establishing an infection model through microinjection of gastric organoids with pathogenic bacteria

Establishing an Infection Model Through Microinjection of Gastric Organoids with Pathogenic Bacteria

To begin microinjection of organoids, harvest H. pylori bacteria, and wash them in basal medium according to standard protocols. Following optical density measurements to determine bacterial number, dilute the culture to 1 x 109 bacteria per milliliter in basal medium.
Pull the glass capillary into two injection needles using a micropipette puller, and store in a clean petri dish. Then using forceps, break the tip of one injection needle to produce an opening that is approximately 10 micrometers wide.
Working at a stereomicroscope, set up inside a sterile culture hood, insert the needle into the injection holder, and fix it to the micromanipulator. Take up approximately 10 microliters of the bacterial solution into the needle. Then, place the 4-well cell culture plate containing gastric organoids under the stereomicroscope.
Navigating with the micromanipulator, position the needle close to an organoid. Insert the needle into the organoid with one swift movement, and then, inject approximately 0.2 microliters of bacterial solution into the center of the organoid. With experience, approximately 30 of the largest organoids in a well can be injected in 5 minutes. Incubate the injected organoids for the desired time in a cell culture incubator.
Often, individuals new to this technique will struggle because they find it difficult to efficiently target the organoids. Mouse gastric organoids grow cystic and are the easiest to target. So, injection of mouse gastric organoids with, for example, a dye, may be a good practice for this technique.

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Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

In Vitro and In Vivo Models

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1. Passage of Organoid Cultures
<ol>
	<li>Add 1 ml of cold basal medium to the basement matrix in the well. Using a 1 ml micropipette, vigorously pipette up and down until the basement matrix is broken up. Transfer to a 15 ml tube. Place on ice.</li>
	<li>Take up organoids in the pipette and vigorously pipette 10x up and down. The breaking of the organoids can be observed by the eye.</li>
</ol>
2. Microinjection of Organoid Culture.
Note: This protocol can be used to microinject bacteria into organoids. It may be helpful to start the injection with organoids that are more permissive to microinjection. For example, mouse gastric organoids can grow into very large cystic organoids that are easy to target.
<ol>
	<li>Preparation of Material
	<ol>
		<li>Pull a glass capillary into two injection needles using a micropipette puller, according to the manufacturers&#39; recommendations. The micropipette puller will heat the glass capillary in the middle and will pull the capillary into two parts, thereby generating two glass needles. It is practical to pull several needles and store them in a clean petri dish.</li>
		<li>To allow bacterial infection, remove the antibiotics from all media for at least 3 medium changes (=one week). This will dilute antibiotics from the basement matrix. If using a bacterial strain that is resistant to a specific antibiotic, add this specific antibiotic to minimize the chances of contamination.</li>
		<li>Prepare the workbench for microinjections. For work at different biosafety levels, it may be necessary to place a stereomicroscope inside a sterile bench. In this protocol, inject H. pylori under biosafety level 2 conditions under a stereomicroscope inside a sterile bench.</li>
		<li>Culture the bacteria according to standard protocols.</li>
		<li>To estimate the multiplicity of infection (MOI), estimate two parameters: The number of bacteria and the number of cells per organoid as outlined below. The MOI can correlate with results, for example with the host&#39;s IL-8 mRNA expression.
		<ol>
			<li>To calculate the bacterial density in the infection solution, first establish the standard curve of optical density and colony forming units (CFU) according to standard protocols. An optical density of 0.1 at 550 nm should yield approximately 107 CFU/ml.</li>
			<li>To estimate the number of cells per organoid, count the number of organoids in 1 well. Disrupt organoids as outlined in steps 1.1-1.4. After centrifugation for 5 min at 200 x g and 4 &#176;C, add 1 ml of enzymatic dissociation solution and resuspend.
			<ol>
				<li>Incubate at 37 &#176;C with repeated shaking for 5-10 min. Determine whether the cells have dissociated into single cells under the microscope (inverted microscope such as Leica DMIL at 10X magnification). If necessary prolong the incubation.</li>
				<li>Count cells per organoid using a hemocytometer and calculate the number of cells per organoid. Human gastric organoids with a diameter of about 200 &#181;m have approximately 4,000 cells. Injection of 0.2 &#181;l of a bacterial solution with 1 x 109 bacteria per ml results in an approximate MOI of 50.</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Injection of Organoids. 
	Note: For microinjection, the glass needle is stabilized in a holder, which can be maneuvered in 3 dimensions by a micromanipulator. Organoids remain within the basement matrix within the well. Due to size and positioning in the well, not all organoids are equally amenable to injection. Usually, the 30 largest organoids in one well can be targeted in 5 min.
	<ol>
		<li>Harvest bacteria and wash them in the basal medium according to standard protocols.</li>
		<li>Calculate the number of bacteria per ml according to the optical density (see step 2.1.5.1). Dilute the bacteria to 1 x 109 bacteria per ml in a basal medium.</li>
		<li>Take a glass needle. Using forceps break the tip so that the opening will be approximately 10 &#181;m wide.</li>
		<li>Insert the needle into the injection holder and fix it to the micromanipulator.</li>
		<li>Take up approximately 10 &#181;l bacterial solution into the needle.</li>
		<li>Place a 4-well plate with low edges containing organoid cultures under the stereomicroscope.</li>
		<li>Navigating with the micromanipulator, target a single organoid with the needle. Position the needle close to the organoid and then insert the needle into the organoid with one swift movement. Inject approximately 0.2 &#181;l bacterial solution into each organoid using the microinjector.</li>
	</ol>
	</li>
</ol>
3. Harvest Organoids for Any Analysis.
<ol>
	<li>For example, fix organoids after 4 hr. Remove supernatant and add 1 ml 2% formaldehyde and incubate 20 min at RT or O/N at 4 &#176;C. Organoids can be processed for immunohistochemistry according to standard procedures.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Invitrogen</td>
			<td>15630-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Invitrogen</td>
			<td>12634-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel, GFR, phenol free</td>
			<td>BD</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Invitrogen</td>
			<td>35050-079</td>
			<td>Stock concentration 200 mM, final concentration 2 mM</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td>Stock concentration 50 x, final concentration 1x</td>
		</tr>
		<tr>
			<td>N-Acetylcysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165-5G</td>
			<td>Stock concentration 500 mM, final concentration 1 mM</td>
		</tr>
		<tr>
			<td>Murine recombinant EGF</td>
			<td>Invitrogen</td>
			<td>PMG8043</td>
			<td>Stock concentration 500 &#181;g/ml, final concentration 50 ng/ml</td>
		</tr>
		<tr>
			<td>Human recombinant FGF10</td>
			<td>Peprotech</td>
			<td>100-26</td>
			<td>Stock concentration 100 &#181;g/ml, final concentration 200 ng/ml</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Neolabs</td>
			<td>2-1033</td>
			<td>Tweezers with fine tips are helpful for the removal of muscle layer from the tissue.</td>
		</tr>
		<tr>
			<td>4 Well Multidishes</td>
			<td>Thermo Scientific</td>
			<td>144444</td>
			<td>You can use other Multidishes. These were particularly helpful for microinjections because they have a low outer rim and allow more mobility for the manipulator.</td>
		</tr>
		<tr>
			<td>Microinjector</td>
			<td>Narishige</td>
			<td>IM-5B</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>MZ75</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>Sutter Instruments</td>
			<td>Flaming Brown Micropipette Puller</td>
			<td></td>
		</tr>
		<tr>
			<td>anti Cag A antibody</td>
			<td>Santa Cruz</td>
			<td>sc-25766</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Bartfeld, S. et al. <a href="https://app.jove.com/v/53359">Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori.</a> J. Vis. Exp.(2015).
This video demonstrates a method to establish a gastric organoid infection model by microinjecting gastric pathogen Helicobacter pylori into the lumen of the organoids. This model helps in studying infection-induced changes in gastric cells.

