Vorremmo ringraziare Brandon Hassett e Carlos Ortiz per la loro assistenza tecnica. Questo lavoro è stato sostenuto dal contributi NSF IOB-0544428, 0951272-IOS, IOS-0925561 e al Dr. Michael Kolomiets, e dalla USDA National Institute of Food and Agriculture (NIFA), la selezione vegetale AFRI e borsa di studio # 2010-85117 -20.539 a Drs. Seth Murray, Thomas Isakeit e Kolomiets Michael.

1. Kernel Bioassay mais <ol><li> Due settimane prima, la cultura dei patogeni fungini, in agar patata destrosio (PDA) a 28 ° C. </li><li> Selezionare kernel con dimensioni e forma simili, preferibilmente appiattito in modo che giaceva a livello con la parte inferiore di fiale bioassay, e il luogo in provette da 50 ml Falcon. Kernel selezionati devono essere stati prodotti in concomitanza stesso ambiente per garantire età simile seme e la composizione metabolita. </li><li> Superficie sterilizzare kernel agitando provette a temperatura ambiente per 5 minuti con il 70% di etanolo, 1 minuto con acqua sterile, e 10 minuti con ipoclorito di sodio al 6%. Risciacquare, tre volte, per 5 minuti ogni volta, con acqua sterile sotto agitazione continua. Kernel Pat secco su asciugamani in autoclave. </li><li> Per facilitare infezione di Aspergillus flavus, crea una piccola ferita sull&#39;embrione lato con un ago 18 G alla profondità di 0,5 cm. Tuttavia, nella nostra esperienza una ferita più grande è necessaria per l&#39;infezione da Fusarium verticillioides. Therefore, utilizzare una lama di rasoio per tagliare nell&#39;embrione lato ad una profondità di 0,5 mm. </li><li> Preparare la sospensione inoculo raschiando coltivate in piastre di circa 5 ml di autoclavato 0,01% Tween-20 la soluzione per liberare le spore e rimuovere micelio per gravità filtrando attraverso almeno 4 strati di garza autoclavato. Calcolare la concentrazione di spore emocitometro e regolare a 10 6 spore / ml per A. flavus o F. verticillioides. Se si utilizzano altre specie di patogeni o di ospite, è importante valutare concentrazioni di inoculo per infezione appropriato. </li><li> Creare una camera umida in un contenitore di plastica (29,2 cm X 18,7 cm X 8,3 cm) foderata con cinque fogli di carta da cucina e 100 ml di acqua sterile. Camera non è acqua o a tenuta d&#39;aria e l&#39;acqua va aggiunto tutto esperimento per mantenere i tovaglioli di carta umida. </li><li> Inserire quattro kernel in un autoclavato fiala di scintillazione 20 ml di vetro e registrare la loro massa. Inoculare con 200 microlitri sospensione di spore,cap, e vortex per rivestire in modo uniforme kernel con sospensioni. Allentare i cappucci per consentire liberamente ricambio d&#39;aria e il luogo in camera di umidità. </li></ol> Nota: Per semi o funghi diversi da quelli descritti in questi metodi, la quantità di inoculo richiesto può essere diverso e dovrebbe essere derivato sperimentalmente. <ol start="8"><li> Incubare kernel sotto un fotoperiodo 12-h-light/12-h-dark a 28 ° C per 7 giorni o fino a quando desiderato. </li></ol> 2. Conidi Enumerazione <ol><li> Dopo il periodo di infezione, aggiungere 5 ml di autoclavato 0,01% Tween-20 a scintillazione fiale contenenti kernel infetti e vortex accuratamente per 1 minuto. </li><li> Utilizzo di un ampio foro punta della pipetta, immediatamente diluire trasferendo due distinte aliquote 200 pl di sospensione di spore in 2.0 ml provette Eppendorf contenenti 1,8 ml di 0,01% Tween-20 (o se necessario diluire seconda infezione). </li><li> Enumerare ogni 200 microlitri aliquota due volte con un emocitometro 4e confrontare i livelli di conidi tra i trattamenti. </li></ol> 3. Aflatossina Quantificazione <ol><li> Aggiungere kernel campione da 1 a 50 ml miscelatore tazza con 20 ml di 80% metanolo e 0,05 g di NaCl, coperchio e la miscela ad alta velocità per 1 min. </li><li> Mettere un filtro a pieghe in un recipiente di raccolta pulito e versare estratto nel filtro. </li><li> Trasferire 10 ml di filtrato in un recipiente pulito, aggiungere 20 ml di acqua distillata H 2 O, e mescolare bene. </li><li> Campione di filtrare attraverso un filtro in microfibra 1,5 micron di vetro in una tazza pulita. </li><li> Applicare 