Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex ...

Northwestern University Feinberg School of Medicine

Many pathogens regulate or modify their immune-stimulating ligands to avoid detection by their infected hosts. Listeria monocytogenes, a facultative intracellular bacterial pathogen, interacts with multiple components of mammalian innate immunity during its infection cycle. During replication within the cytosol of infected cells, L. monocytogenes utilizes two multidrug efflux pumps, MdrM and MdrT, to secrete the small nucleic acid second messenger cyclic-di-AMP (c-di-AMP). Host recognition of c-di-AMP triggers the production of type I interferons, including beta interferon (IFN-Î²), which, surprisingly, promote L. monocytogenes virulence. In this study, we have examined the capacity of multiple laboratory and clinical isolates of L. monocytogenes to stimulate host production of IFN-Î². We have identified the L. monocytogenes strain LO28 as able to hyperinduce IFN-Î² production in infected cells âˆ¼30-fold more than the common laboratory clone L. monocytogenes strain 10403S. Genomic analyses determined that LO28 contains a naturally occurring loss-of-function allele of the transcriptional regulator BrtA and correspondingly derepresses expression of MdrT. Surprisingly, while derepression of MdrT resulted in hyperstimulation of IFN-Î², it results in significant attenuation in multiple mouse models of infection. While type I interferons may promote L. monocytogenes virulence, this study demonstrates that unregulated expression of the c-di-AMP-secreting efflux pump MdrT significantly restricts virulence in vivo by an unknown mechanism.

Hyperinduction of host beta interferon by a Listeria monocytogenes strain naturally overexpressing the multidrug efflux pump MdrT.

De nombreux agents pathog&egrave;nes r&eacute;glementent ou modifier leurs ligands immunostimulantes pour &eacute;viter la d&eacute;tection par leurs h&ocirc;tes infect&eacute;s. Listeria monocytogenes, un bact&eacute;rie pathog&egrave;ne intracellulaire facultatif, interagit avec plusieurs composants de l&#39;immunit&eacute; inn&eacute;e chez les mammif&egrave;res au cours de son cycle d&#39;infection. Lors de la r&eacute;plication dans le cytosol des cellules infect&eacute;es, L. monocytogenes utilise deux pompes d&#39;efflux multir&eacute;sistance, MdrM et MdrT, &agrave; s&eacute;cr&eacute;ter le petit acide nucl&eacute;ique deuxi&egrave;me messager-di-AMP cyclique (c-di-AMP). Reconnaissance h&ocirc;te de c-di-AMP d&eacute;clenche la production d&#39;interf&eacute;rons, y compris l&#39;interf&eacute;ron b&ecirc;ta (IFN-&beta;), qui, &eacute;tonnamment, promouvoir la virulence de L. monocytogenes de type I. Dans cette &eacute;tude, nous avons examin&eacute; la capacit&eacute; du laboratoire multiples et des isolats cliniques de L. monocytogenes pour stimuler la production d&#39;IFN-&beta; h&ocirc;te. Nous avons identifi&eacute; la souche monocytogenes de L. LO28 comme mesure de production hyperinduce IFN-&beta; dans les cellules infect&eacute;es &sim;30-pli, plus la commune laboratoire clone L. monocytogenes souche 10403S. Analyses g&eacute;nomiques d&eacute;termin&eacute; que LO28 contient un all&egrave;le de perte de fonction naturel du r&eacute;gulateur transcriptionnel BrtA et corr&eacute;lativement derepresses expression de MdrT. &eacute;tonnamment, d&eacute;r&eacute;pression de MdrT a donn&eacute; lieu &agrave; l&#39;hyperstimulation des IFN-&beta;, il en r&eacute;sulte une att&eacute;nuation significative dans de multiples mod&egrave;les de souris de l&#39;infection. Certain temps type I interf&eacute;rons peuvent favoriser la virulence de L. monocytogenes, cette &eacute;tude d&eacute;montre que les expression non r&eacute;glement&eacute;e de la pompe &agrave; efflux c-di-AMP-s&eacute;cr&eacute;tant MdrT restreint consid&eacute;rablement la virulence in vivo par un m&eacute;canisme inconnu.

Hyperinduction d&#39;interf&eacute;ron b&ecirc;ta h&ocirc;te par une souche de monocytogenes de Listeria naturellement surexprimant la pompe &agrave; efflux multidrogue MdrT.

Molti agenti patogeni regolano o modificare loro ligands stimolante immunitario per evitare il rilevamento da loro ospiti infetti. Listeria monocytogenes, un patogeno batterico intracellulare facoltativo, interagisce con pi&ugrave; componenti dell&#39;immunit&agrave; innata dei mammiferi durante il ciclo di infezione. Durante la replica all&#39;interno del citosol delle cellule infette, L. monocytogenes utilizza due pompe di efflusso multidrug, MdrM e MdrT, a secernere il piccolo acido nucleico secondo messaggero ciclico-di-AMP (c-di-AMP). Riconoscimento ospite di c-di-AMP innesca la produzione di interferoni, tra cui l&#39;interferone beta (IFN-&beta;), che, sorprendentemente, promuovere la virulenza di L. monocytogenes di tipo I. In questo studio, abbiamo esaminato la capacit&agrave; di laboratorio pi&ugrave; e gli isolati clinici di L. monocytogenes per stimolare la produzione di IFN-&beta; host. Abbiamo identificato ceppo L. monocytogenes LO28 come in grado di produzione di IFN-&beta; hyperinduce in cellule infettate &sim;30-piega pi&ugrave; che il comune laboratorio clone L. monocytogenes strain 10403S. Analisi genomiche determinato che LO28 contiene un allele natura perdita di funzione del regolatore trascrizionale BrtA e corrispondentemente derepresses espressione di MdrT. sorprendentemente, mentre derepression di MdrT ha portato in iperstimolazione di IFN-&beta;, si traduce in attenuazione significativa in pi&ugrave; modelli del mouse dell&#39;infezione. Po di tipo interferoni possono promuovere la virulenza di L. monocytogenes, questo studio dimostra che non regolamentata espressione della pompa efflusso c-di-amplificatore-secernenti MdrT limita notevolmente la virulenza in vivo da un meccanismo sconosciuto.

Hyperinduction di interferone beta host da un ceppo di Listeria monocytogenes naturalmente con sovraesprimono relativa la pompa di efflusso multidrug MdrT.

Stuart D. Rollins

Department of Ophthalmology

Northwestern University Feinberg School of Medicine

Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model

Stuart D. Rollins

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Ophthalmology

Northwestern University Feinberg School of Medicine

Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model

Department of Ophthalmology