Gli autori desiderano ringraziare i membri passati del Goodrich Blair-lab: Samantha Orchard, Kimberly Cowles, Erin Herbert-Tran, Greg Richards, Megan Menard, e Youngjin Parco per il loro contributo allo sviluppo di questo protocollo. Questo lavoro è stato finanziato dalla National Science Foundation concessione IOS-0950873 e il National Institutes of Health NRSA comunione FAI084441Z.

1. Uovo Sterilizzazione Insetti e allevamento <ol><li> Preparare dieta prima autoclave 15 g di agar fornito in 900-1,000 ml H 2 O. Subito dopo la sterilizzazione in autoclave, mescolare con 166 g di dieta germe di grano e mescolate bene in un frullatore laboratorio. Versare in un piatto (o piatti) per raffreddare, poi il trasferimento dieta per un foglio di alluminio, aderire bene, e conservare a 4 ° C. </li><li> All&#39;arrivo, sterilizzare M. uova Sexta con 250 ml di soluzione di candeggina 0,6% per 2-3 minuti in un supporto di vetro e filtro beuta apparato per il vuoto con carta da filtro di 90 millimetri, mescolando occasionalmente. </li><li> Accendere il vuoto per drenare soluzione di candeggina e lavare le uova 3-4 volte con 250 ml sterile distillata H 2 O per lavaggio. </li><li> Trasferimento uova fresche 90 mm carta filtro posto in un coperchio piastra Petri e consentire loro di asciugare fino a che non sono più stretti stretti (circa 20-30 minuti). </li><li> Trasferire le uova al fondo dei contenitori in plastica con dieta insetto, ponendo circa 40 uova in ciascun contenitore(Figura 1A). Le uova sono separati dalla dieta per evitare che l&#39;umidità indotta contaminazione fungina. Mantenere le uova a 26 ° C con una luce 16 ore: 8 ore fotoperiodo scuro. Uova alleggerirà di un colore giallo-colore bianco prima della schiusa. </li><li> Quando schiusa è completa, trasferire accuratamente circa 25 larve per ciascun nuovi contenitori con dieta sul fondo (Figura 1B). E &#39;importante prendere ogni larva dal lungo corno nero con pinze per evitare di danneggiare durante i loro fragili prime fasi di sviluppo. Incubare per 2 giorni come sopra. Mentre la morte delle larve è insolito in questa fase, alcuni morti o, più probabilmente, larve evolutivamente stentata si può osservare. Insetti dovrebbero essere esclusi da ulteriori studi e sacrificato per congelamento. </li><li> Per evitare comportamenti cannibali, larve di trasferire in contenitori singoli con piccoli pezzi di cibo (Figura 1C) e incubare come sopra. Monitorare lo sviluppo degli insetti, feci sostituzione cibo e la pulizia out di contenitori a giorni alterni fino a che non subiscono la terza muta larvale (in genere 6-9 giorni dopo la schiusa). Questo è il 4 ° stadio larvale instar, caratterizzata dalla comparsa di nero ganciformi crochets di ciascun risalto proleg e maggiore di strisce lungo il corpo dell&#39;insetto (Figura 1D). Le larve può variare notevolmente in dimensioni, ma di solito pesano 0,15-0,4 g entrando nella fase 4 ° stadio. Le larve di dimensioni simili dovrebbe essere scelto per l&#39;iniezione. </li></ol> 2. Preparazione di batteri per preparazioni iniettabili <ol><li> Coltivare il ceppo batterico (s) da sottoporre a prova secondo le procedure standard per la fase di crescita che si desidera verificare. </li><li> Pellet 500 pl di ciascun ceppo batterico da testare per la filatura per 2 minuti in una microcentrifuga a 13.000 rpm a temperatura ambiente. </li><li> Risospendere le cellule in 1 ml di fosfato sterile 1x salina tamponata (PBS) e pellet nuovamente come sopra. </li><li> Risospendere le cellule in 0,5 ml di PBS sterile 1x e misurache la densità ottica (OD) della