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Search Results: 1 - 10 of 193439 matches for " Nick D. Read "
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Septins Are Important for Cell Polarity, Septation and Asexual Spore Formation in Neurospora crassa and Show Different Patterns of Localisation at Germ Tube Tips
Adokiye Berepiki, Nick D. Read
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0063843
Abstract: Septins are GTP-binding cytoskeletal proteins that contribute to cell polarity, vesicle trafficking, cytokinesis and cell morphogenesis. Here we have characterised the six septins encoded by the genome of the model filamentous fungus Neurospora crassa. Analysis of septin null mutants demonstrated that septins limit the sites of emergence of germ tubes and are important for septation and conidiation in N. crassa. Septins constituted a range of different higher-order structures in N. crassa – rings, loops, fibres, bands, and caps – which can co-exist within the same cell. They showed different patterns of localisation at germ tube tips, with GFP-CDC-10 and CDC-11-GFP forming a subapical collar with lower signal intensity at the tip apex, CDC-3-GFP and CDC-12-GFP organized as a cap at the tip apex and GFP-ASP-1 forming an extended subapical collar. Purification of the septin complex and mass spectrometry of isolated proteins revealed that the septin complex consists predominantly of CDC-3, CDC-10, CDC-11 and CDC-12. Immunoprecipitation of the putative septin ASP-1 revealed that this protein interacts with the core septin complex.
Importance of MAP Kinases during Protoperithecial Morphogenesis in Neurospora crassa
Alexander Lichius, Kathryn M. Lord, Chris E. Jeffree, Radek Oborny, Patid Boonyarungsrit, Nick D. Read
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0042565
Abstract: In order to produce multicellular structures filamentous fungi combine various morphogenetic programs that are fundamentally different from those used by plants and animals. The perithecium, the female sexual fruitbody of Neurospora crassa, differentiates from the vegetative mycelium in distinct morphological stages, and represents one of the more complex multicellular structures produced by fungi. In this study we defined the stages of protoperithecial morphogenesis in the N. crassa wild type in greater detail than has previously been described; compared protoperithecial morphogenesis in gene-deletion mutants of all nine mitogen-activated protein (MAP) kinases conserved in N. crassa; confirmed that all three MAP kinase cascades are required for sexual development; and showed that the three different cascades each have distinctly different functions during this process. However, only MAP kinases equivalent to the budding yeast pheromone response and cell wall integrity pathways, but not the osmoregulatory pathway, were essential for vegetative cell fusion. Evidence was obtained for MAP kinase signaling cascades performing roles in extracellular matrix deposition, hyphal adhesion, and envelopment during the construction of fertilizable protoperithecia.
Comparative Live-Cell Imaging Analyses of SPA-2, BUD-6 and BNI-1 in Neurospora crassa Reveal Novel Features of the Filamentous Fungal Polarisome
Alexander Lichius, Mario E. Yá?ez-Gutiérrez, Nick D. Read, Ernestina Castro-Longoria
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0030372
Abstract: A key multiprotein complex involved in regulating the actin cytoskeleton and secretory machinery required for polarized growth in fungi, is the polarisome. Recognized core constituents in budding yeast are the proteins Spa2, Pea2, Aip3/Bud6, and the key effector Bni1. Multicellular fungi display a more complex polarized morphogenesis than yeasts, suggesting that the filamentous fungal polarisome might fulfill additional functions. In this study, we compared the subcellular organization and dynamics of the putative polarisome components BUD-6 and BNI-1 with those of the bona fide polarisome marker SPA-2 at various developmental stages of Neurospora crassa. All three proteins exhibited a yeast-like polarisome configuration during polarized germ tube growth, cell fusion, septal pore plugging and tip repolarization. However, the localization patterns of all three proteins showed spatiotemporally distinct characteristics during the establishment of new polar axes, septum formation and cytokinesis, and maintained hyphal tip growth. Most notably, in vegetative hyphal tips BUD-6 accumulated as a subapical cloud excluded from the Spitzenk?rper (Spk), whereas BNI-1 and SPA-2 partially colocalized with the Spk and the tip apex. Novel roles during septal plugging and cytokinesis, connected to the reinitiation of tip growth upon physical injury and conidial maturation, were identified for BUD-6 and BNI-1, respectively. Phenotypic analyses of gene deletion mutants revealed additional functions for BUD-6 and BNI-1 in cell fusion regulation, and the maintenance of Spk integrity. Considered together, our findings reveal novel polarisome-independent functions of BUD-6 and BNI-1 in Neurospora, but also suggest that all three proteins cooperate at plugged septal pores, and their complex arrangement within the apical dome of mature hypha might represent a novel aspect of filamentous fungal polarisome architecture.