1. Passage of Organoid Cultures

	Add 1 ml of cold basal medium to the basement matrix in the well. Using a 1 ml micropipette, vigorously pipette up and ...

Under a stereomicroscope, observe gastric organoids, three-dimensional cellular structures composed of differentiated gastric cells, and stem cells.
Introduce a microinjector loaded with Helicobacter pylori, a gastric pathogen.
Insert the needle into the target organoid and inject the bacterial suspension.&#160;
Observe a cloudy solution develop within the lumen.
H.pylori employs a specific adhesin protein on its surface to bind to the gastric cell receptor, establishing a stable interaction between the bacteria and host cells.
This interaction triggers the elongation of the bacterial secretion pilus, a large tunnel-shaped structure that extends toward the host cell membrane.
H.pylori uses the secretion pilus to inject virulence factors, including cytotoxic associated gene A or CagA into the host cell.
Once inside the cell, CagA undergoes phosphorylation by the host cell kinase.
Phosphorylated CagA deregulates multiple intracellular signaling cascades that alter host cell functions, promoting cancerous transformation.&#160;
Collect the infected organoids for further analysis.

55 Aufrufe. Etablierung eines Infektionsmodells durch Mikroinjektion von Magenorganoiden mit pathogenen Bakterien

Serie: Etablierung eines Infektionsmodells durch Mikroinjektion von Magenorganoiden mit pathogenen Bakterien - Experiment

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2. H. pylori is the primary cause of gastric cancer, the second leading cause of cancer-related deaths worldwide3. One virulence factor that has been associated with increased risk of gastric disease is the Cag-pathogenicity island, a 40-kb region within the chromosome of H. pylori that encodes a type IV secretion system and the cognate effector molecule, CagA4,5. The Cag-T4SS is responsible for translocating CagA and peptidoglycan into host epithelial cells5,6. The activity of the Cag-T4SS results in numerous changes in host cell biology including upregulation of cytokine expression, activation of proinflammatory pathways, cytoskeletal remodeling, and induction of oncogenic cell-signaling networks5-8. The Cag-T4SS is a macromolecular machine comprised of sub-assembly components spanning the inner and outer membrane and extending outward from the cell into the extracellular space. The extracellular portion of the Cag-T4SS is referred to as the &#8220;pilus&#8221;5. Numerous studies have demonstrated that the Cag-T4SS pili are formed at the host-pathogen interface9,10. However, the environmental features that regulate the biogenesis of this important organelle remain largely obscure. Recently, we reported that conditions of low iron availability increased the Cag-T4SS activity and pilus biogenesis. Here we present an optimized protocol to grow H. pylori in varying conditions of iron availability prior to co-culture with human gastric epithelial cells. Further, we present the comprehensive protocol for visualization of the hyper-piliated phenotype exhibited in iron restricted conditions by high resolution scanning electron microscopy analyses.