sospensione. Diluire tutte le sospensioni allo stesso OD, se necessario. Mezzi di crescita sterili possono essere utilizzati anche per la risospensione delle cellule e diluizione in luogo di PBS 1x, se desiderato. </li></ol> 3. Iniezione di larve 4 ° Instar <ol><li> Preparare seriali 10-diluizioni del ceppo batterico prima in 6 pozzetti di una piastra a 96 pozzetti per microtitolazione in 1x PBS sterile, utilizzando un puntale per pipetta per ogni diluizione (Figura 2A). Il volume finale della sospensione di cellule in ciascun pozzetto deve superare il volume richiesto per iniezione (10 microlitri per insetti) più 20 microlitri per l&#39;inoculo piastra (vedere i passi 3,2 e 3,7). </li><li> Spot 10 microlitri ciascuna per 5 diluizioni (10 -2 attraverso 10 -6) lungo la parte superiore di una piastra Petri contenente un appropriato mezzo di crescita. La piastra in modo che le macchie diffondersi al centro (Figura 2B). </li><li> Rimuovere dieta insetti e feci e insetti posto in contenitori in ghiaccio percirca 5 min. </li><li> Sterilizzare l&#39;ago della siringa con il risciacquo 3 volte in 2 tubi di 70-100% etanolo, seguito da una provetta sterile di H 2 O (Figura 2C). Il volume di liquido deve essere sufficiente per sommergere l&#39;ago. </li><li> Quantificare le sospensioni di cellule diluite sotto un microscopio con un emocitometro. A seconda della inoculo desiderato, elaborare 10 microlitri dal pozzetto microtitolo (Figura 2D). </li><li> Tampone superficie insetto con etanolo al 95%, e iniettare il 10 ml di sospensione cellulare in insetto con un angolo (inferiore a 45 °) dietro una delle prolegs addominali. Fare attenzione a non forare l&#39;intestino e iniettare il contenuto appena sotto lo strato epidermico (Figura 2E). Mentre una certa perdita di emolinfa (chiaro, verde-blu liquido) è normale, le particelle e il liquido giallo che emerge dal sito di iniezione sono indicativi di foratura budello. Se ciò si verifica, sacrificare l&#39;insetto e ottenere una nuova larva di riposizionarlo. </li><li> Ripetere il punto 3.6 per ogni insetto restante del gruppo prima iniezione. Dell&#39;ago sterilizzazione non è necessario tra ogni insetto iniettato con la stessa diluizione e ceppo di batteri meno contaminazione. In alternativa, un dispensatore può essere utilizzata per maggiore coerenza nel volume di iniezione tra i campioni. </li><li> Dopo aver iniettato ogni insetto con il ceppo di batteri prima, ripetere la fase 3.2, questa volta avvistare sospensioni cellulari nel centro del piatto e facendo loro flusso verso il basso (Figura 2F). Incubare le piastre a temperatura adeguata e contare le colonie per enumerare l&#39;inoculo. Questo passaggio seconda placcatura viene utilizzato per verificare che i campioni non sono stati contaminati durante il processo di iniezione, poiché ciò comporterebbe residenti sono diversi tra placcature primo e secondo. </li><li> Ripetere i passaggi da 3.1 al 3.8 per ogni ceppo di batteri nell&#39;esperimento. Infine, iniettare 3-5 insetti con sterile 1x PBS usando una sterago ile come gruppo di controllo negativo. Insetti incubare a 26 ° C con una luce 16 ore: 8 ore fotoperiodo scuro. </li><li> Monitorare la sopravvivenza degli insetti nel tempo dopo l&#39;iniezione. La morte degli insetti si caratterizza come una mancanza di movimento volontario dopo stimolazione. Insetti spesso mostrano perdita di appetito, diarrea (acquosa, escrementi giallo), e / o perdita di acqua (&quot;sudare&quot;) durante l&#39;infezione prima della morte, queste caratteristiche può essere interessante notare come bene. </li></ol>