Heterokaryon Incompatibility Is Suppressed Following Conidial Anastomosis Tube Fusion in a Fungal Plant Pathogen
Francine H. Ishikawa, Elaine A. Souza, Jun-ya Shoji, Lanelle Connolly, Michael Freitag, Nick D. Read, M. Gabriela Roca
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0031175
Abstract: It has been hypothesized that horizontal gene/chromosome transfer and parasexual recombination following hyphal fusion between different strains may contribute to the emergence of wide genetic variability in plant pathogenic and other fungi. However, the significance of vegetative (heterokaryon) incompatibility responses, which commonly result in cell death, in preventing these processes is not known. In this study, we have assessed this issue following different types of hyphal fusion during colony initiation and in the mature colony. We used vegetatively compatible and incompatible strains of the common bean pathogen Colletotrichum lindemuthianum in which nuclei were labelled with either a green or red fluorescent protein in order to microscopically monitor the fates of nuclei and heterokaryotic cells following hyphal fusion. As opposed to fusion of hyphae in mature colonies that resulted in cell death within 3 h, fusions by conidial anastomosis tubes (CAT) between two incompatible strains during colony initiation did not induce the vegetative incompatibility response. Instead, fused conidia and germlings survived and formed heterokaryotic colonies that in turn produced uninucleate conidia that germinated to form colonies with phenotypic features different to those of either parental strain. Our results demonstrate that the vegetative incompatibility response is suppressed during colony initiation in C. lindemuthianum. Thus, CAT fusion may allow asexual fungi to increase their genetic diversity, and to acquire new pathogenic traits.
Two Functional Motifs Define the Interaction, Internalization and Toxicity of the Cell-Penetrating Antifungal Peptide PAF26 on Fungal Cells
Alberto Mu?oz, Eleonora Harries, Adriana Contreras-Valenzuela, Lourdes Carmona, Nick D. Read, Jose F. Marcos
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0054813
Abstract: The synthetic, cell penetrating hexapeptide PAF26 (RKKWFW) is antifungal at low micromolar concentrations and has been proposed as a model for cationic, cell-penetrating antifungal peptides. Its short amino acid sequence facilitates the analysis of its structure-activity relationships using the fungal models Neurospora crassa and Saccharomyces cerevisiae, and human and plant pathogens Aspergillus fumigatus and Penicillium digitatum, respectively. Previously, PAF26 at low fungicidal concentrations was shown to be endocytically internalized, accumulated in vacuoles and then actively transported into the cytoplasm where it exerts its antifungal activity. In the present study, two PAF26 derivatives, PAF95 (AAAWFW) and PAF96 (RKKAAA), were designed to characterize the roles of the N-terminal cationic and the C-terminal hydrophobic motifs in PAF26's mode-of-action. PAF95 and PAF96 exhibited substantially reduced antifungal activity against all the fungi analyzed. PAF96 localized to fungal cell envelopes and was not internalized by the fungi. In contrast, PAF95 was taken up into vacuoles of N. crassa, wherein it accumulated and was trapped without toxic effects. Also, the PAF26 resistant Δarg1 strain of S. cerevisiae exhibited increased PAF26 accumulation in vacuoles. Live-cell imaging of GFP-labelled nuclei in A. fumigatus showed that transport of PAF26 from the vacuole to the cytoplasm was followed by nuclear breakdown and dissolution. This work demonstrates that the amphipathic PAF26 possesses two distinct motifs that allow three stages in its antifungal action to be defined: (i) its interaction with the cell envelope; (ii) its internalization and transport to vacuoles mediated by the aromatic hydrophobic domain; and (iii) its transport from vacuoles to the cytoplasm. Significantly, cationic residues in PAF26 are important not only for the electrostatic attraction and interaction with the fungal cell but also for transport from the vacuole to the cytoplasm, which coincides with cell death. Peptide containment within vacuoles preserves fungal cells from peptide toxicity.
