Oxyresveratrol-induced DNA cleavage triggers apoptotic response in Candida albicans.

Oxyresveratrol is a naturally occurring phytoalexin produced by plants in response to infection. Biological activities of oxyresveratrol have been studied such as antioxidant, anticancer and anti-inflammation. However, further antimicrobial activity and its mechanism need to be investigated. This study exhibited growth inhibition against pathogenic fungi and investigated its mode of action. Oxyresveratrol inflicted cleavage on DNA, leading to G2/M phase arrest. DNA damage by oxyresveratrol was not the result of oxidative stress but it was triggered by direct binding to DNA. Oxyresveratrol-treated cells showed an apoptotic pathway characterized by phosphatidylserine exposure, apoptotic volume decrease and metacaspase activation. Mitochondria-associated apoptotic features also appeared. Oxyresveratrol-induced Ca2+ overload led to mitochondrial membrane depolarization and release of cytochrome c from mitochondria to cytosol. In conclusion, oxyresveratrol with DNA-binding affinity induces DNA cleavage, and eventually leads to mitochondria-mediated apoptosis in Candida albicans.

Evaluation of multifarious plant growth promoting traits, antagonistic potential and phylogenetic affiliation of rhizobacteria associated with commercial tea plants grown in Darjeeling, India.

Plant growth promoting rhizobacteria (PGPR) are studied in different agricultural crops but the interaction of PGPR of tea crop is not yet studied well. In the present study, the indigenous tea rhizobacteria were isolated from seven tea estates of Darjeeling located in West Bengal, India. A total of 150 rhizobacterial isolates were screened for antagonistic activity against six different fungal pathogens i.e. Nigrospora sphaerica (KJ767520), Pestalotiopsis theae (ITCC 6599), Curvularia eragostidis (ITCC 6429), Glomerella cingulata (MTCC 2033), Rhizoctonia Solani (MTCC 4633) and Fusarium oxysporum (MTCC 284), out of which 48 isolates were antagonist to at least one fungal pathogen used. These 48 isolates exhibited multifarious antifungal properties like the production of siderophore, chitinase, protease and cellulase and also plant growth promoting (PGP) traits like IAA production, phosphate solubilization, ammonia and ACC deaminase production. Amplified ribosomal DNA restriction analysis (ARDRA) and BOX-PCR analysis based genotyping clustered the isolates into different groups. Finally, four isolates were selected for plant growth promotion study in two tea commercial cultivars TV-1 and Teenali-17 in nursery conditions. The plant growth promotion study showed that the inoculation of consortia of these four PGPR isolates significantly increased the growth of tea plant in nursery conditions. Thus this study underlines the commercial potential of these selected PGPR isolates for sustainable tea cultivation.

Mitochondrial dynamics in the pathogenic mold Aspergillus fumigatus: therapeutic and evolutionary implications.

Mitochondria within eukaryotic cells continuously fuse and divide. This phenomenon is called mitochondrial dynamics and crucial for mitochondrial function and integrity. We performed a comprehensive analysis of mitochondrial dynamics in the pathogenic mold Aspergillus fumigatus. Phenotypic characterization of respective mutants revealed the general essentiality of mitochondrial fusion for mitochondrial genome maintenance and the mold's viability. Surprisingly, it turned out that the mitochondrial rhomboid protease Pcp1 and its processing product, s-Mgm,1 which are crucial for fusion in yeast, are dispensable for fusion, mtDNA maintenance and viability in A. fumigatus. In contrast, mitochondrial fission mutants show drastically reduced growth and sporulation rates and increased heat susceptibility. However, reliable inheritance of mitochondria to newly formed conidia is ensured. Strikingly, mitochondrial fission mutants show a significant and growth condition-dependent increase in azole resistance. Parallel disruption of fusion in a fission mutant partially rescues growth and sporulation defects and further increases the azole resistance phenotype. Taken together, our results indicate an emerging dispensability of the mitochondrial rhomboid protease function in mitochondrial fusion, the suitability of mitochondrial fusion machinery as antifungal target and the involvement of mitochondrial dynamics in azole susceptibility.

