Agradecemos ao Dr. Diane Baronas-Lowell para a leitura crítica do manuscrito. Este trabalho foi apoiado por uma FAU Charles E. Schmidt Faculdade de Ciências Grant Semente e uma bolsa Envelhecimento da Fundação Médica Ellison para KJ

1. Placas XLD (xilose lisina desoxicolato) Agar Ágar XLD é um meio de crescimento seletivo para Salmonella, que aparece como colônias pretas nas placas de ágar XLD. No entanto, se não existem preocupações de contaminação, uma placa LB regular pode ser substituído. <ol><li> Pesar 5,5 g de ágar XLD e ressuspender em 5 ml de água deionizada. </li><li> Misture bem até que todo o agar é molhado. Adicionar 95 ml de água deionizada até que todos os pedaços se foram e o meio é completamente em suspensão. </li><li> Ferva o meio para dissolver completamente (não autoclave). </li><li> Arrefece-se o meio à temperatura ambiente e 50 ° C. </li><li> Verter 25 mL de agar em cada um 95 x 15 mm (diâmetro x altura) da placa (placas seladas com Parafilm pode ser armazenado a 4 ° C durante até 1 mês). </li></ol> 2. Nematode Crescimento Médio (NGM) RNAi Alimentando Plates Preparação de C. elegans placas NGM foi descrito anteriormente 19. Aqui um procedimento é descrito resumidamente a adicionar o antibiótico ampicilina e a RNAi indutor químico isopropílico β-D-1-tiogalactopiranósido (IPTG) para o meio de NGM para fazer as placas de alimentação de RNAi. <ol><li> Dissolve-se 3 g de NaCl e 2,5 g de Bacto peptona em 1 L de água desionizada. </li><li> Adicione 17 g de ágar Bacto na mídia. </li><li> Autoclave a media durante 45 min e arrefecer a mídia para 50 ° C em um banho de água. </li><li> Adicionar as seguintes soluções: 1 ml de colesterol (5 mg / ml em etanol a 95%), 1 mL de 1 M de CaCl2, 1 ml 1 M MgSO4, e tampão de fosfato de potássio 25 ml 1 M (pH 6,0). Misture bem. </li><li> Adicionar 1 ml de 1 M de IPTG e 500 ul de ampicilina (100 mg / ml em água esterilizada). </li><li> Misturar a solução bem e despeje em 60 x 15 mm (diâmetro x altura) placas de Petri usando uma pipeta Aid e 25 ml de pipeta sorológica seguintes procedimentos estéreis. Encha cada placa com 12 ml de ágar. As placas podem ser armazenadas a 4 ° C durante até 1 mês. </li></ol> O gene essencial autofagia bec-1 é utilizado como um exemplo para examinar a função de um gene hospedeiro na defesa contra a infecção por Salmonella. Os procedimentos experimentais são ilustradas na Figura 1 e Tabela 1. O protocolo para a preparação de animais tratados em RNAi para infecção segue, com o dia de cada passo experimental dado entre parênteses. <ol><li> Inocular bec-1 RNAi alimentação e controlar o vector vazio L4440 RNAi bactérias alimentação pela colocação de um floco de -80 ° C bactérias congeladas em 2 ml de meio LB suplementado com 100 mg / ml de ampicilina (Dia 1). Repita este passo uma vez por semana, durante todo o experimento ter bactérias RNAi frescos. Armazenar a cultura no frigorífico a 4 ° C quando não usado. </li><li> Semente 100 ml de cultura bacteriana durante a noite RNAi em placas de RNAi. Prepare três bec-1 RNAi e três controle vazio vEctor placas de RNAi. Incubar as placas a 37 ° C durante a noite (dia 2). </li><li> Remova as placas de RNAi da 37 ° C incubadora e deixe esfriar até a temperatura ambiente no banco. Pegue bem alimentados L4 tipo selvagem hermafroditas N2 e transferi-los para bec-1 RNAi e controlar placas RNAi vetores vazios. Coloque três vermes por placa, em placas triplicadas. No mesmo dia, para preparar as placas de RNAi, como descrito no passo 3.2 (Dia 3). </li><li> Incubar as placas de RNAi com vermes na incubadora a 20 ° C durante 36-40 hr e de transferência de vermes para placas frescas de RNAi correspondentes preparados no passo 3.3. Depois de vermes são transferidos, incubar as placas no 20 ° C incubadora por 64 horas (dia 4). </li></ol> 4. Prepare Salmonella para a Infecção <ol><li> Streak Salmonella -80 ° C lote congelado em 1 placa de ágar XLD e incubar a placa a 37 ° C durante a noite (dia 5 </strong&gt;). </li><li> Escolha bem isolado preto colónia de Salmonella e inocula-lo em 2 ml de meio LB a 37 ° C com agitação durante a noite (dia 6). </li><li> Semente de 80 ml ​​de cultura de uma noite de Salmonella em 1 de C. elegans 60 x 15 mm (diâmetro x altura) placa de ágar NGM e preparar 6 pratos no total. Incubar as placas à temperatura ambiente durante 6 horas. A cultura bacteriana deve secar e formar um relvado sobre a placa (dia 7). </li></ol> 5. Worms tratados com RNAi Infect com Salmonella <ol><li> Transferir bec-1 e controle de vetores vazios tratados com RNAi hermafroditas L4 N2 (progênie de minhocas criadas no passo 3) para placas de Salmonella tratados com RNAi. Colocar 40 vermes por placa em 3 placas para cada grupo. Incubar as placas sem-fim, a 20 ° C durante 48 h (Dia 7). </li><li> Prepare um conjunto de placas de RNAi frescos como descrito nos passos 3.1 e 3.2 (dia 7 e <strong&gt; Dia 8). </li><li> Após 48 horas de infecção, transferir vermes infectadas com Salmonella às placas correspondentes RNAi preparados na etapa 5.2 e incubar a 20 ° C (dia 9). </li></ol> 6. Ensaio de sobrevivência <ol><li> Marque a sobrevivência dos vermes diária e transferir worms para frescas correspondentes placas de RNAi durante o tempo de postura. Preparar um conjunto de novas placas de RNAi, antes de cada transferência de sem-fim, tal como descrito nas etapas 3.1 e 3.2. Toque o corpo verme (cabeça, parte intermediária e cauda) delicadamente com um fio de platina achatado-end. Um worm é marcado como morto se nenhum movimento do corpo verme é observada. </li><li> Marque a sobrevivência dos vermes diárias ou em dias alternados, e transferir para vermes frescas correspondentes placas de RNAi duas vezes por semana depois de vermes parar de pôr ovos. </li><li> Depois de todos os vermes morrem, reunir os dados de sobrevivência de placas triplicadas como um conjunto de dados. Introduza os dados de sobrevivência de cada grupo em software estatístico adequado, como GraphPad PRISM para gerar curvas de sobrevivência e para realizar a análise de sobrevida de Kaplan-Meier. Toda a experiência foi repetida pelo menos uma vez para confirmar a conclusão. </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

