Les auteurs remercient la Fondation médicale Ellison pour leur soutien. Nous tenons également à remercier les membres du laboratoire Abby Dernburg d&#39;assistance à l&#39;utilisation du wormsorter.

Une. L&#39;obtention d&#39;une culture synchronisée des jeunes adultes Animaux <ol><li> Grandir vers sur plusieurs plaques NGM ensemencées avec OP50-1 E. colibacilles (10x concentré d&#39;une culture saturée) jusqu&#39;à ce que de nombreux vers ont atteint le stade gravide. </li><li> Traiter les animaux gravides avec une solution d&#39;oeuf préparation pour obtenir une culture synchronisée (œufs). </li><li> oeufs de plaque sur plusieurs plaques de 60 mm NGM ensemencées avec 10x concentré OP50-1 à une densité d&#39;environ 150-200 oeufs / plaque. </li><li> Incuber les plaques à 25 ° C pendant 2 jours (jusque vers ont atteint le L4 - stade jeune adulte). </li></ol> 2. Préparation Pseudomonas aeruginosa plaques et infecter Worms <ol><li> Le jour de l&#39;oeuf-prep (étape 1.1) inoculer une culture PA14 :: GFP dans 2 ml d&#39;un milieu contenant B de King la rifampicine à une concentration finale de 100 pg / ml. Incuber la culture à 37 ° C avec agitation O / N. </li><li> Plate la PA14 :: GFP culture sur autant de plaques de Killing lent (SKP) comme nécessaire pour le test. Sur chaque 150 mm SKP boîte de Pétri pipette 75-100 ul de culture saturée et répartir uniformément avec une spatule en verre stérile. Incuber les plaques à 37 ° C pendant 20-24 h. </li><li> Retirer les plaques de l&#39;incubateur et de les laisser refroidir à la température ambiante (~ 20 min). En utilisant un tampon M9, laver les vers de l&#39;étape 1.4 sur PA14 :: plaques GFP dans un volume minimal de liquide. Lors de l&#39;utilisation des plaques de 150 mm, plusieurs centaines de vers peuvent être placés sur une seule plaque. </li><li> Incuber les plaques à 25 ° C pendant 18-21 h. Pour P. jeunes vers adultes aeruginosa exposés, c&#39;est la fenêtre de temps l&#39;affichage de la répartition optimale de colonisé vs animaux noncolonized. </li></ol> 3. Mise en place du Wormsorter Avant d&#39;effectuer l&#39;échantillon sorte, s&#39;assurer que la machine fonctionne correctement. Les détails peuvent être obtenus http://www.unionbio.com/support/documents.aspx?id=43, et ee étapes essentielles sont décrites ci-dessous. <ol><li> Régler la pression de la soupape de gaine à environ 5 psi (4,5-5,5 gamme). </li><li> Pour vérifier que le débit de fluide gaine est approprié, placer un tube conique de 15 ml sous le distributeur, tournez la vanne de la gaine, et fermez le robinet de tri. Tenir le tube sous le distributeur pendant 60 secondes. Le volume dans le tube doit être de 9 à 10 ml. Si elle est en dehors de cette gamme, ajuster la pression de la soupape de gaine en conséquence. </li><li> Pour veiller à ce que le débit de fluide gaine (défini comme dans l&#39;étape 3.2) permettra tri précis, tester le taux de tri à l&#39;aide de 40 particules um de contrôle: </li><li> Fermez le robinet de la gaine. Ajouter environ 25 ml de solution de particules de commande au réservoir. </li><li> Ouvrir la valve de la gaine, et la soupape d&#39;échantillon. </li><li> Sur le logiciel de l&#39;ordinateur, accédez à la &quot;mode de particule de contrôle&quot; et cliquez acquérir. </li><li> Assurez-vous que le taux d&#39;écoulement de particules est d&#39;environ 5-8 particules / sec, avec une TOF (Time Of Flight) approximation de la largeurde la particule, de 40 um. Un tel TOF indique que seulement une seule 40 um objet passe à travers à la fois. Si les valeurs sont en dehors de cette plage, régler les paramètres (en suivant les indications ci-dessus) en conséquence. </li></ol> 4. Le tri Colonisée vs Noncolonized Worms <ol><li> Utilisation M9, laver vers dans des tubes coniques de 15 ml. Permettre vers adultes à se déposent au fond du tube par gravité et retirer le surnageant. Remplir le tube avec frais M9. Répétez ce processus 3-4x. Cela supprime une grande partie des larves ainsi que les bactéries en excès et dure environ 10 min. </li><li> Ajouter vers le réservoir de wormsorter avec une petite barre d&#39;agitation en douceur de mélange. </li><li> Régler le gain de signal initial en définissant la PMT vert à 600 et le rouge et jaune PMT à 200. </li><li> Commencer à acquérir des données pour ajuster les réglages. </li><li> Viser un taux de 25-30 événements (vers) par seconde de tri. Si le taux de nombre est trop élevé, diluer les vers dans le réservoir avec M9 en conséquence. Si le taux est deo faible, ajouter des vers dans un petit volume de M9. </li><li> Si l&#39;expérience nécessite une séparation très stricte de colonisé vs vers noncolonized, réglez le &quot;bilan de coïncidence&quot; à &quot;pur&quot;. Cela garantit que dans le cas où deux objets apparaissent trop proches les uns des autres ou dans la même chute, la machine rejette la chute plutôt que de les recueillir. </li><li> Pour trier la population d&#39;intérêt, établir des barrières autour des axes indiquant la taille (pour obtenir des vers adultes) et de l&#39;intensité de fluorescence (c.-haut pour colonisé, faible pour noncolonized). Les valeurs de fluorescence de déclenchement doivent être déterminées de manière empirique. </li><li> Placez une plaque multi-puits ou d&#39;une boîte de Pétri sous le distributeur pour recueillir les vers puis ouvrez la vanne de sorte à commencer le tri vers. </li><li> Recueillir une petite population d&#39;animaux pour vérifier sous le microscope que la population triée est la population d&#39;intérêt. Sinon, ajustez les paramètres de déclenchement en conséquence et poursuivre la collecte de l&#39;échantillon. </li></ol>