Chromosome-level genome map provides insights into diverse defense mechanisms in the medicinal fungus Ganoderma sinense.

Fungi have evolved powerful genomic and chemical defense systems to protect themselves against genetic destabilization and other organisms. However, the precise molecular basis involved in fungal defense remain largely unknown in Basidiomycetes. Here the complete genome sequence, as well as DNA methylation patterns and small RNA transcriptomes, was analyzed to provide a holistic overview of secondary metabolism and defense processes in the model medicinal fungus, Ganoderma sinense. We reported the 48.96 Mb genome sequence of G. sinense, consisting of 12 chromosomes and encoding 15,688 genes. More than thirty gene clusters involved in the biosynthesis of secondary metabolites, as well as a large array of genes responsible for their transport and regulation were highlighted. In addition, components of genome defense mechanisms, namely repeat-induced point mutation (RIP), DNA methylation and small RNA-mediated gene silencing, were revealed in G. sinense. Systematic bioinformatic investigation of the genome and methylome suggested that RIP and DNA methylation combinatorially maintain G. sinense genome stability by inactivating invasive genetic material and transposable elements. The elucidation of the G. sinense genome and epigenome provides an unparalleled opportunity to advance our understanding of secondary metabolism and fungal defense mechanisms.

Yeast Pif1 accelerates annealing of complementary DNA strands.

Pif1 is a helicase involved in the maintenance of nuclear and mitochondrial genomes in eukaryotes. Here we report a new activity of Saccharomyces cerevisiae Pif1, annealing of complementary DNA strands. We identified preferred substrates for annealing as those that generate a duplex product with a single-stranded overhang relative to a blunt end duplex. Importantly, we show that Pif1 can anneal DNA in the presence of ATP and Mg(2+). Pif1-mediated annealing also occurs in the presence of single-stranded DNA binding proteins. Additionally, we show that partial duplex substrates with 3'-single-stranded overhangs such as those generated during double-strand break repair can be annealed by Pif1.

Genotoxicity of tri- and hexavalent chromium compounds in vivo and their modes of action on DNA damage in vitro.

Chromium occurs mostly in tri- and hexavalent states in the environment. Hexavalent chromium [Cr(VI)] compounds are extensively used in diverse industries, and trivalent chromium [Cr(III)] salts are used as micronutrients and dietary supplements. In the present work, we report that they both induce genetic mutations in yeast cells. They both also cause DNA damage in both yeast and Jurkat cells and the effect of Cr(III) is greater than that of Cr(VI). We further show that Cr(III) and Cr(VI) cause DNA damage through different mechanisms. Cr(VI) intercalates DNA and Cr(III) interferes base pair stacking. Based on our results, we conclude that Cr(III) can directly cause genotoxicity in vivo.

Kinetic mechanism of DNA translocation by the RSC molecular motor.

ATP-dependent nucleosome repositioning by chromatin remodeling enzymes requires the translocation of these enzymes along the nucleosomal DNA. Using a fluorescence stopped-flow assay we monitored DNA translocation by a minimal RSC motor and through global analysis of these time courses we have determined that this motor has a macroscopic translocation rate of 2.9 bp/s with a step size of 1.24 bp. From the complementary quantitative analysis of the associated time courses of ATP consumption during DNA translocation we have determined that this motor has an efficiency of 3.0 ATP/bp, which is slightly less that the efficiency observed for several genetically related DNA helicases and which likely results from random pausing by the motor during translocation. Nevertheless, this motor is able to exert enough force during translocation to displace streptavidin from biotinylated DNA. Taken together these results are the necessary first step for quantifying both the role of DNA translocation in nucleosome repositioning by RSC and the efficiency at which RSC couples ATP binding and hydrolysis to nucleosome repositioning.

Molecular identification and characterization of ileal and cecal fungus communities in broilers given probiotics, specific essential oil blends, and under mixed Eimeria infection.