1 ml di estratto filtrato a test colonna Afla e, mediante aria compressa, forzare l&#39;estratto filtrato attraverso la colonna ad una velocità di 1-2 gocce / secondo fino svuotati. </li><li> Lavare la colonna due volte con 1 ml di acqua distillata H 2 O e passare attraverso la colonna a 1-2 goccia / secondo fino a quando l&#39;aria passa attraverso. </li><li> Colonna eluire con metanolo% 1ml di 100 HPLC ad un 1-2 goccia / secondo tasso raccolti in una cuvetta di vetro. </li><li> Aggiungere1 ml di Developer test Afla di eluato e mescolare bene. Luogo in cuvetta fluorimetro tarato e leggere a 60 sec. </li></ol> Nota: Quando si utilizza il protocollo Aflatest FGIS, la misura dal fluorimetro viene calcolata in base al verificarsi di un iniziale 50 grammi di campione estratto in 100 ml. Se si considera la diluizione del campione con acqua, il 1 ml applicato ad una colonna rappresenta 0,166 grammi di campione. Così, quando si modifica il protocollo, è necessario tener conto delle differenze di dimensione del campione e la soluzione di estrazione iniziale. Ad esempio, 2 grammi di kernel sono estratte in 20 ml di metanolo 80%. Questo è 0,1 grammi / ml. Quando 1 ml di questo campione viene miscelato con 2 ml di acqua, la proporzione del campione nel liquido è ora 0,033 grammi / ml. Se questo esempio fornisce una lettura fluorometro di 100 ppb, la concentrazione effettiva è basata sulla percentuale di campione di 0,166, cioè, ppb = (0,166 grammi X 100 ppb) / 0,1 grammo), o 166 ppb. Per le aflatossine quantifi alternativemetodi di cationi, vedere il riferimento 7. 4. Fumonisina B1 (FB1) Analisi <ol><li> Quando fungo è cresciuto su chicchi di mais, estrarre campioni overnight con 10 ml di acetonitrile / acqua (50/50, v / v) a temperatura ambiente senza agitazione. Successivamente, mescolare l&#39;estratto (2 ml) con acqua deionizzata (6 ml), e applicare questa miscela (8 ml) direttamente al di estrazione in fase solida C-18. </li><li> Prima di caricare campione, presupposto il C-18 solida colonna di estrazione in fase di risciacquo con 2 ml di acetonitrile seguito da 2 ml di acqua. </li><li> Dopo aver caricato il campione, la colonna viene lavata con 2 ml di acqua seguita da 2 ml di acetonitrile / acqua (15/85, v / v). Campione per l&#39;analisi HPLC (contenente FB1) viene eluito con 2 ml di acetonitrile / acqua (70/30, v / v). </li><li> Derivatizzare FB1 con o-phthaldehyde (OPA), trasferendo 0,1 ml di eluato colonna ad una fiala contenente 0,1 ml di tampone borato (0,05 M borico acid/0.05 M sodio borato [50/50, v / v], pH 8,5) e 0,1 OPA ml (0,1 mg / ml in acetoneitrile con 0,5% - mercaptoetanolo). </li><li> Arrestare la reazione dopo 10 minuti aggiungendo 0,5 ml di acetonitrile/0.01 M di acido borico (40/60, v / v). </li><li> Analizzare FB1 su una HPLC Shimadzu LC-20at sistema dotato di una colonna analitica Zorbax ODS (4,6 150 mm) e una lunghezza d&#39;onda variabile Shimatzu RF-10Axl fluorescenza (eccitazione 335 nm / emissione 440 nm). </li><li> Un gradiente lineare viene usato (solvente A: acetonitrile/0.1 M fosfato di sodio (40/60), pH 3,3; solvente B: acetonitrile / 0,1 M fosfato di sodio (60/40), pH 3,3) e il programma è il seguente gradiente: A 100% al 100% B in 10 min, 100% B per 5 min. </li><li> Analisi mediante HPLC standard FB1, e misurazioni di area di picco sono utilizzati per generare curva standard. Successivamente, quantificare FB1 livello in un campione confrontando le aree dei picchi con FB1 curva standard. </li></ol> 5. Analisi ergosterolo <ol><li> Cultura il fungo sulla chicchi di mais per 7-14 giorni. </li><li> Dopo il periodo di incubazione, aggiungere 10 ml di cloroformio: methI LAVORI (2:1, v / v) in ciascun flacone. Una volta aggiunto, agitare bene e poi i campioni incubare fiale al buio a temperatura ambiente per 24 ore. </li><li> Dopo 24 ore, centrifugare i campioni, e raccogliere il supernatante e filtrarli attraverso la membrana di nylon 0,45 um. </li><li> Iniettare il campione direttamente in un HPLC Shimadzu LC-20at impianto dotato di 4,6 U ODS-C18 colonna (200 Å, 250 ± 4,6 mm) e uno Shimadzu SPD-20A UV / VIS rilevatore