dna-based-fish-species-identification-protocol

DNA-based Fish Species Identification Protocol

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. ...

Crystal (Cry) proteins made by the bacterium Bacillus thuringiensis are pore-forming toxins that specifically target insects and nematodes and are used around the world to kill insect pests. To better understand how pore-forming toxins interact with their host, we have screened for Caenorhabditis elegans mutants that resist Cry protein intoxication. We find that Cry toxin resistance involves the loss of two glycosyltransferase genes, bre-2 and bre-4. These glycosyltransferases function in the intestine to confer susceptibility to toxin. Furthermore, they are required for the interaction of active toxin with intestinal cells, suggesting they make an oligosaccharide receptor for toxin. Similarly, the bre-3 resistance gene is also required for toxin interaction with intestinal cells. Cloning of the bre-3 gene indicates it is the C. elegans homologue of the Drosophila egghead (egh) gene. This identification is striking given that the previously identified bre-5 has homology to Drosophila brainiac (brn) and that egh-brn likely function as consecutive glycosyltransferases in Drosophila epithelial cells. We find that, like in Drosophila, bre-3 and bre-5 act in a single pathway in C. elegans. bre-2 and bre-4 are also part of this pathway, thereby extending it. Consistent with its homology to brn, we demonstrate that C. elegans bre-5 rescues the Drosophila brn mutant and that BRE-5 encodes the dominant UDP-GlcNAc:Man GlcNAc transferase activity in C. elegans. Resistance to Cry toxins has uncovered a four component glycosylation pathway that is functionally conserved between nematodes and insects and that provides the basis of the dominant mechanism of resistance in C. elegans.

Resistance to a bacterial toxin is mediated by removal of a conserved glycosylation pathway required for toxin-host interactions.

Der Widerstand gegen ein bakterielles Toxin wird durch Entfernen eines konservierten Glycosylierungswegs f&#xFC;r Toxin-Wirt-Wechselwirkungen vermittelt erforderlich.

The interaction of pathogenic bacteria with a target host is regulated both by bacterial virulence factors and by host components that either protect the host or that promote pathogenesis. The soil nematode Caenorhabditis elegans is a host for a number of bacterial pathogens, as briefly reviewed here. Bacillus thuringiensis (Bt) is a pathogenic bacteria that C. elegans is likely to encounter naturally in the soil. The pore-forming Crystal (Cry) toxins made by Bt are recognized as the dominant virulence factor in this host-pathogen interaction. Forward genetic screens for C. elegans mutants resistant to the Cry toxin, Cry5B, have identified a host carbohydrate structure that promotes pathogenesis. Data suggest this structure is likely to be a Cry5B receptor expressed in the host intestine. This finding is discussed in light of other carbohydrate receptors for bacterial toxins. To investigate host-toxin interactions on a global level, the response of C. elegans to the pore-forming Cry5B is also being investigated by gene transcription profiling (microarrays). These data are beginning to reveal a diverse intracellular response to toxin exposure. To put these investigations in perspective, host responses to other pore-forming toxins are discussed. Investigations with Cry5B in C. elegans show a promising beginning in helping to elucidate host-toxin and host-pathogen interactions.

Pore worms: using Caenorhabditis elegans to study how bacterial toxins interact with their target host.

Pore &#x200B;&#x200B;W&#xFC;rmer: mit Caenorhabditis elegans zu untersuchen, wie bakterielle Toxine mit ihren Ziel-Host interagieren.

Cytolytic pore-forming toxins are important for the virulence of many disease-causing bacteria. How target cells molecularly respond to these toxins and whether or not they can mount a defense are poorly understood. By using microarrays, we demonstrate that the nematode Caenorhabditis elegans responds robustly to Cry5B, a member of the pore-forming Crystal toxin family made by Bacillus thuringiensis. This genomic response is distinct from that seen with a different stressor, the heavy metal cadmium. A p38 mitogen-activated protein kinase (MAPK) kinase and a c-Jun N-terminal-like MAPK are both transcriptionally up-regulated by Cry5B. Moreover, both MAPK pathways are functionally important because elimination of either leads to animals that are (i) hypersensitive to a low, chronic dose of toxin and (ii) hypersensitive to a high, brief dose of toxin such that the animal might naturally encounter in the wild. These results extend to mammalian cells because inhibition of p38 results in the hypersensitivity of baby hamster kidney cells to aerolysin, a pore-forming toxin that targets humans. Furthermore, we identify two downstream transcriptional targets of the p38 MAPK pathway, ttm-1 and ttm-2, that are required for defense against Cry5B. Our data demonstrate that cells defend against pore-forming toxins by means of conserved MAPK pathways.

