Nous remercions le Dr Diane Baronas-Lowell pour la lecture critique du manuscrit. Ce travail a été soutenu par un Charles E. Schmidt College FAU des sciences Subvention de démarrage et une bourse d&#39;études du vieillissement de la Fondation médicale Ellison à KJ

1. XLD (xylose-lysine-désoxycholate) des plaques de gélose gélose XLD est un milieu de croissance sélective de Salmonella, qui apparaît comme des colonies noires sur des plaques de gélose XLD. Toutefois, s&#39;il n&#39;y a pas des préoccupations de contamination, une plaque régulière LB peut être substitué. <ol><li> Peser 5,5 g XLD agar et remettre en suspension dans 5 ml d&#39;eau déminéralisée. </li><li> Bien mélanger jusqu&#39;à ce que tous agar est humide. Ajouter 95 ml d&#39;eau déminéralisée jusqu&#39;à ce que tous les grumeaux et le milieu est complètement remises en suspension. </li><li> Faire bouillir le milieu pour dissoudre complètement (ne pas l&#39;autoclave). </li><li> Refroidir le milieu à la température ambiante à 50 ° C. </li><li> Verser 25 ml d&#39;agar dans chacune 95 x 15 mm (diamètre x hauteur) plaque (plaques scellées avec du Parafilm peuvent être conservés à 4 ° C pendant 1 mois). </li></ol> 2. Nématode Croissance moyenne (NGM) ARNi alimentation Plaques C. Préparation d&#39; des plaques NGM elegans a été décrit précédemment une9. Voici une procédure est décrite brièvement à ajouter l&#39;antibiotique ampicilline et l&#39;ARNi inducteur chimique isopropyl β-D-1-thiogalactopyranoside (IPTG) dans le support NGM pour fabriquer les plaques d&#39;alimentation d&#39;ARNi. <ol><li> Dissoudre 3 g de NaCl et 2,5 g Bacto peptone dans 1 L d&#39;eau déminéralisée. </li><li> Ajouter 17 g de gélose Bacto dans les médias. </li><li> Autoclaver le support pendant 45 minutes et refroidir le support à 50 ° C dans un bain d&#39;eau. </li><li> Ajouter les solutions suivantes: 1 ml de cholestérol (5 mg / ml dans de l&#39;éthanol à 95%), 1 ml de 1 M CaCl2, 1 ml 1 M MgSO 4, et 25 ml de 1 M de tampon phosphate de potassium (pH 6,0). Mélangez bien. </li><li> Ajouter 1 ml de 1 M d&#39;IPTG et 500 ul d&#39;ampicilline (100 mg / ml dans de l&#39;eau stérile). </li><li> Bien mélanger la solution et verser dans les 60 x 15 mm (diamètre x hauteur) des plaques de Pétri en utilisant une aide Pipet et 25 ml de pipettes sérologiques suivants procédures stériles. Remplir chaque plaque avec 12 ml de gélose. Les plaques peuvent être conservées à 4 ° C pendant jusqu&#39;à 1 mois. </li></ol> Le gène de l&#39;autophagie essentiel bec-1 est utilisé comme un exemple d&#39;examiner la fonction d&#39;un gène de l&#39;hôte dans la défense contre l&#39;infection à Salmonella. Les procédures expérimentales sont illustrées à la Figure 1 et le Tableau 1. Le protocole de préparation des animaux RNAi-traités pour une infection résulte, avec le jour de chaque étape expérimentale donnée entre parenthèses. <ol><li> Inoculer bec-1 ARNi alimentation et de contrôler le vecteur vide L4440 ARNi nourrir les bactéries en plaçant un flocon de -80 ° C bactéries congelées dans 2 ml de milieu LB additionné de 100 mg / ml d&#39;ampicilline (Jour 1). Répétez cette étape une fois par semaine pendant toute l&#39;expérience d&#39;avoir des bactéries ARNi frais. Rangez la culture dans le réfrigérateur à 4 ° C lorsqu&#39;il n&#39;est pas utilisé. </li><li> Ensemencer 100 ml de culture bactérienne nuit ARNi sur