms of expression stability. We anticipate that the PlantRGS will provide a platform for the identification of most suitable reference gene(s) under given experimental conditions and facilitate quantitative gene expression studies in plants.     

Design, total synthesis, and functional overexpression of the Candida rugosa lip1 gene coding for a major industrial lipase.

The dimorphic yeast Candida rugosa has an unusual codon usage that hampers the functional expression of genes derived from this yeast in a conventional heterologous host. Commercial samples of C. rugosa lipase (CRL) are widely used in industry, but contain several different isoforms encoded by the lip gene family, among which the isoform encoded by the gene lip1 is the most prominent. In a first laborious attempt, the lip1 gene was systematically modified by site-directed mutagenesis to gain functional expression in Saccharomyces cerevisiae. As alternative approach, the gene (1647 bp) was completely synthesized with an optimized nucleotide sequence in terms of heterologous expression in yeast and simplified genetic manipulation. The synthetic gene was functionally expressed in both hosts S. cerevisiae and Pichia pastoris, and the effect of heterologous leader sequences on expression and secretion was investigated. In particular, using P. pastoris cells, the synthetic gene was functionally overexpressed, allowing for the first time to produce recombinant Lipl of high purity at a level of 150 U/mL culture medium. The physicochemical and catalytic properties of the recombinant lipase were compared with those of a commercial, nonrecombinant C. rugosa lipase preparation containing lipase isoforms.     

Mitogen activated protein kinase-1 and cell division control protein-42 are putative targets for the binding of novel natural lead molecules: a therapeutic intervention against Candida albicans

Abstract

Candida albicans, fungal yeast causes several lethal infections in immune-suppressed patients and recently emerged as drug-resistant pathogens worldwide. The present study aimed to screen putative drug targets of Candia albicans and to study the binding potential of novel natural lead compounds towards these targets by computational virtual screening and molecular dynamic (MD) simulation. Through extensive analysis of mitogen-activated protein kinase (MAPK) signalling pathways, mitogen-activated protein kinase-1 (HOG1) and cell division control protein-42 (CDC42) genes were prioritized as putative targets based on their virulent functions. The three-dimensional structures of these genes, not available in their native forms, were computationally modeled and validated. 76 lead molecules from various natural sources were screened and their drug likeliness and pharmacokinetic features were predicted. Among these ligands, two lead molecules that demonstrated ideal drug-likeliness and pharmacokinetic features were docked against HOG1 and CDC42 and their binding potential was compared with the binding of conventional drug Fluconazole with their usual target. The prediction was computationally validated by MD simulation. The current study revealed that Cudraxanthone-S present in Cudrania cochinchinensis and Scutifoliamide-B present in Piper scutifolium exhibited ideal drug likeliness, pharmacokinetics and binding potential to the prioritized targets in comparison with the binding of Fluconazole and their usual target. MD simulation showed that CDC42-Cudraxanthone-S and HOG1-Scutifoliamide-B complexes were exhibited stability throughout MD simulation. Thus, the study provides significant insight into employing HOG1 and CDC42 of MAPK as putative drug targets of C. albicans and Cudraxanthone-S and Scutifoliamide-B as potential inhibitors for drug discovery.

Communicated by Ramaswamy H. Sarma     

Cloning and characterization of an acyl-CoA-dependent diacylglycerol acyltransferase 1 (DGAT1) gene from Tropaeolum majus, and a study of the functional motifs of the DGAT protein using site-directed mutagenesis to modify enzyme activity and oil content

SUMMARY: A full-length cDNA encoding a putative diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) was obtained from Tropaeolum majus (garden nasturtium). The 1557-bp open reading frame of this cDNA, designated TmDGAT1, encodes a protein of 518 amino acids showing high homology to other plant DGAT1s. The TmDGAT1 gene was expressed exclusively in developing seeds. Expression of recombinant TmDGAT1 in the yeast H1246MATalpha quadruple mutant (DGA1, LRO1, ARE1, ARE2) restored the capability of the mutant host to produce triacylglycerols (TAGs). The recombinant TmDGAT1 protein was capable of utilizing a range of (14)C-labelled fatty acyl-CoA donors and diacylglycerol acceptors, and could synthesize (14)C-trierucin. Collectively, these findings confirm that the TmDGAT1 gene encodes an acyl-CoA-dependent DGAT1. In plant transformation studies, seed-specific expression of TmDGAT1 was able to complement the low TAG/unusual fatty acid phenotype of the Arabidopsis AS11 (DGAT1) mutant. Over-expression of TmDGAT1 in wild-type Arabidopsis and high-erucic-acid rapeseed (HEAR) and canola Brassica napus resulted in an increase in oil content (3.5%-10% on a dry weight basis, or a net increase of 11%-30%). Site-directed mutagenesis was conducted on six putative functional regions/motifs of the TmDGAT1 enzyme. Mutagenesis of a serine residue in a putative SnRK1 target site resulted in a 38%-80% increase in DGAT1 activity, and over-expression of the mutated TmDGAT1 in Arabidopsis resulted in a 20%-50% increase in oil content on a per seed basis. Thus, alteration of this putative serine/threonine protein kinase site can be exploited to enhance DGAT1 activity, and expression of mutated DGAT1 can be used to enhance oil content.     

Development of a system for the on‐line measurement of carbon dioxide production in microbioreactors: Application to aerobic batch cultivations of Candida utilis

We developed and applied a conductometric method for the quantitative online measurement of the carbon dioxide (CO₂) production during batch cultivations of Candida utilis on a 100‐μL scale. The applied method for the CO₂ measurement consisted of absorption of the produced CO₂ from the exhaust gas of the microbioreactor in an alkali solution, of which the conductivity was measured on‐line. The measured conductivity change of the alkali solution showed a linear relation with the total amount of CO₂ absorbed. After calibration of the CO₂ measurement system, it was connected to a well of a 96‐well microtiter plate. The mixing in the well was achieved by a magnetic stirrer. Using online measurement of the CO₂ production during the cultivation, we show reproducible exponential batch growth of C. utilis on a 100‐μL scale. The CO₂ production measurements obtained from the microcultivation were compared with the CO₂ production measurement in a 4‐L bioreactor equipped with a conventional off‐gas analyzer. The measurements showed that on‐line measurement of the CO₂ production rate in microbioreactors can provide essential data for quantitative physiological studies and provide better understanding of microscale cultivations. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009