impostato per monitorare a 282 nm. </li><li> Utilizzare metanolo (100%) ad una portata di 1,5 mL / min come fase mobile. Determinare quantificazione dell&#39;ergosterolo in campioni confrontando aree di picco di campioni ad una curva standard generata da HPLC ergosterolo. </li></ol> 6. Risultati rappresentativi A seguito di inoculo e l&#39;incubazione in una camera umida per due o tre giorni, la crescita di funghi dovrebbero cominciare ad apparire sul kernel. Sette giorni dopo il trattamento, la crescita vegetativa sulle piante trattate devono essere chiaramente visibili, whilcontrolli elettronici finte dovrebbe essere infetto (Figura 1B, in alto). I periodi di incubazione più sono favorevoli a più copiosa crescita vegetativa (Figura 1B, in basso). Nelle condizioni descritte nel presente documento (Figura 2G), A. flavus wild-type NRRL 3357 valori visualizzati massimi di colonizzazione, l&#39;accumulo di aflatossina, e la produzione di conidi a 8, 6, e 8 giorni, rispettivamente (Figura 2, AC). Tuttavia, quando la contaminazione da micotossine e conidiation sono stati confrontati per unità di ergosterolo, i livelli più elevati sono stati osservati a 4 e 6 giorni, rispettivamente (Figura 2, DE). Figura 2F riassume queste biomasse fungine-dipendenti il massimo valore osservazioni. È interessante notare che tra 4-6 giorni dopo l&#39;inoculazione, i gherigli di subire la degradazione degli acidi nucleici, visto da RNA totale da campioni sul tempo-corso (Figura 2H, in basso). Diversi studi, compresa la nostra, hanno esaed F. verticilliodies infezione chicchi di mais 2,8,9,10,11,12 e utilizzato con successo 7-13 giorni alla scadenza di punti per le osservazioni. Usando i metodi descritti in questa sede, la fumonisina gamma dei livelli da 3,500-8,000 ng / g gamma kernel 4,13 e livelli di ergosterolo da 5.000-10.000 ng / g kernel 14,13. La figura 3 mostra picchi rappresentativi per fumonisina (in alto) e dell&#39;ergosterolo (in basso ) da cromatogrammi HPLC. Misurazione dell&#39;ergosterolo può anche essere effettuata tramite spettri di assorbanza 15. <img alt="Figura 1" src="/files/ftp_upload/3727/3727fig1.jpg" /> Figura 1. Diagramma di flusso per la quantificazione della biomassa fungina, sporogenesi, e la produzione di micotossine e dei risultati rappresentativi per la crescita vegetativa da biotest del kernel. A) Diagramma di flusso che illustra il metodo descritto per la quantificazione dei fondamentali parametri biologici utilizzati per valutare la patogenesi fungina sul chicchi di mais. B) Rappresentanterisultati di test biologici del kernel. Top, A. flavus (NRRL3357) la crescita vegetativa a chicchi di mais in background genetico B73. I semi sono stati inoculati con 200 microlitri di 10 6 spore / mL e la foto sono state scattate sette giorni dopo l&#39;inoculazione. In basso, F. verticillioides inoculato kernel B73 in fondo dopo 13 giorni dall&#39;infezione. Kernels sono state inoculate con 200 pl di 10 6 spore / mL. <img alt="Figura 2" src="/files/ftp_upload/3727/3727fig2.jpg" /> Figura 2. Aspergillus flavus biotest kernel time-course. B73 kernel sono state inoculate con 200 microlitri di 10 6 spore / ml sospensione Aspergillus flavus conidi e incubati per 2-8 giorni (G). Tutti i valori sono stati determinati dalla media peso a secco di 4 kernel (n = 3-4; media ± SE). A) colonizzazione (basato su ergosterolo), (B) aflatossina, e (C) conidiation sono stati quantificati usando i metodi descritti sopra. E) L&#39;aflatossina e (F) conidiation vengono visualizzatiin funzione di biomassa fungina come misurata attraverso ergosterolo h. F) i valori massimi per ergosterolo, l&#39;accumulo di aflatossina e conidiation in funzione della biomassa fungina - valori massimi per ogni quantità osservato è stato fissato al 100%. (H) 1 mg per ogni corsia di RNA totale di time-course. <img alt="Figura 3" src="/files/ftp_upload/3727/3727fig3.jpg" /> Figura 3. Rappresentativi High Performance cromatografia liquida (HPLC) cromatogrammi per la fumonisina B1 (in alto) e dell&#39;ergosterolo (in basso) isolato dal kernel con infezione da Fusarium verticillioides (M3125).