des plaques de RNAi. Préparer trois bec-1 ARNi et commande trois vide vEctor plaques ARNi. Incuber les plaques à 37 ° C pendant la nuit (Jour 2). </li><li> Retirer les plaques d&#39;ARNi de 37 ° C incubateur et les laisser refroidir à la température ambiante sur le banc. Pick-up bien nourris L4 type sauvage hermaphrodites N2 et les transfèrent à Bec-1 ARNi et contrôlent vides vecteur plaques de RNAi. Placez trois vers par plaque, sur des plaques en triple. Le même jour, préparer des plaques ARNi comme décrit dans l&#39;étape 3.2 (Jour 3). </li><li> Incuber les plaques d&#39;ARNi avec des vers dans l&#39;incubateur à 20 ° C pour 36-40 h et de transfert vers Fresh plaques d&#39;ARNi correspondants préparés à l&#39;étape 3.3. Après les vers sont transférés, incuber les plaques à 20 ° C incubateur pendant 64 heures (jour 4). </li></ol> 4. Préparer Salmonella de l&#39;infection <ol><li> Streak Salmonella -80 ° C stock congelé sur une plaque de gélose XLD et incuber la plaque à 37 ° C pendant la nuit (Jour 5 </strong&gt;). </li><li> Choisissez une colonie de Salmonella noir bien isolé et l&#39;inoculer dans 2 ml de milieu LB à 37 ° C avec agitation pendant la nuit (Jour 6). </li><li> Seed 80 ml ​​Salmonella culture de la nuit sur ​​une C. elegans 60 x 15 mm (diamètre x hauteur) plaque de gélose NGM et préparer 6 plaques au total. Incuber les plaques à température ambiante pendant 6 heures. La culture bactérienne doit sécher et former une pelouse sur la plaque (jour 7). </li></ol> 5. Worms Infect ARNi traités avec Salmonella <ol><li> Transfert bec-1 ARNi traités et témoins vides vecteur ARNi traité hermaphrodites L4 N2 (de la descendance des vers mis en place à l&#39;étape 3) des plaques de Salmonella. Placez 40 vers par plaque 3 plaques pour chaque groupe. Incuber les plaques à vis sans fin à 20 ° C pendant 48 heures (Jour 7). </li><li> Préparer un ensemble de plaques d&#39;ARNi frais comme décrit dans les étapes 3.1 et 3.2 (Jour 7 et <strong&gt; Jour 8). </li><li> Après 48 heures de l&#39;infection, transférer vers infectés par Salmonella sur les plaques d&#39;ARNi correspondants préparés à l&#39;étape 5.2 et incuber à 20 ° C (Jour 9). </li></ol> 6. Survival Assay <ol><li> Note la survie des vers tous les jours et transférer vers les plaques ARNi correspondant fraîches pendant la période de ponte. Préparer une série de plaques de RNAi frais avant chaque transfert de ver comme décrit dans les étapes 3.1 et 3.2. Toucher le corps de vis sans fin (de la tête, partie centrale et la queue) délicatement avec un fil de platine fin aplatie. Un ver est marqué comme mort si aucun mouvement du corps du ver est observée. </li><li> Note la survie des vers par jour ou tous les deux jours, et de les transférer vers des plaques ARNi correspondant frais deux fois par semaine après les vers ne pondent. </li><li> Après tous les vers meurent, regrouper les données de survie à partir de plaques en triple comme un ensemble de données. Entrez les données de survie de chaque groupe dans le logiciel statistique approprié tel que GraphPad Prisme pour générer des courbes de survie et d&#39;effectuer de Kaplan-Meier analyse de survie. L&#39;expérience est répétée au moins une fois pour confirmer la conclusion. </li></ol>

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Les auteurs déclarent qu&#39;ils n&#39;ont aucun intérêt financier concurrents.