Broiler digestive tract fungal communities have gained far less scrutiny than that given corresponding bacterial communities. Attention given poultry-associated fungi have focused primarily on feed-associated toxin-producers, yeast, and yeast products. The current project focused on the use of pyrosequencing and denaturing gradient gel electrophoresis (DGGE) to identify and monitor broiler digestive fungal communities. Eight different treatments were included. Four controls were an Uninfected-Unmedicated Control, an Unmedicated-Infected Control, the antibiotic bacitracin methylene disalicylate plus the ionophore monensin as Positive Control, and the ionophore monensin alone as a Negative Control. Four treatments were two probiotics (BC-30 and Calsporin) and two specific essential oil blends (Crina Poultry Plus and Crina Poultry AF). All chickens except the Unmedicated-Uninfected Control were given, at 15 days of age, a standard oral Eimeria inoculum of sporulated oocysts. Ileal and cecal digesta were collected at pre-Eimeria infection at 14 days of age and at 7 days post-Eimeria infection at 22 days of age. Extracted cecal DNA was analyzed by pyrosequencing to examine the impact of diet supplements and Eimeria infection on individual constituents in the fungal community, while DGGE was used to compare more qualitative changes in ileal and cecal communities. Pyrosequencing identified three phyla, seven classes, eight orders, 13 families, 17 genera, and 23 fungal species. Ileal and cecal DGGE patterns showed fungal communities were clustered mainly into pre- and post-infection patterns. Post-infection Unmedicated-Uninfected patterns were clustered with pre-infection groups demonstrating a strong effect of Eimeria infection on digestive fungal populations. These combined techniques offered added versatility towards unraveling the effects of enteropathogen infection and performance enhancing feed additives on broiler digestive microflora.

Antifungal activity of Coriandrum sativum essential oil, its mode of action against Candida species and potential synergism with amphotericin B.

The increasing incidence of drug-resistant pathogens and toxicity of existing antifungal compounds has drawn attention towards the antimicrobial activity of natural products. The aim of the present study was to evaluate the antifungal activity of coriander essential oil according to classical bacteriological techniques, as well as with flow cytometry. The effect of the essential oil upon germ tube formation, seen as an important virulence factor, and potential synergism with amphotericin B were also studied. Coriander essential oil has a fungicidal activity against the Candida strains tested with MLC values equal to the MIC value and ranging from 0.05 to 0.4% (v/v). Flow cytometric evaluation of BOX, PI and DRAQ5 staining indicates that the fungicidal effect is a result of cytoplasmic membrane damage and subsequent leakage of intracellular components such as DNA. Also, concentrations bellow the MIC value caused a marked reduction in the percentage of germ tube formation for C. albicans strains. A synergetic effect between coriander oil and amphotericin B was also obtained for C. albicans strains, while for C. tropicalis strain only an additive effect was observed. This study describes the antifungal activity of coriander essential oil on Candida spp., which could be useful in designing new formulations for candidosis treatment.

False-positive Aspergillus real-time PCR assay due to a nutritional supplement in a bone marrow transplant recipient with GVH disease.

PCR screening for circulating DNA, especially when combined with antigen testing, has shown promise for the definitive diagnosis of invasive aspergillosis. False positives for Aspergillus real-time PCR assays have been described in several reports, but no sources of fungal DNA contamination could be clearly identified. We report a false-positive case for both galactomannan (GM) antigenemia and Aspergillus PCR due to nutritional supplement intake in a bone marrow transplant recipient with digestive graft-versus-host disease. Our case report also suggests that fungal DNA can pass into the serum from the intestinal tract in the same way as fungal GM. Clinicians should be aware of this possibility, so that the administration of costly, unnecessary antifungal treatments with potential adverse side-effects can be avoided.

Movement of newly assimilated 13C carbon in the grass Lolium perenne and its incorporation into rhizosphere microbial DNA.

