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EoE Sub Articles

1. Bacterial Culture
<ol>
	<li>Generate isogenic mutant strains to be used for the study of the specific secreted factor of interest. 
	Note: The GAS (Group A Streptococcus) M1T1 5448 strains utilized for these experiments included WT (Wild type) GAS&#39;, a sagA&#916;cat SLS-deficient (sag = Streptolysin S associated gene, SLS = Streptolysin S) mutant (abbreviated in text as &#916;sagA), and a sagA complemented strain (abbreviated in text as &#916;sagA+sagA).</li>
	<li>Prepare overnight liquid cultures of bacterial strains of interest.&#160; Grow GAS M1T1 5448 strains in 10 ml of Todd-Hewitt broth at 37 &#176;C for 16-20 hr prior to infecting human cells.</li>
	<li>Centrifuge overnight bacterial cultures (2,400 rcf for 10 min) and resuspend in fresh bacterial medium. 
	Note: Resuspension in the same volume as the overnight cultures were grown in (5-10 ml) generally produces a desirable initial optical density.</li>
	<li>Normalize bacterial cultures to equal starting concentrations by diluting the re-suspended cultures in additional bacterial medium until the same optical density at 600 nm (OD600) is obtained for all strains to be tested (OD600 of approximately 1.0 is recommended for convenience). 
	Note: In these studies, stationary phase bacterial cultures were used to infect host cells immediately following normalization. However, as some strains of bacteria exit stationary phase nonsynchronously it may be more appropriate to begin host infections with log phase cultures if this is found to be the case for the strains of interest.</li>
	<li>Prior to further analysis, perform a colony counting assay to determine the colony forming units (CFU) per milliliter present in the starting cultures so that the multiplicity of infection (MOI), or ratio of bacteria per host cell, can be determined.
	<ol>
		<li>Prepare 1 ml serial dilutions of bacterial cultures that have been normalized to the desired optical density in phosphate-buffered saline (PBS) or appropriate bacterial growth medium (Todd-Hewitt broth was used in these studies). Prepare dilutions ranging from 10-1 to 10-6 to produce at least one set of agar plates with countable colonies (30-300 colonies). Perform all dilutions in triplicate.</li>
		<li>Transfer 100 &#181;l of each serial dilution onto an agar plate containing the appropriate medium for the bacterial species being analyzed. 
		Note: Todd-Hewitt agar was used in these studies.</li>
		<li>Use a glass or plastic bacterial spreader to evenly distribute the 100 &#181;l aliquot across the entire surface of the agar plate, and continue spreading until the liquid has been absorbed into the agar. Perform under flame using a sterile technique.</li>
		<li>Incubate the agar plates at 37 &#176;C overnight or until countable colonies appear.</li>
		<li>Count the colonies that grow on each plate and calculate the CFU/ml in the original culture by the following formula: number of colonies x inverse of the serial dilution/culture volume plated in milliliters. 
		e.g. (200 colonies x 1/10-5 dilution) / 0.1 ml plated = 2.0 x 108 CFU/ml.</li>
	</ol>
	</li>
</ol>
2. Host Cell Culture
<ol>
	<li>Obtain an appropriate host cell line for the desired analyses. 
	Note: HaCaT human epithelial keratinocytes, a kind gift from V. Nizet, were used for these studies.
	<ol>
		<li>Maintain HaCaT cells in 100 mm culture dishes in Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) with 10% heat-inactivated fetal bovine serum (FBS). Incubate at 37 &#176;C with 5% CO2.</li>
	</ol>
	</li>
</ol>
3. Host Cell Infection with Permeable Membrane Insert System
<ol>
	<li>Plate cells in appropriate tissue culture dishes and allow them to grow until they reach 90% confluency. 
	Note: The HaCaT cells used in these studies typically reach confluency within 2-3 days after plating.
	<ol>
		<li>For lysate collection (e.g. signaling analysis and adenosine triphosphate (ATP) determination assays), plate HaCaT cells in 6 well dishes at a seeding density of approximately 3 x 105 cells/well.</li>
		<li>For immunofluorescence imaging experiments, plate cells on sterile glass coverslips within 6 well dishes at a seeding density of approximately 3 x 105 cells/well.</li>
		<li>For ethidium homodimer membrane permeabilization and lactate dehydrogenase (LDH) release assays, plate HaCaT cells in 24 well dishes at a seeding density of approximately 5 x 104 cells/well.</li>
	</ol>
	</li>
	<li>Immediately prior to treatment, wash cells with 1x sterile PBS.</li>
	<li>Apply fresh growth medium to cells. For the conditions described here, apply 2 ml medium per well for 6 well plates or 0.5 ml medium per well for 24 well plates. If pharmacological treatments are being tested, mix with fresh medium and apply during this step. 
	Note: Some assays may require alternative media. For example, as phenol red and high serum concentrations interfere with the LDH release assay, phenol red-free Dulbecco&#39;s modified eagle&#39;s medium (DMEM) supplemented with 1% bovine serum albumin (BSA) (w/v), 2 mM L-glutamine, and 1 mM sodium pyruvate was utilized for these experiments.</li>