<ol>
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<p class="jove_content"&gt; Non ho nulla da rivelare.</p

<p class="jove_content"&gt; I metodi descritti qui sono stati ampiamente testati e di comprovata robustezza nella generazione di risultati quantificabili per la colonizzazione fungina, sporogenesi, e la produzione di micotossine. Inoltre, questi metodi dovrebbe essere applicabile a semi di altre specie di piante che sono sensibili alla contaminazione da funghi micotossigeni (es. arachidi, grano, cotone, pistacchi, ecc.) Per competenti pianta-patogeno analisi di interazione, è imperativo che i semi essere mantenuto in vita. Un sito piccola feri...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Catalogo # </td></tr><tr><td> Agar Potato Destrosio </td><td> Fisher Scientifico </td><td> S71659A </td></tr><tr><td> Tween-20 </td><td> Fisher Scientifico </td><td> BP337-100 </td></tr><tr><td> Incubazione contenitore di plastica </td><td> Sterilite </td><td> 1713LAB06 </td></tr><tr><td> Miscelatore </td><td> VICAM </td><td> 20200 </td></tr><tr><td> 24 centimetri filtro pieghettato Papers </td><td> VICAM </td><td> 31240 </td></tr><tr><td> 1,5 um microfibra di vetro </td><td> VICAM </td><td> 31955 </td></tr><tr><td> Afla test della colonna </td><td> VICAM </td><td> G1024 </td></tr><tr><td> Afrla test Developer </td><td> VICAM </td><td> 32010 </td></tr><tr><td> Metanolo </td><td> VICAM </td><td> 35016 </td></tr><tr><td> Acetonitrile </td><td> Fisher Scientifico </td><td> AC14952-0025 </td></tr><tr><td> Etanolo </td><td> Fisher Scientifc </td><td> AC39769-0025 </td></tr><tr><td> C-18 estrazione colonna a fase solida (Prep settembre colonna SPE C18) </td><td> Fisher Scientifico </td><td> 60108-304 </td></tr><tr><td> O-ftalaldeide (OPA) </td><td> Sigma Chemical Co </td><td> 79760-5g </td></tr><tr><td> Acido borico </td><td> Fisher Scientifico </td><td> BP168-500 </td></tr><tr><td> Sodio borato </td><td> Fisher Scientifico </td><td> RDCS0330500 </td></tr><tr><td> Mercaptoetanolo </td><td> Fisher Scientifico </td><td> 45-000-231 </td></tr><tr><td> HPLC Shimadzu LC-20at (Pump) </td><td> Shimadzu Scientific Instruments, Inc. </td><td> LC-20at </td></tr><tr><td> Zorbax ODS colonna (4.6x150mm) </td><td> Agilent Technologies </td><td> 443905-902 </td></tr><tr><td> Shimatzu RF-10Axl rivelatore a fluorescenza </td><td> Shimadzu Scientific Instruments, Inc. </td><td> RF-10AXL </td></tr><tr><td> Fosfato di sodio </td><td> Fisher Scientifico </td><td> AC38987-0010 </td></tr><tr><td> FB1 standard </td><td> Sigma Chemical Co. </td><td> F1147-1mg </td></tr><tr><td> Cloroformio </td><td> VWR </td><td> MK444410 </td></tr><tr><td> 13 mm con filtro per siringa membrana di nylon 0,45 um (HPLC) </td><td> Pall Life Science </td><td> 4426 </td></tr><tr><td> Ergosterolo </td><td> Sigma-Aldrich </td><td> 45480-50G-F </td></tr><tr><td> Scintillazione fiale </td><td> VWR </td><td> 66021-602 </td></tr><tr><td> Cloruro di sodio </td><td> VICAM </td><td> G1124 </td></tr></tbody></table>