One of the key processes that drives rhizosphere microbial activity is the exudation of soluble organic carbon (C) by plant roots. We describe an experiment designed to determine the impact of defoliation on the partitioning and movement of C in grass (Lolium perenne L.), soil and grass-sterile sand microcosms, using a (13)CO(2) pulse-labelling method. The pulse-derived (13)C in the shoots declined over time, but that of the roots remained stable throughout the experiment. There were peaks in the atom% (13)C of rhizosphere CO(2) in the first few hours after labelling probably due to root respiration, and again at around 100 h. The second peak was only seen in the soil microcosms and not in those with sterilised sand as the growth medium, indicating possible microbial activity. Incorporation of the (13)C label into the microbial biomass increased at 100 h when incorporation into replicating cells, as indicated by the amounts of the label in the microbial DNA, started to increase. These results indicate that the rhizosphere environment is conducive to bacterial growth and replication. The results also show that defoliation had no impact on the pattern of movement of (13)C from plant roots into the microbial population in the rhizosphere.

Transcriptional profiling reveals genes in the human pathogen Trichophyton rubrum that are expressed in response to pH signaling.

Trichophyton rubrum is a dermatophyte that infects human skin and nails. Its growth on keratin as its carbon source shifts the ambient pH from acidic to alkaline, which may be an efficient strategy for its successful infection and maintenance in the host. In this study, we used suppression subtractive hybridization to identify genes preferentially expressed in T. rubrum incubated at either pH 5.0 or pH 8.0. The functional grouping of the 341 overexpressed unigenes indicated proteins putatively involved in diverse cellular processes, such as membrane remodeling, cellular transport, metabolism, cellular protection, fungal pathogenesis, gene regulation, interaction with the environment, and iron uptake. Although the basic metabolic machinery identified under both growth conditions seems to be functionally similar, distinct genes are upregulated at acidic or alkaline pHs. We also isolated a large number of genes of unknown function, probably unique to T. rubrum or dermatophytes. Interestingly, the transcriptional profiling of several genes in a pacC(-) mutant suggests that, in T. rubrum, the transcription factor PacC has a diversity of metabolic functions, in response to either acidic or alkaline ambient pH.

Salicylic acid and salicylic acid glucoside in xylem sap of Brassica napus infected with Verticillium longisporum.

Salicylic acid (SA) and its glucoside (SAG) were detected in xylem sap of Brassica napus by HPLC-MS. Concentrations of SA and SAG in xylem sap from the root and hypocotyl of the plant, and in extracts of shoots above the hypocotyl, increased after infection with the vascular pathogen Verticillium longisporum. Both concentrations were correlated with disease severity assessed as the reduction in shoot length. Furthermore, SAG levels in shoot extracts were correlated with the amount of V. longisporum DNA in the hypocotyls. Although the concentration of SAG (but not SA) in xylem sap of infected plants gradually declined from 14 to 35 days post infection, SAG levels remained significantly higher than in uninfected plants during the whole experiment. Jasmonic acid (JA) and abscisic acid (ABA) levels in xylem sap were not affected by infection with V. longisporum. SA and SAG extend the list of phytohormones potentially transported from root to shoot with the transpiration stream. The physiological relevance of this transport and its contribution to the distribution of SA in plants remain to be elucidated.

Linking uracil base excision repair and 5-fluorouracil toxicity in yeast.

5-fluorouracil (5-FU) is a widely used anticancer drug that disrupts pyrimidine nucleotide pool balances and leads to uracil incorporation in DNA, which is then recognized and removed by the uracil base excision repair (BER) pathway. Using complementary biochemical and genetic approaches we have examined the role of uracil BER in the cell killing mechanism of 5-FU. A yeast strain lacking the enzyme uracil DNA glycosylase (Ung1), which excises uracil from the DNA backbone leaving an abasic site, showed significant protection against the toxic effects of 5-FU, a G1/S cell cycle arrest phenotype, and accumulated massive amounts of U/A base pairs in its genome (approximately 4% of T/A pairs were now U/A). A strain lacking the major abasic site endonuclease of Saccharomyces cerevisiae (Apn1) showed significantly increased sensitivity to 5-FU with G2/M arrest. Thus, efficient processing of abasic sites by this enzyme is protective against the toxic effects of 5-FU. However, contrary to expectations, the Apn1 deficient strain did not accumulate intact abasic sites, indicating that another repair pathway attempts to process these sites in the absence Apn1, but that this process has catastrophic effects on genome integrity. These findings suggest that new strategies for chemical intervention targeting BER could enhance the effectiveness of this widely used anticancer drug.