C. elegans est un simple organisme modèle génétique qui mange des bactéries comme source d&#39;éléments nutritifs. Ainsi, il est aisé de substituer sa nourriture bactérienne normale avec un agent pathogène intestinal pour enquêter sur les interactions entre C. elegans et l&#39;agent pathogène choisi. Ici, un protocole est décrit à combiner infection à Salmonella et C. elegans ARNi alimentation de traitement pour examiner le rôle des gènes de l&#39;hôte dans la défens...

Le nématode du sol vivant librement Caenorhabditis elegans est un organisme simple et génétiquement traitable modèle utilisé pour étudier plusieurs questions biologiques. C. elegans existe dominante comme hermaphrodites auto-fertilisation. Les mâles sont spontanément générées par des non-disjonction du chromosome X durant la gamétogenèse 1,2. En présence d&#39;une alimentation abondante, C. elegans développer en continu à travers quatre stades larvaires à l&#39;âge adulte. La température influe aussi C. développement elegans; On observe un développement plus rapide à des températures plus élevées. En laboratoire, C. elegans est cultivé à une température standard de 20 ° C sur gélose ensemencée avec la bactérie Escherichia coli (souche OP50) que la nourriture 1,2. Dans la dernière décennie, C. elegans est devenu un organisme invertébré pour étudier les interactions hôte-pathogène 3-5. Dans la nature, C. elegans mange des bactéries comme nutrient la source 1,2. Sa nourriture de laboratoire bactérienne normale, OP50, peut être facilement remplacé par d&#39;autres agents pathogènes d&#39;étudier les interactions entre C. elegans et n&#39;importe quel agent pathogène choisi. Dans ces conditions, l&#39;intestin est le site primaire de l&#39;infection. En effet, une large gamme de pathogènes bactériens a été montré pour infecter léthalement C. elegans 5.3. La bactérie Salmonella Gram négatif est un agent pathogène gastro-intestinal qui provoque une maladie d&#39;origine alimentaire humaine dans le monde 6,7. C. elegans est un bon hôte de modèle pour Salmonella typhimurium que cette bactérie se réplique et présente infections intestinales persistantes 8-10. C. elegans a été utilisé pour identifier à la fois roman et les facteurs précédemment connu virulence de Salmonella 11. Fait intéressant, l&#39;C. système immunitaire elegans limite efficacement la réplication de la bactérie Salmonella. Il a été rapporté précédemment que inhibition des gènes de l&#39;autophagie rend réplication accrue de Salmonella dans C. elegans, entraînant la mort précoce de vers infectées 10. Macroautophagie (ci-après appelé autophagie) est un processus dynamique impliquant le réarrangement des membranes intracellulaires de séquestrer cytoplasme et des organites pour la livraison vers le lysosome pour dégradation 12. Autophagy a été rapportée pour limiter la réplication de Salmonella dans C. elegans et chez les mammifères 10,13. Le C. génome elegans a été le premier génome eucaryote multicellulaire séquencé; il est sensible à l&#39;ARNi traitement 14-16. En outre, l&#39;ARNi peut être administré efficacement en soumettant les vers à ingérer des bactéries contenant de l&#39;ARN à double brin du gène cible, connu sous le nom de RNAi alimentation 16,17. Bibliothèques d&#39;alimentation ARNi du génome entier ont été générés pour l&#39;ARNi du génome de dépistage 16,18. Ici, une infection pro Salmonellaprotocole est couplé avec l&#39;ARNi alimentation pour permettre de tester C. gènes elegans d&#39;intérêt pour leur capacité à protéger contre l&#39;infection à Salmonella.