Mitogen-activated protein kinase pathways defend against bacterial pore-forming toxins.

Mitogen-aktivierten Protein-Kinase-Signalwege gegen bakterielle porenbildende Toxine zu verteidigen.

Caenorhabditis elegans is well suited for toxicological studies owing to its established biology, short generation time, large brood size, and readily scorable life traits. Quantitative parameters of C. elegans that can be assayed include growth, size, progeny production, behavior, and mortality. Qualitative parameters of toxicity, such as changes in appearance or movement, can also be determined. This chapter describes four assays we have used for analyzing the toxic effects of Bacillus thuringiensis crystal proteins toward C. elegans. The assays are quantitative growth measurement, brood size measurement, and determination of lethal concentration, as well as a qualitative health assay based on worm appearance. Although these assays are described for crystal proteins, the approaches are suited for the studies of other toxins with C. elegans.

Assays for toxicity studies in C. elegans with Bt crystal proteins.

Caenorhabditis Elegans eignet sich gut f&uuml;r toxikologische Untersuchungen aufgrund seiner etablierten Biologie, kurze Generationszeit, Brut gro&szlig; und leicht bewertbare Leben Z&uuml;ge. Quantitative Parametern von C. Elegans, die gepr&uuml;ft werden k&ouml;nnen geh&ouml;ren Wachstum, Gr&ouml;&szlig;e, Nachkommen Produktion, Verhalten und Sterblichkeit. Qualit&auml;tsparameter der Toxizit&auml;t, wie etwa &Auml;nderungen in der Darstellung oder Bewegung k&ouml;nnen auch bestimmt werden. Dieses Kapitel beschreibt vier Proben, die wir verwendet haben, f&uuml;r die Analyse der toxischen Wirkung von Bacillus Thuringiensis Kristall Proteine in Richtung C. Elegans. Die Assays sind quantitatives Wachstum Messung, Brut Gr&ouml;&szlig;enmessung und Bestimmung der letale Konzentration sowie eine qualitative Gesundheit-Assay basiert auf Wurm aussehen. Obwohl diese Proben f&uuml;r Kristall Proteine beschrieben sind, eignen sich die Ans&auml;tze f&uuml;r die Studien von anderen Toxinen mit C. Elegans.

Assays f&uuml;r Toxizit&auml;tsstudien in C. Elegans mit Bt-Kristall-Proteine.

Here we present the first global functional analysis of cellular responses to pore-forming toxins (PFTs). PFTs are uniquely important bacterial virulence factors, comprising the single largest class of bacterial protein toxins and being important for the pathogenesis in humans of many Gram positive and Gram negative bacteria. Their mode of action is deceptively simple, poking holes in the plasma membrane of cells. The scattered studies to date of PFT-host cell interactions indicate a handful of genes are involved in cellular defenses to PFTs. How many genes are involved in cellular defenses against PFTs and how cellular defenses are coordinated are unknown. To address these questions, we performed the first genome-wide RNA interference (RNAi) screen for genes that, when knocked down, result in hypersensitivity to a PFT. This screen identifies 106 genes (âˆ¼0.5% of genome) in seven functional groups that protect Caenorhabditis elegans from PFT attack. Interactome analyses of these 106 genes suggest that two previously identified mitogen-activated protein kinase (MAPK) pathways, one (p38) studied in detail and the other (JNK) not, form a core PFT defense network. Additional microarray, real-time PCR, and functional studies reveal that the JNK MAPK pathway, but not the p38 MAPK pathway, is a key central regulator of PFT-induced transcriptional and functional responses. We find C. elegans activator protein 1 (AP-1; c-jun, c-fos) is a downstream target of the JNK-mediated PFT protection pathway, protects C. elegans against both small-pore and large-pore PFTs and protects human cells against a large-pore PFT. This in vivo RNAi genomic study of PFT responses proves that cellular commitment to PFT defenses is enormous, demonstrates the JNK MAPK pathway as a key regulator of transcriptionally-induced PFT defenses, and identifies AP-1 as the first cellular component broadly important for defense against large- and small-pore PFTs.