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M., Mylonakis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+albicans+infection+of+Caenorhabditis+elegans+induces+antifungal+immune+defenses.">Candida albicans infection of Caenorhabditis elegans induces antifungal immune defenses.</a> PLoS Pathog. , (2011).</li><li>Shapira, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+role+for+a+GATA+transcription+factor+in+regulating+epithelial+innate+immune+responses.">A conserved role for a GATA transcription factor in regulating epithelial innate immune responses.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 14086-14091 (2006).</li><li>Troemel, E. 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Les auteurs n&#39;ont rien à révéler.

La méthode que nous décrivons tire parti de marquage fluorescent des entités à l&#39;extérieur de la vis sans fin, pour suivre les interactions entre la vis sans fin et de son environnement. Dans le cas que nous présentons, la séparation a été basée sur l&#39;étiquetage d&#39;un agent pathogène et a été utilisé pour les vers séparées avec lourde charge pathogène de ceux qui n&#39;ont pas (ou lumière) charge. Après l&#39;analyse de l&#39;expression du gène n&#39;a trouvé aucune différence dans les ...

Tri à vis sans fin automatique est un peu comme FACS, fonctionnant en mesurant un signal fluorescent dans une vis sans fin (typiquement fournie par l&#39;expression transgénique de protéines rapporteurs) lors de son passage redressé dans un tube, ce qui permet la redirection soit à un tube de collecte / puits ou à un conteneur de déchets selon l&#39; à gating paramètres fixés par le chercheur 1. Le wormsorter peut faciliter la recherche de plusieurs façons, un exemple de l&#39;employer comme outil d&#39;analyse est une étude qui a suivi les modèles spatio-temporelles de l&#39;activité du promoteur pour près de 1000 gènes 2. Cependant, l&#39;utilisation principale de la wormsorter est dans cribles génétiques, à la suite des niveaux ou des gènes localisation de la protéine fluorescente selon l&#39;axe de la vis sans fin 3-5 expression cibles. Ici, nous décrivons une nouvelle application pour la wormsorter, en suite à la colonisation de la vis sans fin par un agent pathogène fluorescence marqués. Avec comme un outil nous nous sommes concentrés sur la relation entre pathogfr colonisation / charge et la réponse immunitaire, à acquérir de nouvelles connaissances sur les mécanismes responsables de l&#39;initiation de la réponse immunitaire dans le ver. Dans pratiquement tous les organismes étudiés à ce jour, l&#39;initiation des réponses immunitaires innées à des agents pathogènes microbiens dépend de la reconnaissance de motifs moléculaires associés à des pathogènes (PAMP), et / ou motifs moléculaires danger / dommages-associé (Damps) 6,7. Les structures microbiennes premiers sont conservés qui comprennent les éléments de la paroi cellulaire microbienne, son flagelle, ou son bicouche lipidique 6; la seconde, comprennent deux molécules libérées (par exemple ATP 8), protéines modifiées ou d&#39;autres marqueurs de processus cellulaires modifiés 9,10. Les deux types de signaux sont reconnus par des protéines désignées comme des récepteurs