<table border="0" cellpadding="0" cellspacing="0" width="621">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LB Broth</td>
			<td style="width:119px;">Fisher</td>
			<td style="width:119px;">BP9723-500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XLD agar</td>
			<td>EMD Chemicals</td>
			<td>1.05287.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto Agar</td>
			<td>Fisher</td>
			<td>DF0140-01-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peptone</td>
			<td>Fisher</td>
			<td>BP1420-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride</td>
			<td>Fisher</td>
			<td>S671-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium Chloride</td>
			<td>Fisher</td>
			<td>C69-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Sulfate</td>
			<td>Fisher</td>
			<td>M65-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IPTG</td>
			<td>Gold Biotechnology</td>
			<td>12481C50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cholesterol</td>
			<td>Sigma</td>
			<td>C8667-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicillin</td>
			<td>Fisher&#160;</td>
			<td>BP1760-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Salmonella typhimurium</td>
			<td>ATCC</td>
			<td>ATCC14028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dish 95 x 15mm</td>
			<td>Fisher</td>
			<td>FB0875714G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dish 60 x 15mm&#160;</td>
			<td>Fisher</td>
			<td>08-757-13A&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Serological pipet</td>
			<td>Fisher</td>
			<td>13-668-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Express Pipet-Aid</td>
			<td>Fisher</td>
			<td>13-675-42</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MaxQ6000 shaking incubator&#160;</td>
			<td>Thermo Scientific</td>
			<td>SHKE6000-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td>Percival</td>
			<td>I-36DL</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol to infect caenorhabditis elegans with salmonella typhimurium

Dans la dernière décennie, C. elegans est devenu un organisme invertébré pour étudier les interactions entre hôtes et pathogènes, y compris la défense de l&#39;hôte contre les bactéries gram-négatif bactérie Salmonella typhimurium. Salmonella établit une infection persistante dans l&#39;intestin de C. elegans et entraîne la mort précoce des animaux infectés. Un certain nombre de mécanismes de l&#39;immunité ont été identifiés en C. elegans pour se défendre contre les infections à Salmonella. Autophagy, une voie de dégradation lysosomale évolutif conservé, a été montré pour limiter la réplication de Salmonella dans C. elegans et chez les mammifères. Ici, un protocole est décrit à infecter C. elegans avec Salmonella typhimurium, dans laquelle les vers sont exposés à Salmonella pendant une durée limitée, semblable à une infection à Salmonella chez l&#39;homme. infection à Salmonella raccourcit considérablement la durée de vie de C. elegans &lt;/ Em&gt;. Utilisation du gène de l&#39;autophagie essentiel bec-1, par exemple, nous avons combiné cette méthode d&#39;infection à C. elegans ARNi alimentation approche et montré ce protocole peut être utilisé pour examiner la fonction de C. gènes de l&#39;hôte elegans dans la défense contre l&#39;infection à Salmonella. Depuis C. elegans génome des bibliothèques entières de RNAi sont disponibles, ce protocole permet à l&#39;écran complètement pour C. gènes elegans qui protègent contre les salmonelles et d&#39;autres agents pathogènes intestinaux utilisant des bibliothèques de l&#39;ARNi du génome entier.

A 20 ° C, la durée de vie médiane de type sauvage vers N2 est de 17 jours (figure 2A et Tableau 2). L&#39;infection à Salmonella diminue de manière significative la durée de vie médiane de vers N2 à 10,5 jours (p = 0,0002, log-rank test) (figure 2A ). Si un C. gène elegans joue un rôle important dans la défense contre l&#39;infection à Salmonella, il est prévu que son inhibition peut ...

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A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans a émergé comme un nouveau modèle génétique pour étudier les interactions hôte-pathogène. Ici, nous décrivons un protocole pour infecter C. elegans avec Salmonella typhimurium couplé avec la technique d&#39;interférence double-brin ARNi pour étudier le rôle des gènes de l&#39;hôte dans la défense contre l&#39;infection à Salmonella.