C. elegans é um simples organismo modelo genético que se alimenta de bactérias como fonte de nutrientes. Assim, é fácil de substituir a sua comida bacteriana normal com um patógeno intestinal para investigar as interações entre C. elegans e o agente patogénico escolhido. Nisto um protocolo é descrito combinar infecção por Salmonella e C. elegans RNAi alimentando tratamento para examinar o papel dos genes do hospedeiro na defesa contra a infecção por Salmonella. ...

O solo de vida livre Caenorhabditis elegans nematóide é um organismo modelo simples e geneticamente tratável usada para estudar muitas questões biológicas. C. elegans dominantemente existe como hermafroditas auto-fertilização. Os machos são gerados de forma espontânea por não-disjunção do cromossomo X durante a gametogênese 1,2. Na presença de alimento abundante, C. elegans desenvolver continuamente através de quatro fases larval ao adulto. A temperatura também influencia C. desenvolvimento elegans; desenvolvimento mais rápido é observada a temperaturas mais elevadas. No laboratório, C. elegans é cultivada a uma temperatura normal de 20 ° C em placas de agar com semeado bactéria Escherichia coli (estirpe OP50) como alimento de 1,2. Na última década, C. elegans tem emergido como um organismo de invertebrados para estudar as interações patógeno-hospedeiro 3-5. Na natureza, C. elegans come bactérias como nutrient 1,2 fonte. Sua comida laboratório bacteriana normal, OP50, pode ser facilmente substituído por outros agentes patogénicos para examinar as interações entre C. elegans e qualquer agente patogénico escolhido. Sob estas condições, o intestino é o principal local da infecção. De facto, uma grande variedade de agentes patogénicos bacterianos tem sido mostrado para infectar letalmente C. elegans 3-5. A bactéria Salmonella gram-negativo é um patógeno gastrointestinal que causa doenças de origem alimentar humana em todo o mundo 6,7. C. elegans é um bom anfitrião modelo para Salmonella typhimurium como esta bactéria replica e apresenta infecções intestinais persistentes 8-10. C. elegans foi utilizado para identificar novas e anteriormente conhecida virulência de Salmonella factores 11. Curiosamente, a C. sistema imunitário elegans limita com sucesso a replicação de Salmonella. Foi relatado anteriormente que inibidoition de genes autofagia torna o aumento da replicação Salmonella em C. elegans, resultando em morte precoce de vermes infectados 10. Macroautofagia (aqui referido como autofagia) é um processo dinâmico, que envolve o rearranjo de membranas intracelulares para sequestrar citoplasma e organelas para entrega do lisossoma para degradação 12. Autophagy foi reportado para limitar a replicação de Salmonella em C. elegans e em mamíferos 10,13. O C. elegans genoma foi o primeiro genoma eucarionte multicelular seqüenciado; ele é sensível ao tratamento 14-16 RNAi. Além disso, o RNAi pode ser administrado de forma eficaz, sujeitando vermes ingerir bactérias contendo o ARN de cadeia dupla do gene alvo, conhecido como RNAi alimentação 16,17. Genoma bibliotecas inteiras de alimentação RNAi foram gerados por RNAi do genoma triagem 16,18. Nisto, uma infecção pro Salmonellatocolo está acoplado com o RNAi alimentação para permitir o teste C. elegans genes de interesse para a sua capacidade de proteger contra infecção por Salmonella.