de reconnaissance des formes (PRR), qui, lors de la liaison spécifique d&#39;une molécule modèle activer une chaîne d&#39;événements conduisant à une réponse protectrice. C. elegans a été extrêmement utile comme Tractable modèle de disséquer les différents aspects des interactions hôte-pathogène, mais une chose qui n&#39;est pas bien compris comment les réponses immunitaires sont initiées dans le ver. Aucun des récepteurs putatifs qui sont orthologues de reconnaissance de formes récepteurs (PRR) dans d&#39;autres organismes ont été montré pour lier PAMP, et la plupart des orthologues de PRR qui sont essentielles pour les réponses immunitaires dans d&#39;autres organismes montrent une contribution étonnamment limité de réponses ver pathogènes et la résistance. Par exemple, le récepteur de type Toll de Drosophila, ce qui est essentiel pour la résistance à des agents pathogènes Gram positifs, est représenté en C. elegans par un seul homologue, tol-1, ce qui contribue à la protection contre l&#39;agent pathogène à Gram négatif Salmonella Typhimurium 11, mais pas par les autres agents pathogènes testés positifs ou Gram-négatifs, 11,12. Ces observations, combinées avec des données indiquant que les réponses immunitaires pourrait être induite par perturber protéine cellulaire traduction hque conduit certains à suggérer que C. elegans détecte principalement Damps 9,13,14. Néanmoins, les rapports décrivant la capacité des agents pathogènes morts à induire des réponses immunitaires suggérer que la liaison PAMP peut avoir un rôle important dans la reconnaissance de l&#39;agent pathogène dans C. elegans 15,16. Des travaux antérieurs mettant l&#39;accent sur ​​les réponses immunitaires de l&#39;âge synchronisée génétiquement identiques C. elegans populations, ont montré une grande variabilité individuelle dans la colonisation intestinale par le Gram négatif bactérienne pathogène Pseudomonas aeruginosa. Cependant, des études de profilage transcriptionnel traités ces populations variable colonisés comme une seule entité 17,18. Profitant de cette variabilité, nous avons élaboré un protocole en se concentrant un wormsorter automatisé pour séparer les populations colonisées différentielle de Caenorhabditis elegans exposés à la GFP exprimant P. aeruginosa. Examen expression des gènes dans les populations différemment colonisés Faciévaluation litated de la relation entre la charge de pathogènes (et les dommages associés) et les réponses immunitaires et fourni de nouvelles informations sur la reconnaissance des agents pathogènes dans C. elegans 19. Ci-dessous nous décrivons le protocole, qui pourrait être appliquée pour trier vers infectées par l&#39;agent pathogène tout marqué par fluorescence. Pour les utilisateurs potentiels, il convient de noter que le nombre de vers qui doivent être triés dépend de la nature des analyses ultérieures et les protocoles utilisés. Par exemple, dans le cas de l&#39;analyse de l&#39;expression des gènes de microréseau,&gt; 1000 vers devront obtenir suffisamment ARN, si les protocoles standards sont utilisés, mais ~ 100 vers suffirait si l&#39;amplification est utilisé, permettant la collecte rapide de matériel et de minimiser ainsi le stress de les vers.