	<li>After fresh medium has been applied, carefully place a sterile 0.4 &#181;m permeable membrane insert in each well. Perform this step in a laminar flow hood, and use sterile forceps to transfer the permeable membrane insert into each well.</li>
	<li>Apply fresh cell culture medium to the upper chamber of each well according to the manufacturer&#39;s instructions. For the conditions described here, apply 1 ml medium per well for 6 well plates or 0.1 ml medium per well for 24 well plates.</li>
	<li>Apply an appropriate volume of normalized bacterial cultures (see section 1) to the upper chamber of the permeable membrane insert system. Use a bacterial medium for uninfected control treatments. For the results described here, add 25 &#181;l of the normalized cultures per chamber for 24 well plates and 100 &#181;l of the cultures per chamber for 6 well plates. 
	Note: In these studies, wild-type or mutant GAS was applied to host cells at a MOI of 10. MOI was calculated based on the final eukaryotic cell numbers (e.g. 2 x 106 cells/well), such that 10 times as many bacteria were added per host cell at the beginning of the infection period (e.g. 2 x 107 CFU per well). Appropriate MOI will vary with different bacterial species and with the desired follow-up analyses. 
	Note: In addition to using bacterial medium for uninfected controls, other useful controls for certain applications of this technique may include heat-killed bacteria or spent bacterial medium for comparison.</li>
	<li>Incubate infected cells at 37 &#176;C with 5% CO2.</li>
</ol>
4. Sample Collection
<ol>
	<li>To collect cell culture medium, host cell lysates, or glass coverslips with intact cells for follow-up analyses, begin by carefully removing the permeable membrane insert with sterile forceps.</li>
	<li>For Assessment of Secreted Host Proteins:
	<ol>
		<li>Collect the medium from the lower chamber into 1.5 ml tubes. Avoid disturbing the monolayer during collection.</li>
		<li>Centrifuge samples at 14,000 rcf for 10 min at 4 &#176;C to remove cellular debris.</li>
		<li>Remove all but 50 &#181;l of the supernatant to a fresh 1.5 ml tube and store at -20 &#176;C or use immediately.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Todd-Hewitt broth</td>
			<td>Acumedia</td>
			<td>7161A</td>
			<td>Use appropriate broth for bacterial species of interest.</td>
		</tr>
		<tr>
			<td>BioSpectrometer</td>
			<td>Eppendorf</td>
			<td>basic model</td>
			<td>Any UV-Vis spectrophotometer is suitable.</td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td>Other brands are suitable.</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma</td>
			<td>A1296-1kg</td>
			<td>Other brands are suitable.</td>
		</tr>
		<tr>
			<td>HaCaT human epithelial keratinocytes</td>
			<td></td>
			<td></td>
			<td>Gift from lab of Victor Nizet.</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Life Technologies</td>
			<td>11995-073</td>
			<td>Use appropriate media for cell line of interest.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Biowest</td>
			<td>S162H</td>
			<td>Use appropriate media additives for cell line of interest.</td>
		</tr>
		<tr>
			<td>10cm tissue culture dishes</td>
			<td>Nunc</td>
			<td>150350</td>
			<td>Other brands are suitable.</td>
		</tr>
		<tr>
			<td>6-well tissue culture dishes</td>
			<td>CytoOne</td>
			<td>cc7682-7506</td>
			<td>Other brands are suitable but need to be compatible with the Corning insert.</td>
		</tr>
		<tr>
			<td>24-well tissue culture dishes</td>
			<td>CytoOne</td>
			<td>cc7682-7524</td>
			<td>Other brands are suitable but need to be compatible with the Corning insert.</td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td>Fisherbrand</td>
			<td>12-541-B</td>
			<td>These must be autoclaved prior to use for cell plating.</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red-Free DMEM</td>
			<td>Life Technologies</td>
			<td>31053028</td>
			<td>Phenol Red-Free media is only required for the LDH release assay.</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Fisher Scientific</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030149</td>
			<td>Only required to supplement cell culture media for LDH release assay.</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Gibco</td>
			<td>BP356100</td>
			<td>Only required to supplement cell culture media for LDH release assay.</td>
		</tr>
		<tr>
			<td>Collagen-coated 0.4&#956;m Transwell inserts (6 well)</td>
			<td>Corning</td>
			<td>3540</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen-coated 0.4&#956;m Transwell inserts (24 well)</td>
			<td>Corning</td>
			<td>3595</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher</td>
			<td>3120018</td>
			<td>These must be sterilized with ethanol prior to handeling Transwell inserts.</td>
		</tr>
		<tr>
			<td>Cell scrapers</td>
			<td>Fisherbrand</td>
			<td>08-100-241</td>
			<td>These are only required for the collection of cell lysates.</td>
		</tr>
	</tbody>
</table>