Un protocole d&#39;infecter<em&gt; Caenorhabditis elegans</em&gt; Avec<em&gt; Salmonella typhimurium</em

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

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Immunology and Infection

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

11.5K vues.  Florida Atlantic University.  C. elegans a émergé comme un nouveau modèle génétique pour étudier les interactions hôte-pathogène. Ici, nous décrivons un protocole pour infecter C. elegans avec Salmonella typhimurium couplé avec la technique d'interférence double-brin ARNi pour étudier le rôle des gènes de l'hôte dans la défense contre l'infection à Salmonella. 

Vidéo: A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus. However, there are two species, six subspecies, and over 2,500 different Salmonella serovars, and not all are equal in their significance to public health. For example, finding S. enterica subspecies IIIa Arizona on a table egg layer farm is insignificant compared to the isolation of S. enterica subspecies I serovar Enteritidis, the leading cause of salmonellosis linked to the consumption of table eggs. Serovars are identified based on antigenic differences in lipopolysaccharide (LPS)(O antigen) and flagellin (H1 and H2 antigens). These antigenic differences are the outward appearance of the diversity of genes and gene alleles associated with this phenotype.
We have developed an allelotyping, multiplex PCR that keys on genetic differences between four major S. enterica subspecies I serovars found in poultry and associated with significant human disease in the US. The PCR primer pairs were targeted to key genes or sequences unique to a specific Salmonella serovar and designed to produce an amplicon with size specific for that gene or allele. Salmonella serovar is assigned to an isolate based on the combination of PCR test results for specific LPS and flagellin gene alleles. The multiplex PCRs described in this article are specific for the detection of S. enterica subspecies I serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.
Here we demonstrate how to use the multiplex PCRs to identify serovar for a Salmonella isolate.

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

We describe a multiplex PCR for the rapid detection of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. Specific Salmonella serovars can be identified by targeting a multiplex PCR to genes and sequences unique to the O-antigen biosynthesis cluster and flagellin of a given serovar. Serovar is assigned then to a Salmonella isolate based on the appearance of specific, size amplicons (PCR product) corresponding to the target allele.

Une PCR pour l&#39;identification allélotypage Salmonella enterica Sérovars Enteritidis, Hadar, Heidelberg et Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus.  However, there are two species, six subspecies, and over ...

Recherche

Immunologie et infection

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

À haut débit de dosage de phénotype<em&gt; Salmonella enterica</em&gt; Association Typhimurium, Invasion, et la réplication dans les macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Isolement de Salmonella typhimurium-contenant des Phagosomes des Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. Among them, reversible lysine acetylation is one of the most widely distributed in all domains of life. Although numerous mass spectrometry-based acetylome studies have been performed, further characterization of these putative acetylation targets has been limited. One possible reason is that it is difficult to generate purely acetylated proteins at desired positions by most classic biochemical approaches. To overcome this challenge, the genetic code expansion technique has been applied to use the pair of an engineered pyrrolysyl-tRNA synthetase variant, and its cognate tRNA from Methanosarcinaceae species, to direct the cotranslational incorporation of acetyllysine at the specific site in the protein of interest. After first application in the study of histone acetylation, this approach has facilitated acetylation studies on a variety of proteins. In this work, we demonstrated a facile protocol to produce site-specifically acetylated proteins by using the model bacterium Escherichia coli as the host. Malate dehydrogenase was used as a demonstration example in this work.

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Genetic code expansion serves as a powerful tool to study a wide range of biological processes, including protein acetylation. Here we demonstrate a facile protocol to exploit this technique for generating homogeneously acetylated proteins at specific sites in Escherichia coli cells.