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Nessun conflitto di interessi dichiarati.

L&#39;iniezione diretta di M. larve sexta con batteri entomopatogeni, come descritto qui, serve come un mezzo semplice ed efficace per analizzare la virulenza batterica. Il metodo è anche forte adattabilità a diversi soggetti sperimentali e / o condizioni. I batteri possono essere preparati in diversi modi prima dell&#39;iniezione. Nel caso di X. nematophila, le cellule coltivate in di tipo selvatico ricco di sostanze nutritive Luria-Bertani (LB) medio a metà-log fase sono in genere i più ...

<table border="1"><tbody><tr><td> Reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> 90 mm Carta filtro </td><td> Whatman </td><td> 1001 090 </td><td></td></tr><tr><td> Vetro portafiltri </td><td> Millipore </td><td> XX1004700 </td><td></td></tr><tr><td> Manduca sexta uova </td><td> Carolina Biological Supply </td><td> 143880 </td><td></td></tr><tr><td> Dieta Gypsy Moth + agar </td><td> MP Biomedicals </td><td> 0296029301 </td><td></td></tr><tr><td> 5,5 once contenitori di plastica e coperchi </td><td> Solo Cup Società </td><td> URC55-0090-0090 PL4 </td><td></td></tr><tr><td> 1 oncia contenitori di plastica e coperchi </td><td> DART Container Corporation </td><td> 100PC 100PCL25 </td><td></td></tr><tr><td> 1x PBS </td><td></td&gt; <td></td><td> 137 millimetri NaCl, 2,7 mM KCl, 8 mM Na 2 HPO 4, 1,46 mM KH 2 PO 4, pH 7.4 </td></tr><tr><td> Siringa </td><td> Hamilton </td><td> 80208 </td><td> 30 gauge, 0,375 &quot;lunghezza, stile, punto 2, </td></tr></tbody></table>

rearing and injection of manduca sexta larvae to assess bacterial virulence

Manduca sexta, comunemente noto come il hornworm tabacco, è considerato un parassita agricolo significativo, nutrendosi di piante solanacee tra cui tabacco e pomodoro. La suscettibilità di M. larve sexta ad una varietà di specie di batteri entomopatogeni 1-5, così come la ricchezza di informazioni disponibili riguardo al sistema immunitario dell&#39;insetto 6-8, e la sequenza del genoma in attesa di 9 ne fanno un organismo buon modello per l&#39;uso in studio delle interazioni ospite-microbo durante la patogenesi. Inoltre, M. larve sexta sono relativamente grandi e facili da manipolare e mantenere nel relativo laboratorio per altre specie sensibili insetti. Loro grandi dimensioni facilita anche efficiente tessuto / emolinfa estrazione per analisi della risposta dell&#39;ospite all&#39;infezione. Il metodo qui presentato descrive l&#39;iniezione diretta di batteri nella hemocoel (cavità sangue) di M. Sexta larve. Questo approcciopuò essere utilizzato per analizzare e confrontare le caratteristiche di virulenza di varie specie batteriche, ceppi mutanti, o semplicemente controllare il tempo di morte insetto dopo l&#39;iniezione. Questo metodo è stato sviluppato per studiare la patogenicità di Xenorhabdus e specie Photorhabdus che associano di norma con i vettori di nematodi come mezzo per entrare in l&#39;insetto. Nematodi entomopatogeni in genere infettano le larve tramite digestivo naturale o aperture respiratorie, e di rilasciare il loro contenuto simbiotici batteriche nella emolinfa insetto (sangue) poco dopo 10. Il metodo descritto qui di iniezione evita il bisogno di un vettore nematode, quindi sganciare gli effetti dei batteri e nematodi sul insetto. Questo metodo permette di conteggio accurato del materiale infetto (cellule o proteine) entro l&#39;inoculo, che non è possibile utilizzare altri metodi esistenti per analizzare entomopathogenesis, compresi nick 11 e saggi di tossicità orale 12 <em&gt;. Inoltre, test di tossicità orale affrontare la virulenza di tossine secrete introdotte nel sistema digerente delle larve, mentre il metodo di iniezione diretta affronta la virulenza di cellule intere inoculi. L&#39;utilità del metodo di iniezione diretta, come descritto qui è analizzare patogenesi batterica monitorando mortalità insetto. Tuttavia, questo metodo può essere facilmente espanso per uso a studiare gli effetti di infezione sul M. sexta sistema immunitario. L&#39;insetto risponde alle infezioni tramite sia le risposte umorali e cellulari. La risposta umorale comprende il riconoscimento di batteri associati modelli e conseguente produzione di vari peptidi antimicrobici 7, l&#39;espressione di geni che codificano questi peptidi possono essere monitorati dopo infezione diretta tramite estrazione di RNA e PCR quantitativa 13. La risposta cellulare di infezione coinvolge nodulazione, incapsulamento, e fagocitosi di agenti infettivi attraverso emociti 6 </sup&gt;. Per analizzare queste risposte, insetti iniettato può essere sezionato e visualizzati mediante microscopia 13, 14.

Un esempio rappresentativo di un saggio mortalità insetto è illustrato in Figura 3. In questo esperimento, gli insetti sono stati iniettati con circa 50 unità formanti colonie (CFU) sia di tipo selvatico (ATCC19061) o un ceppo mutante attenuato (LRP 13) di Xenorhabdus nematophila cresciuto a metà fase log (n = 6 insetti per ceppo). Insetti sono stati osservati per circa 72 ore, e la percentuale di insetti iniettati ancora vivi a ciascun timepoint registrata. In questo ca...