Evaluation of differential gene expression in fluconazole-susceptible and -resistant isolates of Candida albicans by cDNA microarray analysis.

The opportunistic fungal pathogen Candida albicans is the major causative agent of oropharyngeal candidiasis (OPC) in AIDS. The development of azoles, such as fluconazole, for the treatment of OPC has proven effective except in cases where C. albicans develops resistance to fluconazole during the course of treatment. In the present study, we used microarray technology to examine differences in gene expression from a fluconazole-susceptible and a fluconazole-resistant well-characterized, clinically obtained matched set of C. albicans isolates to identify genes which are differentially expressed in association with azole resistance. Among genes found to be differentially expressed were those involved in amino acid and carbohydrate metabolism; cell stress, cell wall maintenance; lipid, fatty acid, and sterol metabolism; and small molecule transport. In addition to CDR1, which has previously been demonstrated to be associated with azole resistance, the drug resistance gene RTA3, the ergosterol biosynthesis gene ERG2, and the cell stress genes CRD2, GPX1, and IFD5 were found to be upregulated. Several genes, such as the mitochondrial aldehyde dehydrogenase gene ALD5, the glycosylphosphatidylinositol synthesis gene GPI1, and the iron transport genes FET34 and FTR2 were found to be downregulated. Further study of these differentially regulated genes is warranted to evaluate how they may be involved in azole resistance. In addition to these novel findings, we demonstrate the utility of microarray analysis for studying the molecular mechanisms of drug resistance in pathogenic organisms.

Interaction of the GTS1 gene product with glyceraldehyde- 3-phosphate dehydrogenase 1 required for the maintenance of the metabolic oscillations of the yeast Saccharomyces cerevisiae.

We previously reported that GTS1 is involved in regulating ultradian oscillations of the glycolytic pathway induced by cyanide in cell suspensions as well as oscillations of energy metabolism in aerobic continuous cultures. Here, we screened a yeast cDNA library for proteins that bind to Gts1p using the yeast two-hybrid system and cloned multiple TDH cDNAs encoding the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found that the zinc-finger and dimerization sites of Gts1p were required for full ability to bind GAPDH, and Gts1ps mutated at these sites lost the ability to regulate both aerobic and unaerobic ultradian oscillations of energy metabolism. Of the three TDH genes, only TDH1 fluctuated at the mRNA level in continuous culture and its deletion resulted in the disappearance of the oscillation without any affect on growth rate. This loss of biological rhythms in the TDH1-deleted mutant was rescued by the expression of TDH1 but not of TDH2 or TDH3 under the control of the TDH1 promoter. Thus, we hypothesized that Gts1p plays a role in the regulation of metabolic oscillation by interacting with the TDH1 product, GAPDH1, in yeast.

Identification and characterization of transcription factor IIIA from Schizosaccharomyces pombe.