Un protocole Facile à générer des protéines relativement acétylées chez Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Une plate-forme haut débit pour la détection de Salmonella spp /Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human diseases have been reported to be associated with four species of bacteria: Aeromonas dhakensis, Aeromonas hydrophila, Aeromonas veronii, and Aeromonas caviae. The model organism Caenorhabditis elegans is a bacterivore that provides an excellent infection model by which to study the bacterial pathogenesis of Aeromonas. Here, we introduce three different experiments to study Aeromonas infection using a C. elegans model, including survival, liquid toxicity, and muscle necrosis assays. The results of the three methods determining the virulence of Aeromonas were consistent. A. dhakensis was shown to be the most toxic among the 4 major Aeromonas species causing clinical infections. These methods are shown to be a convenient way to evaluate the toxicity among and within Aeromonas species and contribute to our understanding of the pathogenesis of Aeromonas infection.

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

Here, we introduce three different experiments to study Aeromonas infection in C. elegans. Using these convenient methods, it is easy to evaluate the toxicity among and within Aeromonas species.

Évaluer la Virulence et la pathogenèse de l’Infection à Aeromonas dans un modèle de Caenorhabditis elegans

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human ...

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of massage on skeletal muscles, the molecular mechanisms behind are poorly understood. We have recently developed a simple device to apply local cyclical compression (LCC), which can generate intramuscular pressure waves with varying amplitudes. Using this device, we have demonstrated that LCC modulates inflammatory responses of macrophages in situ and alleviates immobilization-induced muscle atrophy. Here, we describe protocols for the optimization and application of LCC as a massage-like intervention against immobilization-induced inflammation and atrophy of skeletal muscles of mouse hindlimbs. The protocol that we have developed can be useful for investigating the mechanism underlying beneficial effects of physical exercise and massage. Our experimental system provides a prototype of the analytical approach to elucidate the mechanical regulation of muscle homeostasis, although further development needs to be made for more comprehensive studies.

Here we describe the protocols for applying defined mechanical loads to mouse calves and for monitoring the concomitant intramuscular pressure changes. The experimental systems that we have developed can be useful for investigating the mechanism behind the beneficial effects of physical exercise and massage.

Application de perturbations de massage cohérentes sur les veaux de souris et surveillance des changements de pression intramusculaire qui en résultent

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from human induced pluripotent stem cells (hiPSCs) and maintained in culture, provides an exciting opportunity to facilitate the modeling of the epithelial response to enteric infections. In vivo, intestinal epithelial cells (IECs) play a key role in regulating intestinal homeostasis and may directly inhibit pathogens, although the mechanisms by which this occurs are not fully elucidated. The cytokine interleukin-22 (IL-22) has been shown to play a role in the maintenance and defense of the gut epithelial barrier, including inducing a release of antimicrobial peptides and chemokines in response to infection.
We describe the differentiation of healthy control hiPSCs into iHOs via the addition of specific cytokine combinations to their culture medium before embedding them into a basement membrane matrix-based prointestinal culture system. Once embedded, the iHOs are grown in media supplemented with Noggin, R-spondin-1, epidermal growth factor (EGF), CHIR99021, prostaglandin E2, and Y-27632 dihydrochloride monohydrate. Weekly passages by manual disruption of the iHO ultrastructure lead to the formation of budded iHOs, with some exhibiting a crypt/villus structure. All iHOs demonstrate a differentiated epithelium consisting of goblet cells, enteroendocrine cells, Paneth cells, and polarized enterocytes, which can be confirmed via immunostaining for specific markers of each cell subset, transmission electron microscopy (TEM), and quantitative PCR (qPCR). To model infection, Salmonella enterica serovar Typhimurium SL1344 are microinjected into the lumen of the iHOs and incubated for 90 min at 37 &#176;C, and a modified gentamicin protection assay is performed to identify the levels of intracellular bacterial invasion. Some iHOs are also pretreated with recombinant human IL-22 (rhIL-22) prior to infection to establish whether this cytokine is protective against Salmonella infection.

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids offer exciting opportunities to model enteric diseases in vitro. We demonstrate the differentiation of hiPSCs into intestinal organoids (iHOs), the stimulation of these iHOs with cytokines, and the microinjection of Salmonella Typhimurium into the iHO lumen, enabling the study of an epithelial invasion by this pathogen.