Microbial genome jambalaya
Timothy D Read
Genome Biology , 2003, DOI: 10.1186/gb-2003-4-4-314
Abstract: In January this year, the horde that descended on New Orleans' French Quarter spoke a language quite different from the local patois, with words such as "glycome" and "pathogenomic". The third ASM/TIGR conference was an opportunity to assess progress in microbial genomics. Perhaps the most striking theme to emerge at the conference was the prevalence of comparative genomic analysis: with whole-genome sequencing becoming accessible to an increasing number of institutions, many bacterial species now have multiple complete sequences. The adaptations of pathogenic microorganisms and how hosts respond to them was another key theme.Julian Parkhill (Sanger Centre, Hinxton, UK) gave a description of the comparison of three closely related, completely sequenced genomes: Bordetella bronchiseptica (5.34 megabases, Mb), which causes respiratory disease in a range of animals, and B. pertussis (4.03 Mb) and B. parapertussis (4.73 Mb), both of which cause whooping cough. The more virulent strains show symptoms of recent genome change, especially an increased proportion of pseudogenes and insertion sequences; for example, there are more than 230 almost identical copies of the insertion element IS481 in the B. pertussis genome. Many changes involve loss of genes encoding cell-surface proteins, which may explain the reduced host range of B. pertussis and B. parapertussis compared with B. bronchiseptica.Joseph Heitman (Duke University, Durham, USA) presented an initial look at the basidiomycete fungus Cryptococcus neoformans, three distinct serotypes of which are currently being sequenced at different genome centers. Among the features emerging from comparative genomic analysis mentioned by Heitman was the role of extended mating-type loci in virulence: pathogenesis of C. neoformans is associated with the α mating type, which is controlled by a key gene, Sxi1α. Several posters included analyses of three or more closely-related bacterial genome sequences, including Xanthomonas (D. Park
Prevalence of childhood disability and the characteristics and circumstances of disabled children in the UK: secondary analysis of the Family Resources Survey
Clare M Blackburn, Nick J Spencer, Janet M Read
BMC Pediatrics , 2010, DOI: 10.1186/1471-2431-10-21
Abstract: Data were generated from secondary analysis of the Family Resources Survey, a national UK cross-sectional survey, (2004/5) which had data on 16,012 children aged 0-18 years. Children were defined as disabled if they met the Disability Discrimination Act (DDA) definition (1995 and 2005). Frequency distributions and cross-tabulations were run to establish prevalence estimates, and describe the circumstances of disabled children. To establish the association between individual social and material factors and childhood disability when other factors were controlled for, logistic regression models were fitted on the dependent variable 'DDA defined disability'.7.3% (CI 6.9, 7.7) of UK children were reported by as disabled according to the DDA definition. Patterns of disability differed between sexes with boys having a higher rate overall and more likely than girls to experience difficulties with physical coordination; memory, concentration and learning; communication. Disabled children lived in different personal situations from their non-disabled counterparts, and were more likely to live with low-income, deprivation, debt and poor housing. This was particularly the case for disabled children from black/minority ethnic/mixed parentage groups and lone-parent households. Childhood disability was associated with lone parenthood and parental disability and these associations persisted when social disadvantage was controlled for.These analyses suggest that UK disabled children experience higher levels of poverty and personal and social disadvantage than other children. Further research is required to establish accurate prevalence estimates of childhood disability among different black and minority ethnic groups and to understand the associations between childhood disability and lone parenthood and the higher rates of sibling and parental disability in households with disabled children.There is considerable global concern to reduce the prevalence of childhood disability and to
Shuffling bacterial metabolomes
Brendan Thomason, Timothy D Read
Genome Biology , 2006, DOI: 10.1186/gb-2006-7-2-204