<table border="0" cellpadding="0" cellspacing="0" width="927">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">M9 Buffer</td>
			<td style="width:139px;">Prepared in house</td>
			<td style="width:527px;">Recipe at wormbook.org</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rifampicin&#160;</td>
			<td>Sigma</td>
			<td>R3501</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Egg prep solution&#160;</td>
			<td>Prepared in house</td>
			<td>50ml water ; 40ml bleach ; 10ml of 10N Sodium Hydroxide&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NGM plates&#160;</td>
			<td>Prepared in house</td>
			<td>Recipe at wormbook.org</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SKP plates</td>
			<td>Prepared in house</td>
			<td>Recipe same as NGM only 0.35% peptone instead of 0.25%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Control test particles&#160;</td>
			<td>Union Biometrica&#160;</td>
			<td>310-5071-001</td>
		</tr>
	</tbody>
</table>

automated separation of c. elegans variably colonized by a bacterial pathogen

Le wormsorter est un instrument analogue à une machine FACS qui est utilisé dans les études de Caenorhabditis elegans, typiquement pour trier vers basés sur l&#39;expression d&#39;un rapporteur fluorescent. Ici, nous mettons en évidence une utilisation alternative de cet instrument, pour trier vers en fonction de leur degré de colonisation par un agent pathogène exprimant la GFP. Cette nouvelle utilisation nous a permis d&#39;aborder la relation entre la colonisation de l&#39;intestin du ver et de l&#39;induction de réponses immunitaires. Alors que C. réponses immunitaires elegans à différents agents pathogènes ont été documentés, il est encore inconnue ce qui les déclenche. Les deux possibilités (qui ne sont pas mutuellement exclusives) sont la reconnaissance de motifs moléculaires associés à des pathogènes et la détection des dommages causés par une infection. Pour différencier entre les deux possibilités, l&#39;exposition à l&#39;agent pathogène doit être dissociée de la dommages qu&#39;il cause. La séparation de wormsorter permis de vers qui ont été largement colonisée par le Gram-npathogène egative Pseudomonas aeruginosa, avec les dommages susceptibles provoquée par la charge pathogène, des vers qui ont été exposées de façon similaire, mais pas, ou peu, colonisé. Ces populations distinctes ont été utilisés pour évaluer la relation entre la charge de l&#39;agent pathogène et l&#39;induction de réponses immunitaires de transcription. Les résultats suggèrent que les deux sont dissociées, confirmant la possibilité de reconnaissance de l&#39;agent pathogène.

Lorsque appariés pour l&#39;âge, génétiquement identiques C. elegans sont exposés à P. aeruginosa, une large distribution est observée dans les niveaux de colonisation (Figure 1A). Avec l&#39;aide du protocole décrit ci-séparation efficace contre les vers de noncolonized colonisés peut être réalisé (figure 1B). Contrairement vers noncolonized, vers colonisés montrent des signes de dommages, comme la lenteur et la défécation réduit 19. Ce der...