Transcription factor IIIA (TFIIIA) is specifically required for transcription of 5S rRNA genes and is the archetypal C2H2 zinc finger protein. All known vertebrate TFIIIAs have a similar organization: nine zinc fingers, followed by a C-terminal domain of unknown structure. The zinc fingers of Saccharomyces cerevisiae TFIIIA are interrupted between fingers eight and nine by an 81-amino acid spacer. Aside from the amino acids required for zinc finger folding, TFIIIAs from different species are remarkably divergent, whereas the natural binding site, the internal control region of the 5S rRNA gene, is well conserved. We now describe the identification and characterization of TFIIIA from Schizosaccharomyces pombe. This protein is organized differently from its known homologs, in that it contains eight closely spaced zinc fingers, a ninth zinc finger missing a C-terminal Zn2+-coordinating histidine, a 53- amino acid spacer, and an unprecedented tenth zinc finger. We have confirmed the identity of this divergent protein as TFIIIA by showing that it binds specifically and with high affinity to the S.pombe 5S rRNA gene. Comparison of DNase I protection patterns produced by TFIIIA from multiple species suggests a novel mode of DNA recognition by the S.pombe protein. Recombinant S.pombe TFIIIA was also shown to support specific transcription of the 5S rRNA gene in vitro.

[Effects of lead and selenium on telomere binding protein Rap1p, telomerase and telomeric DNA in Saccharomyces cerevisiae].

The effects on S.cerevisiae telomere binding protein Rap1p, telomerase and telomeric DNA by the lead (Pb), the selenium (Se) and Pb + Se were tested respectively in this study. Compared with the control S.cerevisiae after 100 gene rations, the mean telomere length shortened, Rap1p concentration was significantly lower and the secondary structure of Rap1p was disturbed, the telomerase activity was reduced in Pb treated cells. In Se treated cells, telomere length was significantly longer, and telomerase activity expressed higher. The concentration and secondary structure of Rap1p were similar to that of the control. Further more, the viability of Pb treated cells were significantly reduced while cells undergone other three treatments were similar and normal. These results suggest that Pb could damage Rap1p, reduce telomerase activity, resulting in the telomer length shortening and cell death. On the other hand, Se could protect and repair the damage in Rap1p and telomere caused by Pb to some extent.

DNA postreplication repair and mutagenesis in Saccharomyces cerevisiae.

DNA postreplication repair (PRR) is defined as an activity to convert DNA damage-induced single-stranded gaps into large molecular weight DNA without actually removing the replication-blocking lesions. In bacteria such as Escherichia coli, this activity requires RecA and the RecA-mediated SOS response and is accomplished by recombination and mutagenic translesion DNA synthesis. Eukaryotic cells appear to share similar DNA damage tolerance pathways; however, some enzymes required for PRR in eukaryotes are rather different from those of prokaryotes. In the yeast Saccharomyces cerevisiae, PRR is centrally controlled by RAD6 and RAD18, whose products form a stable complex with single-stranded DNA-binding, ATPase and ubiquitin-conjugating activities. PRR can be further divided into translesion DNA synthesis and error-free modes, the exact molecular events of which are largely unknown. This error-free PRR is analogous to DNA damage-avoidance as defined in mammalian cells, which relies on recombination processes. Two possible mechanisms by which recombination participate in PRR to resolve the stalled replication folk are discussed. Recombination and PRR are also genetically regulated by a DNA helicase and are coupled to the cell-cycle. The PRR processes appear to be highly conserved within eukaryotes, from yeast to human.

Indirect micromanipulation of single molecules in water-in-oil emulsion.

Based on real-time observation and micromanipulation, analytical methods for single DNA molecules have been under development for some time. Precise manipulation, however, is still difficult because single molecules are too small for conventional techniques. We have developed a chemical reaction system that uses water droplets in oil as containers of materials. The water droplets can be manipulated by optical force. The manipulation of the water droplets permits the fusion of two selected droplets. This process corresponds to mixing of different samples. We designate this system as "w/o (water-in-oil emulsion) microreactor system", and each droplet can be thought of as a "microreactor". In this system, single molecules can be manipulated readily, as a molecule can be contained in a microm-sized microreactor. The microreactor utilizes extremely small quantities of samples, therefore, reactions are rapid, as diffusion times in the microreactor are very short. The manipulation technique of the microreactors based on optical force has been applied to induce fusion between microreactors loaded with DNA and YOYO, a fluorescent dye that binds to DNA. This fusion induced a rapid binding of YOYO.