a permeable membrane insert-based infection system to study the impact of bacterial toxins

Source: Flaherty, R. A., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54406">Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells.</a> J. Vis. Exp. (2016).
This video demonstrates a permeable membrane insert-based infection system to assess the impact of secreted bacterial toxins. By eliminating direct contact between the bacterial pathogen and the host cells using a permeable membrane, this technique allows for studying the effect of toxins secreted by pathogenic bacteria on mammalian host cells.

A Permeable Membrane Insert-Based Infection System to Study the Impact of Bacterial Toxins

1. Bacterial Culture

	Generate isogenic mutant strains to be used for the study of the specific secreted factor of interest.
	Note: The GAS (Group A ...

Take a two-chambered infection system separated by a permeable membrane insert. The bottom compartment contains adherent human keratinocytes, or skin cells.
Add bacterial pathogens in the upper chamber, and incubate. The membrane prevents direct contact of the pathogens with the keratinocytes. Pathogens secrete streptolysin S or SLS &#8212; a small toxin &#8212; that crosses the membrane to reach the cells.
The sodium bicarbonate co-transporter on keratinocytes mediates the influx of bicarbonate and sodium ions, maintaining intracellular pH. SLS binds to the co-transporter and disrupts ion transport, leading to intracellular osmotic stress.
The stress-induced downstream signaling causes degradation of the inhibitor bound to sequestered Nuclear Factor-Kappa B, or NF-&#954;B, leading to its activation. The activated NF-&#954;B translocates to the nucleus, leading to the upregulation of pro-inflammatory cytokine production.
Upon extracellular secretion, the cytokines bind to death receptors on the host cell, initiating necrosis. Post-incubation, harvest the culture supernatant to assess the impact of the bacterial toxin.

a-permeable-membrane-insert-based-infection-system-to-study-impact

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Host Defense Mechanism

80 次观看. 来源:Flaherty， R. A.， et al.实施基于可渗透膜插入物的感染系统，以研究分泌的细菌毒素对哺乳动物宿主细胞的影响。J. Vis. Exp.（2016 年）。 该视频演示了一种基于渗透性膜插入物的感染系统，用于评估分泌的细菌毒素的影响。通过使用可渗透膜消除细菌病原体和宿主细胞之间的直接接触，该技术允许研究病原菌分泌的毒素对哺乳动物宿主细胞的影响。 