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

a protocol to infect caenorhabditis elegans with salmonella typhimurium

Na última década, C. elegans tem emergido como um organismo de invertebrados para estudar interações entre hospedeiros e patógenos, incluindo a defesa do hospedeiro contra bactérias gram-negativas bactéria Salmonella typhimurium. Salmonella estabelece infecção persistente no intestino de C. elegans e resulta na morte prematura dos animais infectados. Um número de mecanismos de imunidade foram identificados em C. elegans para se defender contra infecções por Salmonella. Autophagy, uma via de degradação lisossomal evolutivamente conservadas, foi mostrado para limitar a replicação de Salmonella em C. elegans e nos mamíferos. Aqui, é descrito um protocolo para infectar C. elegans com Salmonella typhimurium, em que os vermes são expostos a Salmonella por um tempo limitado, semelhante à infecção por Salmonella em humanos. infecção por Salmonella encurta significativamente o tempo de vida de C. elegans &lt;/ Em&gt;. Usando o gene essencial autofagia bec-1 como exemplo, que combinado este método de infecção com C. elegans ARNi alimentação abordagem e mostraram que este protocolo pode ser utilizado para examinar a função de C. genes do hospedeiro elegans em defesa contra a infecção por Salmonella. Desde C. elegans genoma bibliotecas inteiras RNAi estão disponíveis, este protocolo torna possível para a tela de forma abrangente para C. elegans genes que protegem contra Salmonella e outros patógenos intestinais usando bibliotecas RNAi genômicos.

A 20 ° C, o tempo de vida médio de tipo selvagem vermes N2 de 17 dias (Figura 2A e Tabela 2). Infecção por Salmonella diminui significativamente o tempo de vida médio de vermes N2 a 10,5 dias (p = 0,0002, teste de log-rank) (Figura 2A ). Se um C. elegans gene desempenha um papel importante na defesa contra a infecção por Salmonella, prevê-se que a sua inibição irá conferir susceptibilidade à infecção ...

Watch this Scientific Journal Video about A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium at JoVE.com

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans tem emergido como um novo modelo genético para estudar as interações patógeno-hospedeiro. Aqui nós descrevemos um protocolo para infectar C. elegans com Salmonella typhimurium, juntamente com a técnica de interferência dupla vertente RNAi para examinar o papel dos genes do hospedeiro na defesa contra a infecção por Salmonella.

Um protocolo para infectar<em&gt; Caenorhabditis elegans</em&gt; Com<em&gt; Salmonella typhimurium</em

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

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Research

JoVE Journal

Immunology and Infection

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

11.5K visualizações.  Florida Atlantic University.  C. elegans tem emergido como um novo modelo genético para estudar as interações patógeno-hospedeiro. Aqui nós descrevemos um protocolo para infectar C. elegans com Salmonella typhimurium, juntamente com a técnica de interferência dupla vertente RNAi para examinar o papel dos genes do hospedeiro na defesa contra a infecção por Salmonella. 

Vídeo: A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

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

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

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

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

Uma PCR Allelotyping para Identificar Salmonella enterica Sorovares Enteritidis, Hadar, Heidelberg, e Typhimurium

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

Imunologia e Infecção

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

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

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

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

De alta capacidade: Ensaio para Fenótipo<em&gt; Salmonella enterica</em&gt; Typhimurium Association, invasão e replicação em macrófagos

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

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

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

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

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

Isolamento de Salmonella typhimurium-contendo Phagosomes de macrófagos

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

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

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

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

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

Um protocolo Facile para gerar proteínas Site-Specifically acetiladas em Escherichia Coli

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

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

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

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

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

Uma plataforma de alto rendimento para o rastreio de Salmonella spp. /Shigella spp.

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

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

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

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

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

Avaliando virulência e patogênese da infecção por Aeromonas em um modelo de Caenorhabditis elegans

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

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

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

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

Aplicação de consistente massagem-como perturbações em bezerros do mouse e monitoramento das mudanças de pressão intramuscular resultante

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

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

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

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

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

Usando organóides intestinais derivados de células-tronco pluripotentes induzidas por humanos para estudar e modificar a proteção de células epiteliais contra Salmonella e outros patógenos

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

Chronic Salmonella Infection Induced Intestinal Fibrosis

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

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

Infecção crônica por Salmonella induzida por fibrose intestinal

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

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

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

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

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

NF-κB-dependente Luciferase Ativação e Quantificação da Expressão Gênica em Salmonella Células Cultura dos Tecidos Infectados

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