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Here we describe a method to visualize the oncogenic bacterial organelle known as the Cag Type IV Secretion System (Cag-T4SS). We find that the Cag-T4SS is differentially produced on the surface of H. pylori in response to varying conditions of iron availability.

Hohe Auflösung Elektronenmikroskopie der<em&gt; Helicobacter pylori</em&gt; Cag Typ IV-Sekretionssystem Pili Produziert in variierenden Bedingungen von Iron Verfügbarkeit

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2.  H. pylori is ...

Forschung

JoVE Journal

Immunologie und Infektion

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. pylori-induced gastric inflammation by activating several innate leukocytes including neutrophils, monocytes, and mast cells. The immunogenic and immunomodulatory properties of HP-NAP make it a potential diagnostic and vaccine candidate for H. pylori and a new drug candidate for cancer therapy. In order to obtain substantial quantities of purified HP-NAP used for its clinical applications, an efficient method to purify this protein with high yield and purity needs to be established.
In this protocol, we have described a method for one-step negative chromatographic purification of recombinant HP-NAP overexpressed in Escherichia coli (E. coli) by using diethylaminoethyl (DEAE) ion-exchange resins (e.g., Sephadex) in batch mode. Recombinant HP-NAP constitutes nearly 70% of the total protein in E. coli and is almost fully recovered in the soluble fraction upon cell lysis at pH 9.0. Under the optimal condition at pH 8.0, the majority of HP-NAP is recovered in the unbound fraction while the endogenous proteins from E. coli are efficiently removed by the resin.
This purification method using negative mode batch chromatography with DEAE ion-exchange resins yields functional HP-NAP from E. coli in its native form with high yield and purity. The purified HP-NAP could be further utilized for the prevention, treatment, and prognosis of H. pylori-associated diseases as well as cancer therapy.

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

A high yield method for one-step negative purification of recombinant Helicobacter pylori neutrophil-activating protein (HP-NAP) overexpressed in Escherichia coli by using diethylaminoethyl resins in batch mode is described. HP-NAP purified by this method is beneficial for the development of vaccines, drugs, or diagnostics for H. pylori-associated diseases.

Ein-Schritt-Negative chromatographischer Reinigung von<em&gt; Helicobacter pylori</em&gt; Neutrophil-aktivierendes Protein überexprimiert in<em&gt; Escherichia coli</em&gt; Im Batch-Modus

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. ...

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in humans. Several mouse models of gastric Helicobacter infection have been developed to study the molecular and cellular mechanisms whereby H. pylori bacteria colonize the stomach of human hosts and cause disease. Herein, we describe protocols to: 1) prepare bacterial suspensions for the in vivo infection of mice via intragastric gavage; 2) determine bacterial colonization levels in mouse gastric tissues, by polymerase chain reaction (PCR) and viable counting; and 3) assess pathological changes, by histology. To establish Helicobacter infection in mice, specific pathogen-free (SPF) animals are first inoculated with suspensions (containing &#8805;105 colony-forming units, CFUs) of mouse-colonizing strains of either Helicobacter pylori or other gastric Helicobacter spp. from animals, such as Helicobacter felis. At the appropriate time-points post-infection, stomachs are excised and dissected sagittally into two equal tissue fragments, each comprising the antrum and body regions. One of these fragments is then used for either viable counting or DNA extraction, while the other is subjected to histological processing. Bacterial colonization and histopathological changes in the stomach may be assessed routinely in gastric tissue sections stained with Warthin-Starry, Giemsa or Haematoxylin and Eosin (H&#38;E) stains, as appropriate. Additional immunological analyses may also be undertaken by immunohistochemistry or immunofluorescence on mouse gastric tissue sections. The protocols described below are specifically designed to enable the assessment in mice of gastric pathologies resembling those in human-related H. pylori diseases, including inflammation, gland atrophy and lymphoid follicle formation. The inoculum preparation and intragastric gavage protocols may also be adapted to study the pathogenesis of other enteric human pathogens that colonize mice, such as Salmonella Typhimurium or Citrobacter rodentium.

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Mice represent an invaluable in vivo model to study infection and diseases caused by gastrointestinal microorganisms. Here, we describe the methods used to study bacterial colonization and histopathological changes in mouse models of Helicobacter pylori-related disease.

Maus-Modellen der Helicobacter -Infektion und Magen-Erkrankungen

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in ...

Establishing an Infection Model Through Microinjection of Gastric Organoids with Pathogenic Bacteria

Transkript

Establishing an Infection Model Through Microinjection of Gastric Organoids with Pathogenic Bacteria