Abstract: The generation of bacterial genetic diversity through both vertical and horizontal gene transmission is well documented, but the relative contribution of each process has been a subject of much debate [1-7]. Now, because of the availability of many whole-genome sequences, biologists can perform unprecedented comparative analyses, looking at issues ranging from global genomic evolution to the roles of individual genes. A study published recently in Nature Genetics by Pál et al. [8] demonstrates how the powerful methodology of comparative genomics can be applied to the thorny issue of the evolution of bacterial metabolism.The work by Pál et al. [8] is the first large-scale analysis performed so far to elucidate the effect of horizontal gene transfer (HGT) on bacterial metabolic networks. The subject of the study was the model organism Escherichia coli K12, which has the best-characterized metabolism for a bacterium, several tools for in silico metabolic reconstruction, and an abundance of proteobacterial relatives whose genomes have been fully sequenced. Whether E. coli is typical of all, or even most, free-living bacteria will have to be determined in subsequent work, but there is no reason at present to believe otherwise. Pál et al. [8] identified HGT events by determining the presence and absence of genes by comparison with a phylogenetic tree of conserved proteins in 51 proteobacterial species. Unlike the model eukaryote Saccharomyces cerevisiae, the genome of E. coli K12 contains only a few duplicated genes for enzymes in metabolic pathways, and almost all these duplications appear to be ancient [8]. In fact, only one E. coli duplication event is predicted to have occurred since the divergence from the Salmonella lineage, whereas 15-32 genes are estimated to have been horizontally transferred during the same period. The likely reasons for this difference are the large number of mechanisms that facilitate gene transfer within and between bacteria (such as plasmids
De novo Assembly of a 40 Mb Eukaryotic Genome from Short Sequence Reads: Sordaria macrospora, a Model Organism for Fungal Morphogenesis
Minou Nowrousian,Jason E. Stajich,Meiling Chu,Ines Engh,Eric Espagne,Karen Halliday,Jens Kamerewerd,Frank Kempken,Birgit Knab,Hsiao-Che Kuo,Heinz D. Osiewacz,Stefanie P?ggeler,Nick D. Read,Stephan Seiler,Kristina M. Smith,Denise Zickler,Ulrich Kück ,Michael Freitag
PLOS Genetics , 2010, DOI: 10.1371/journal.pgen.1000891
Abstract: Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30–90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in ~4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology.
The pH-Responsive PacC Transcription Factor of Aspergillus fumigatus Governs Epithelial Entry and Tissue Invasion during Pulmonary Aspergillosis
Margherita Bertuzzi equal contributor,Markus Schrettl equal contributor,Laura Alcazar-Fuoli,Timothy C. Cairns,Alberto Mu?oz,Louise A. Walker,Susanne Herbst,Maryam Safari,Angela M. Cheverton,Dan Chen,Hong Liu,Shinobu Saijo,Natalie D. Fedorova,Darius Armstrong-James,Carol A. Munro,Nick D. Read,Scott G. Filler,Eduardo A. Espeso,William C. Nierman,Hubertus Haas,Elaine M. Bignell
PLOS Pathogens , 2014, DOI: doi/10.1371/journal.ppat.1004413
Abstract: Destruction of the pulmonary epithelium is a major feature of lung diseases caused by the mould pathogen Aspergillus fumigatus. Although it is widely postulated that tissue invasion is governed by fungal proteases, A. fumigatus mutants lacking individual or multiple enzymes remain fully invasive, suggesting a concomitant requirement for other pathogenic activities during host invasion. In this study we discovered, and exploited, a novel, tissue non-invasive, phenotype in A. fumigatus mutants lacking the pH-responsive transcription factor PacC. Our study revealed a novel mode of epithelial entry, occurring in a cell wall-dependent manner prior to protease production, and via the Dectin-1 β-glucan receptor. ΔpacC mutants are defective in both contact-mediated epithelial entry and protease expression, and significantly attenuated for pathogenicity in leukopenic mice. We combined murine infection modelling, in vivo transcriptomics, and in vitro infections of human alveolar epithelia, to delineate two major, and sequentially acting, PacC-dependent processes impacting epithelial integrity in vitro and tissue invasion in the whole animal. We demonstrate that A. fumigatus spores and germlings are internalised by epithelial cells in a contact-, actin-, cell wall- and Dectin-1 dependent manner and ΔpacC mutants, which aberrantly remodel the cell wall during germinative growth, are unable to gain entry into epithelial cells, both in vitro and in vivo. We further show that PacC acts as a global transcriptional regulator of secreted molecules during growth in the leukopenic mammalian lung, and profile the full cohort of secreted gene products expressed during invasive infection. Our study reveals a combinatorial mode of tissue entry dependent upon sequential, and mechanistically distinct, perturbations of the pulmonary epithelium and demonstrates, for the first time a protective role for Dectin-1 blockade in epithelial defences. Infecting ΔpacC mutants are hypersensitive to cell wall-active antifungal agents highlighting the value of PacC signalling as a target for antifungal therapy.
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