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Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

Le wormsorter facilite écrans génétiques dans Caenorhabditis elegans par tri vers selon l&#39;expression de journalistes fluorescentes. Ici, nous décrivons un nouvel usage: le tri selon la colonisation par un agent pathogène exprimant la GFP, et nous l&#39;employons pour examiner le rôle mal compris de la reconnaissance de l&#39;agent pathogène dans l&#39;initiation des réponses immunitaires.

Séparation automatique des<em&gt; C. elegans</em&gt; Façon variable colonisé par une bactérie pathogène

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

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JoVE Journal

Immunology and Infection

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

8.3K vues.  University of California, Berkeley.  Le wormsorter facilite écrans génétiques dans Caenorhabditis elegans par tri vers selon l'expression de journalistes fluorescentes. Ici, nous décrivons un nouvel usage: le tri selon la colonisation par un agent pathogène exprimant la GFP, et nous l'employons pour examiner le rôle mal compris de la reconnaissance de l'agent pathogène dans l'initiation des réponses immunitaires. 

Vidéo: Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

L&#39;invasion des cellules humaines par une bactérie pathogène

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

Recherche

Immunologie et infection

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

Schéma de flux de travail d&#39;une journée pour la détection de pathogènes et des bactéries tests de résistance antimicrobienne à partir d&#39;hémocultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

La salive, les glandes salivaires, et Collection hémolymphe de tiques Ixodes scapularis

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

Visualisation des réponses toxine bactérienne induite en utilisant direct microscopie de fluorescence cellulaire

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

Imagerie RICL sécrétion d&#39;enquêter sur l&#39;infection cellulaire par le pathogène bactérien<em&gt; Listeria monocytogenes</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Génération et multi-phénotypique criblage à haut contenu de<em&gt; Coxiella burnetii</em&gt; Mutants de transposon

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

Démêler la fonction d&#39;un effecteur bactérien à partir d&#39;un non-cultivable Pathogen usine utilisant une levure de l&#39;écran à deux hybrides

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their significance to both cell physiology and immunology, much remains to be discovered about novel lipoprotein forms, how they are synthesized, and the effect of the various forms on host immunity. To enable thorough studies on lipoproteins, this protocol describes a method for bacterial lipoprotein enrichment and preparation of N-terminal tryptic lipopeptides for structural determination by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Expanding on an established Triton X-114 phase partitioning method for lipoprotein extraction and enrichment from the bacterial cell membrane, the protocol includes additional steps to remove non-lipoprotein contaminants, increasing lipoprotein yield and purity. Since lipoproteins are commonly used in Toll-like receptor (TLR) assays, it is critical to first characterize the N-terminal structure by MALDI-TOF MS. Herein, a method is presented to isolate concentrated hydrophobic peptides enriched in N-terminal lipopeptides suitable for direct analysis by MALDI-TOF MS/MS. Lipoproteins that have been separated by Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) are transferred to a nitrocellulose membrane, digested in situ with trypsin, sequentially washed to remove polar tryptic peptides, and finally eluted with chloroform-methanol. When coupled with MS of the more polar trypsinized peptides from wash solutions, this method provides the ability to both identify the lipoprotein and characterize its N-terminus in a single experiment. Intentional sodium adduct formation can also be employed as a tool to promote more structurally informative fragmentation spectra. Ultimately, enrichment of lipoproteins and determination of their N-terminal structures will permit more extensive studies on this ubiquitous class of bacterial proteins.

The enrichment of bacterial lipoproteins using a non-ionic surfactant phase partitioning method is described for direct use in TLR assays or other applications. Further steps are detailed to prepare N-terminal tryptic lipopeptides for structural characterization by mass spectrometry.

Enrichissement des lipoprotéines bactériennes et préparation des Lipopeptides N-terminal pour la détermination de structure par spectrométrie de masse

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their ...

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

Un haut débit et haute teneur, liquide à base de c. elegans pathosystème

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

Analyse comportementale des grands c. elegans des Populations à l’aide d’un champ de vision large suivi plate-forme automatisée

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...