Watch this Scientific Journal Video about Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence at JoVE.com

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Il metodo qui descritto utilizza l&#39;iniezione diretta di batteri entomopatogeni nella hemocoel di<em&gt; Manduca sexta</em&gt; Larve di insetti.<em&gt; M. Sexta</em&gt; È un insetto disponibile in commercio e ben studiata. Quindi, questo metodo rappresenta un approccio semplice per analizzare interazioni host-batteriche dalla prospettiva di uno o entrambi i partner.

Allevamento e iniezione di<em&gt; Manduca sexta</em&gt; Le larve per valutare virulenza batterica

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and ...

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Immunology and Infection

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

12.6K Views.  University of Wisconsin-Madison. Il metodo qui descritto utilizza l'iniezione diretta di batteri entomopatogeni nella hemocoel di Manduca sexta Larve di insetti. M. Sexta È un insetto disponibile in commercio e ben studiata. Quindi, questo metodo rappresenta un approccio semplice per analizzare interazioni host-batteriche dalla prospettiva di uno o entrambi i partner.

Video: Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

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.

Saliva, le ghiandole salivari, e Collection emolinfa dalle zecche 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 ...

Ricerca

Immunologia e infezioni

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

Monitoraggio parte dell&#39;Assemblea di una Secreted batterica Virulence Factor Uso site-specific reticolazione

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

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.

Generation e Multi-fenotipica Screening-Alto contenuto di<em&gt; Coxiella burnetii</em&gt; Mutants transposon

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

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

L&#39;applicazione di un sistema di espressione inducibile per studiare interferenza di fattori batterici di virulenza con segnalazione intracellulare

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can be utilized as an alternative animal model for the study of virulence, host-pathogen interaction, innate immune response, and drug efficacy. Details for the rearing, care and breeding of the hissing cockroach are provided. We also illustrate how it can be infected with bacteria such as the intracellular pathogens Burkholderia mallei, B. pseudomallei, and B. thailandensis. Use of the hissing cockroach is inexpensive and overcomes regulatory issues dealing with the use of mammals in research. In addition, results found using the hissing cockroach model are reproducible and similar to those obtained using mammalian models. Thus, the Madagascar hissing cockroach represents an attractive surrogate host that should be explored when conducting animal studies.

We present a protocol to utilize the Madagascar hissing cockroach as an alternative non-mammalian animal model to conduct bacterial virulence, pathogenesis, drug toxicity, drug efficacy, and innate immune response studies.

Il Madagascar sibilo scarafaggio come modello alternativo Non mammiferi animali per studiare la virulenza, patogenesi e l'efficacia dei farmaci

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can ...

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability to multiply inside such cells. These virulence traits render M. abscessus pathogenic, especially in vulnerable hosts with underlying structural lung disease, such as cystic fibrosis, bronchiectasis or tuberculosis. How patients become infected with M. abscessus remains unclear. Unlike many mycobacteria, M. abscessus is not found in the environment but might reside inside amoebae, environmental phagocytes that represent a potential reservoir for M. abscessus. Indeed, M. abscessus is resistant to amoebal phagocytosis and the intra-amoeba life seems to increase M. abscessus virulence in an experimental model of infection. However, little is known about M. abscessus virulence in itself. To decipher the genes conferring an advantage to M. abscessus intracellular life, a screening of a M. abscessus transposon mutant library was developed. In parallel, a method of RNA extraction from intracellular Mycobacteria after co-culture with amoebae was developed. This method was validated and allowed the sequencing of whole M. abscessus transcriptomes inside the cells; providing, for the first time, a global view on M. abscessus adaptation to intracellular life. Both approaches give us an insight into M. abscessus virulence factors that enable M. abscessus to colonize the airways in humans.

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

Here, we present two protocols to study Phagocyte-Mycobacterium abscessus interactions: the screening of a transposon mutant library for bacterial intracellular deficiency and the determination of bacterial intracellular transcriptome from RNA sequencing. Both approaches provide insight into the genomic advantages and transcriptomic adaptations enhancing intracellular bacteria fitness.

Identificazione di fattori di virulenza di Mycobacterium abscessus per replica intracellulare nei fagociti

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability ...

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.

Valutazione di virulenza e patogenesi dell'infezione da Aeromonas in un modello di Caenorhabditis elegans

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

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Un modello di topo per valutare risposta immunitaria innata per infezione da Staphylococcus aureus

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Un High-throughput Shigella-specifica analisi battericida

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Anticorpli uccisione test per valutare le risposte immunologiche contro batteri patogeni

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...