视频: 一种基于渗透膜插入物的感染系统，用于研究细菌毒素的影响 - 概念

Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated biological pathways to the benefit of the bacterium. Identification of putative bacterial effector proteins has become more manageable due to advances in bacterial genome sequencing and the advent of algorithms that allow in silico identification of genes encoding secretion candidates and/or eukaryotic-like domains. However, identification of these important virulence factors is only an initial step. Naturally, the goal is to determine the molecular function of effector proteins and elucidate how they interact with the host. In recent years, techniques like the yeast two-hybrid screen and large-scale immunoprecipitations coupled with mass spectrometry have aided in the identification of protein-protein interactions. Although identification of a host binding partner is the crucial first step toward elucidating the molecular function of a bacterial effector protein, sometimes the host protein is found to have multiple biological functions (e.g., actin, clathrin, tubulin), or the bacterial protein may not physically bind host proteins, depriving the researcher of crucial information about the precise host pathway being manipulated. A modified yeast toxicity screen coupled with a suppressor screen has been adapted to identify host pathways impacted by bacterial effector proteins. The toxicity screen relies on a toxic effect in yeast caused by the effector protein interfering with the host biological pathways, which often manifests as a growth defect. Expression of a yeast genomic library is used to identify host factors that suppress the toxicity of the bacterial effector protein and thus identify proteins in the pathway that the effector protein targets. This protocol contains detailed instructions for both the toxicity and suppressor screens. These techniques can be performed in any lab capable of molecular cloning and cultivation of yeast and Escherichia coli.

Bacterial pathogens secrete proteins into the host that target crucial biological processes. Identifying the host pathways targeted by bacterial effector proteins is key to addressing molecular pathogenesis. Here, a method using a modified yeast suppressor and toxicity screen to elucidate host pathways targeted by toxic bacterial effector proteins is described.

使用酵母毒性和抑制器屏幕识别细菌效应蛋白的宿主通路

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated ...

科研

JoVE Journal

遗传学

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

人类舱内单聚物研究病原体、共脉和宿主肠道上皮细胞之间的相互作用

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

免疫与感染

Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins Using Leishmania major

Understanding the function and mechanism of pore-forming toxins (PFTs) is challenging because cells resist the membrane damage caused by PFTs. While biophysical approaches help understand pore formation, they often rely on reductionist approaches lacking the full complement of membrane lipids and proteins. Cultured human cells provide an alternative system, but their complexity and redundancies in repair mechanisms make identifying specific mechanisms difficult. In contrast, the human protozoan pathogen responsible for cutaneous leishmaniasis, Leishmania major,&#160;offers an optimal balance between complexity and physiologic relevance. L. major is genetically tractable and can be cultured to high density in vitro, and any impact of perturbations on infection can be measured in established murine models. In addition, L. major synthesizes lipids distinct from their mammalian counterparts, which could alter membrane dynamics. These alterations in membrane dynamics can be probed with PFTs from the best-characterized toxin family, cholesterol-dependent cytolysins (CDCs). CDCs bind to ergosterol in the Leishmania membrane and can kill L. major promastigotes, indicating that L. major is a suitable model system for determining the cellular and molecular mechanisms of PFT function. This work describes methods for testing PFT function in L. major promastigotes, including parasite culture, genetic tools for assessing lipid susceptibility, membrane binding assays, and cell death assays. These assays will enable the rapid use of L. major as a powerful model system for understanding PFT function across a range of evolutionarily diverse organisms and commonalities in lipid organization.

Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins Using Leishmania major

Presented here is a protocol using Leishmania major promastigotes to determine the binding, cytotoxicity, and signaling induced by pore-forming toxins. A proof-of-concept with streptolysin O is provided. Other toxins can also be used to leverage the genetic mutants available in L. major to define new mechanisms of toxin resistance.

利用利什曼原虫破译毛孔形成毒素的分子机制和功能

Understanding the function and mechanism of pore-forming toxins (PFTs) is challenging because cells resist the membrane damage caused by PFTs. While ...

A Permeable Membrane Insert-Based Infection System to Study the Impact of Bacterial Toxins

成績單

A Permeable Membrane Insert-Based Infection System to Study the Impact of Bacterial Toxins