quantification of fungal colonization, sporogenesis, and production of mycotoxins using kernel bioassays

La putrefazione di cereali da seme-infettanti funghi rappresenta una delle più grandi sfide economiche a livello mondiale di produzione di cereali, per non parlare di gravi rischi per la salute umana e animale. Tra la produzione di cereali, il mais è probabilmente la coltura più colpita, a causa di agenti patogeni indotte perdite di integrità del grano e di semi di contaminazione da micotossine. I due micotossine più diffuse e problematiche per i coltivatori di mais e degli alimenti e dei mangimi sono processori aflatossina e fumonisina, prodotta da Aspergillus flavus e Fusarium verticillioides, rispettivamente. Recenti studi molecolari delle interazioni pianta-patogeno hanno dimostrato promessa la comprensione dei meccanismi specifici associati con le risposte delle piante alle infezioni fungine e contaminazione da micotossine 1,2,3,4,5,6. Poiché molti laboratori utilizzando saggi kernel per studiare le interazioni pianta-patogeno, vi è la necessità di un metodo standard per quantificare diversi parametri biologici, cosìrisultati dei diversi laboratori possono essere cross-interpretato. Per un mezzo robusto e riproducibili per le analisi quantitative sui semi, abbiamo sviluppato in laboratorio e metodi di test del kernel successivi per quantificare la crescita di funghi, biomasse e contaminazione da micotossine. Quattro chicchi di mais sterilizzati vengono inoculati in fiale di vetro con una sospensione fungina (10 6) e incubati per un periodo predeterminato. Vial vengono scelti per il conteggio di conidi mediante analisi biomassa emocitometro, ergosterolo basato mediante cromatografia liquida ad alta prestazione (HPLC), quantificazione aflatossine utilizzando un metodo AflaTest fluorometro, e quantificazione fumonisina mediante HPLC.