Utilisation d’organoïdes intestinaux dérivés de cellules souches pluripotentes induites par l’homme pour étudier et modifier la protection des cellules épithéliales contre la salmonelle et d’autres agents pathogènes

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from ...

Chronic Salmonella Infection Induced Intestinal Fibrosis

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and dysregulated repair. In inflammatory bowel disease (IBD), fibrosis leads to recurrent stricture formations for which there is no effective therapy other than surgical resection. Due to its late onset, the processes that drive fibrosis is less studied and largely unknown. Therefore, fibrotic complications represent a major challenge in IBD. In this protocol, a robust in vivo model of intestinal fibrosis is described where streptomycin pre-treatment of C57Bl/6 mice followed by oral gavage with vaccine grade Salmonella Typhimurium &#916;AroA mutant leads to persistent pathogen colonization and fibrosis of the cecum. Methodologies for preparing S. Typhimurium &#916;AroA for inoculation, quantifying pathogen loads in the cecum and spleen, and evaluating collagen deposition in intestinal tissues are explained. This experimental disease model is useful for examining host factors that either enhance or exacerbate CD-like intestinal fibrosis.

This protocol describes a mouse model of Salmonella driven intestinal fibrosis that resembles key pathological hallmarks of Crohn&#8217;s disease including transmural inflammation and fibrosis. This method can be used to evaluate host factors that alter fibrotic outcomes using mutant mice maintained on a C57Bl/6 genetic background.

Fibrose intestinale induite par l'infection chronique à Salmonella

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and ...

NF-&#954;B-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells

The dimeric transcription factor NF-&#954;B regulates many cellular response pathways, including inflammatory pathways by inducing the expression of various cytokines and chemokines. NF-&#954;B is constitutively expressed and is sequestered in the cytosol by the inhibitory protein nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (I&#954;B&#945;). Activation of NF-&#954;B requires the degradation of I&#954;B&#945;, which then exposes a nuclear localization signal on NF-&#954;B and promotes its trafficking to the nucleus. Once in the nucleus, NF-&#954;B binds to the promotor region of NF-&#954;B target genes such as interleukin 6 (IL-6) and IL-23, to promote their expression.
The activation of NF-&#954;B occurs independently of transcription or translation. Therefore, the activation state of NF-&#954;B must be measured either by quantifying NF-&#954;B specifically in the nucleus, or by quantifying expression of NF-&#954;B target genes. In this protocol, cells stably transfected with an NF-&#954;B::luciferase reporter construct are assayed for NF-&#954;B activation using in vitro tissue culture techniques. These cells are infected with Salmonella Typhimurium to activate NF-&#954;B, which traffics to the nucleus and binds to &#954;B sites in the promoter region of luciferase, inducing its expression. Cells are lysed and analyzed with the luciferase assay system. The amount of luciferase produced by the cells correlates with the intensity of the luminescence signal, which is detected by a plate reader. The luminescence signal generated by this procedure provides a quick and highly sensitive method by which to assess NF-&#954;B activation under a range of conditions. This protocol also utilizes quantitative reverse transcription PCR (RT-qPCR) to detect relative mRNA levels that are indicative of gene expression.

NF-&amp;#954;B-dependent Luciferase Activation and Quantification of Gene Expression in &lt;em&gt;Salmonella&lt;/em&gt; Infected Tissue Culture Cells

Here, we present a protocol to quickly and easily measure nuclear factor kappa-light-chain-enhancer of activated B cells (NF-&#954;B) activation in cell lines expressing NF-&#954;B::luciferase reporter constructs, via measurements of luminescence in the cell lysate. Additionally, gene expression is determined via RT-qPCR isolated from cells infected with Salmonella Typhimurium.

Activation et quantification de l'expression génique dans les cellules de culture tissulaire infectées par Salmonella

The dimeric transcription factor NF-&#954;B regulates many cellular response pathways, including inflammatory pathways by inducing the expression of ...