Global functional analyses of cellular responses to pore-forming toxins.

Hier pr&auml;sentieren wir die erste globale Funktionsanalyse von zellul&auml;ren Reaktionen auf die Pore-bildenden Toxine (PFTs). PFTs sind eindeutig wichtiger bakterieller Virulenzfaktoren, bestehend aus die einzelne gr&ouml;&szlig;te Klasse bakterielle Protein Toxine und Bedeutung f&uuml;r die Pathogenese f&uuml;r viele Gram-Positive und Gram-negative Bakterien des Menschen. Ihre Wirkungsweise ist tr&uuml;gerisch einfach, L&ouml;cher in der Plasmamembran von Zellen zu stossen. Die verstreuten Studien bisher PFT-Host Zelle Interaktionen zeigen eine Handvoll Genen zellul&auml;ren Abwehr zu PFTs beteiligt sind. Zellul&auml;ren Abwehrmechanismen gegen PFTs und wie zellul&auml;ren Abwehrmechanismen wie viele Gene beteiligt sind, sind aufeinander abgestimmt, sind unbekannt. Um diese Fragen zu l&ouml;sen, f&uuml;hrten wir den ersten genomweiten RNA-Interferenz (RNAi)-Bildschirm f&uuml;r Gene, die, wenn unten, klopfte &Uuml;berempfindlichkeit gegen eine PFT zur Folge. Dieser Bildschirm identifiziert 106 Gene (&sim;0.5 % des Genoms) in sieben Funktionsgruppen, die Caenorhabditis Elegans von PFT-Angriff zu sch&uuml;tzen. Interaktom Analysen dieser 106 Gene schlagen vor, da&szlig; zwei zuvor identifizierten Mitogen-activated Protein Kinase (MAPK) Wege, eine (p38) studierte im Detail und die anderen (JNK) nicht, ein Kernnetz PFT Verteidigung bilden. Zus&auml;tzliche Microarray, Real-Time PCR und funktionelle Studien zeigen, dass die JNK MAPK-Pathway, aber nicht bei der p38 MAPK Weg, ist ein wichtiger zentralen Regulator der PFT-induzierte transkriptionelle und funktionale Antworten. Finden wir C. Elegans Activator Protein 1 (AP-1, c-jun, c-fos) ist ein nachgeschalteter Ziel des Signalweges Schutz JNK-vermittelte PFT, sch&uuml;tzt gegen klein-Pore und gro&szlig;e Poren PFTs C. Elegans und menschliche Zellen gegen eine gro&szlig;e Pore-PFT sch&uuml;tzt. In-vivo RNAi genomische Studie von PFT Antworten beweist, dass zellul&auml;re Engagement f&uuml;r PFT-Verteidigung enorm ist, die JNK MAPK-Pathway als wichtiger Regulator der transcriptionally induziert PFT Verteidigungsanlagen veranschaulicht und AP-1 als erste zellul&auml;re Komponente weitgehend wichtig f&uuml;r die Verteidigung gegen gro&szlig; - und klein-Pore PFTs identifiziert.

Article	Year
Resistance to a bacterial toxin is mediated by removal of a conserved glycosylation pathway required for toxin-host interactions. The Journal of biological chemistry\| PubMed ID: 12944392	2003
Pore worms: using Caenorhabditis elegans to study how bacterial toxins interact with their target host. International journal of medical microbiology : IJMM\| PubMed ID: 15149037	2004
Mitogen-activated protein kinase pathways defend against bacterial pore-forming toxins. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 15256590	2004
Assays for toxicity studies in C. elegans with Bt crystal proteins. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 16988432	2006
Global functional analyses of cellular responses to pore-forming toxins. PLoS pathogens\| PubMed ID: 21408619	2011

Danielle Huffman

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