Watch this Scientific Journal Video about Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays at JoVE.com

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

La devastazione delle colture di cereali da semi-infezione funghi ha spinto numerosi sforzi di ricerca per comprendere meglio le interazioni pianta-patogeno. Per studiare le interazioni semi-fungine in un ambiente di laboratorio, abbiamo sviluppato un metodo affidabile per la quantificazione della riproduzione fungina, la biomassa, e la contaminazione da micotossine mediante biotest del kernel.

Quantificazione di colonizzazione fungina, sporogenesi, e la produzione di micotossine Uso Bioassays Kernel

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

quantification-fungal-colonization-sporogenesis-production-mycotoxins

Research

JoVE Journal

Immunology and Infection

18.0K Views.  Texas A&M University. La devastazione delle colture di cereali da semi-infezione funghi ha spinto numerosi sforzi di ricerca per comprendere meglio le interazioni pianta-patogeno. Per studiare le interazioni semi-fungine in un ambiente di laboratorio, abbiamo sviluppato un metodo affidabile per la quantificazione della riproduzione fungina, la biomassa, e la contaminazione da micotossine mediante biotest del kernel.

Video: Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

La colonizzazione delle Euprymna scolopes Squid da Vibrio fischeri</em

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

Ricerca

Immunologia e infezioni

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Live-cellula Microscopia Video di funghi patogeni fagocitosi

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Caratterizzazione delle risposte infiammatorie durante la colonizzazione intranasale con streptococco pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

Isolamento, la purificazione e della dicitura del mouse Bone Marrow neutrofili per studi funzionali ed Esperienze trasferimento adottivo

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

Istituzione di un<em&gt; In vitro</em&gt; Sistema per studiare intracellulare Comportamento<em&gt; Candida glabrata</em&gt; In umani THP-1 macrofagi

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Indagare gli effetti dei probiotici sul pneumococcico colonizzazione utilizzo di un<em&gt; In Vitro</em&gt; Aderenza Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

Valutazione dell'invasione batterica delle cellule cardiache in coltura e colonizzazione cardiaca nei topi infetti utilizzando listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Un modello murino di Gruppo B<em&gt; Streptococcus</em&gt; Colonizzazione vaginale

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse models of pulmonary fungal infection. Although multiple methods are used to assess fungal burden, quantitative polymerase chain reaction (qPCR) of fungal DNA has emerged as a technique with several advantages over previous culture-based methods. Currently, a comprehensive assessment of lung pathology, leukocyte recruitment, fungal burden, and gene expression in mice with invasive aspergillosis (IA) necessitates the use of a significant number of experimental and control animals. Here the quantification of lung histological staining to determine fungal burden using a reduced number of animals was examined in detail. Lung sections were stained to identify fungal structures with Gomori&#39;s modified methanamine silver (GMS) staining. Images were taken from the GMS-stained sections from 4 discrete fields of each formalin-fixed paraffin-embedded lung. The GMS stained areas within each image were quantified using an image analysis program, and from this quantification, the mean percentage of stained area was determined for each sample. Using this strategy, eosinophil-deficient mice exhibited decreased fungal burden and disease with caspofungin therapy, while wild-type mice with IA did not improve with caspofungin. Similarly, fungal burden in mice lacking &#947;&#948; T cells were also improved by caspofungin, as measured by qPCR and GMS quantification. GMS quantification is therefore introduced as a method for the determination of relative lung fungal burden that may ultimately reduce the quantity of experimental animals required for comprehensive studies of invasive aspergillosis.

Here we describe a protocol to determine pulmonary fungal burden in mice with invasive aspergillosis by quantification of Gomori&#39;s modified methanamine silver staining in histological sections. Use of this method resulted in comparable results with less animals compared to assessment of fungal burden by quantitative PCR of lung fungal DNA.

Istologico quantificazione per determinare il fardello fungine del polmone nell'aspergillosi sperimentale

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

Imaging in tempo reale e quantificazione dello sviluppo di Biofilm fungine utilizza un sistema bifase di flusso a ricircolo

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...