Thank you to Dr. Christian Braendle and the Centre Nationale de la Recherche Scientifique (CNRS) Nouragues Field Station.

1. Inducing dauer formation for wild nematodes on NGM plates
<ol>
	<li>Grow the wild Caenorhabditis strain on standard NGM plates with OP50-1 E. coli as a food source after obtaining the worms with an unculturable microbe of interest. Incubate the nematodes&#160;at 20 &#176;C. 
	NOTE: The standard temperature to grow Caenorhabditis nematodes is between 15-25 &#176;C, but should be determined empirically to prevent loss of the microbe of interest.</li>
	<li>Starve the plate of animals at 20 &#176;C for 4-5 days until all the OP50-1 is consumed and the majority have reached the dauer stage. 
	&#8203;NOTE: Dauers are long-lived larvae that develop due to the absence of nutrition and have protective cuticles.</li>
</ol>
2. Washing the nematodes
<ol>
	<li>Add 5 mL of M9 minimal salts media (42 mM Na2HPO4, 22 mM KH2PO4, 8.6 mM NaCl, 19 mM NH4Cl) to a 6 cm plate of starved worms.</li>
	<li>Using a sterile glass pipette and bulb, pipette up the M9 and worms from the plate and transfer them to a sterile 15 mL centrifuge tube.</li>
	<li>Using a clinical centrifuge, spin down the worms in the tube at 1000 x g for 30 s at room temperature.</li>
	<li>Using a sterile 15 mL pipette, remove and discard the M9 supernatant above the pellet of worms. Do not disturb the live worms at the bottom of the tube by leaving approximately 50 &#181;L of M9 above the worms.</li>
	<li>Add 10 mL of M9 + 0.05% of Triton X-100 to the same centrifuge tube and adequately tighten the tube&#39;s cap.</li>
	<li>Incubate the tube on a nutator for 20 min at room temperature to remove the external microbes. Remove the tube from the nutator and spin down the worms at 1000 x g for 30 s.</li>
	<li>Using a sterile pipette, remove and discard the M9 supernatant above the pellet of worms. Do not disturb the live worms at the bottom of the tube by leaving approximately 50 &#181;L of M9 above the worms.</li>
	<li>Follow and repeat steps 2.5-2.8 three more times.</li>
</ol>
3. Disinfecting the nematodes
<ol>
	<li>Prepare an antibiotic and SDS solution in 10 mL of M9 buffer by adding 0.25% SDS (250 &#181;L of 10% SDS), 50 &#181;g/mL of carbenicillin, 25 &#181;g/mL of kanamycin, 12.5 &#181;g/mL of tetracycline, 100 &#181;g/mL of gentamycin, 50 &#181;g/mL of streptomycin, 37.5 &#181;g/mL of chloramphenicol, and 200 &#181;g/mL of cefotaxime (see Table of Materials).</li>
	<li>Incubate the tube containing nematodes in the antibiotic and SDS solution on a nutator at room temperature overnight. 
	&#8203;NOTE: All non-dauer worms and embryos will die, but many of the dauer worms will survive this cleaning process.</li>
</ol>
4. Removing the antibiotic and SDS solution
<ol>
	<li>Spin down the worms in the tube at 1000 x g for 1 min at room temperature. Using a sterile pipette, remove the supernatant from the centrifuge tube without disturbing the worms at the bottom of the tube.</li>
	<li>Add 10 mL of M9 + 0.05% Triton X-100 and adequately tighten the cap of the tube. Incubate the tube on a nutator for 20 min at room temperature.</li>
	<li>Remove the tube from the nutator and spin down the worms at 1000 x g for 1 min. Follow and repeat steps 4.2-4.3 three times.</li>
	<li>After the last wash, leave the worm pellet undisturbed at the bottom in 100 &#181;L of the solution and discard the rest.</li>
</ol>
5. Propagate a clean nematode strain
<ol>
	<li>Using a sterile glass pipette, transfer 100 &#181;L of the supernatant and the pellet to the center of a 10 cm NGM plate seeded with OP50-1. Allow the plate to dry without disturbing the liquid at the center.</li>
	<li>Allow the dauers to crawl out of the center and through the OP50-1 lawn for 5-10 min.</li>
	<li>Carefully pick up one single dauer and plate it onto a 6 cm NGM seeded plate. In total, pick at least 10 dauers and plate them in individual 6 cm NGM plates seeded with OP50-1 (10 plates total).</li>
	<li>Incubate the plates for 4-5 days at 20 &#176;C until dauers have grown and the following generation (F1) has reached the adult stage. Visually check for contamination on all the plates, seen as non-OP50-1 microbial growth.</li>
	<li>For each clean plate, verify the propagation of the microbe of interest via Normarski or fluorescent microscopy, such as fluorescent in situ hybridization (FISH)12, depending on the phenotype of interest.</li>
</ol>
6. Intestinal dissection and PCR identification of microbial species
<ol>
	<li>Grow animals at 20 &#176;C for 3-4 days to allow them to starve and reduce the amount of OP50-1 bacteria. Add 5 mL of M9 media to the plate of starved worms.</li>
	<li>Using a sterile glass pipette and bulb, pipette up the M9 + worms from the plate and transfer them to a sterile 15 mL centrifuge tube.</li>
	<li>Using a clinical centrifuge, spin down the worms in the tube at 1000 x g for 30 s at room temperature.</li>
	<li>Using a sterile 15 mL pipette, remove the supernatant from the centrifuge tube without disturbing the live worms at the bottom of the tube.</li>
	<li>Add 10 mL of M9 + 0.05% Triton X-100 and incubate the tube on a nutator for 20 min to remove external microbes. Repeat four times to wash the worms.</li>
	<li>After the last wash, using a sterile pipette tip, remove the supernatant without disturbing the pellet at the bottom in 100 &#181;L of the solution.</li>
	<li>Using a sterile pipette tip, transfer M9 and the worms to an unseeded NGM plate and let the plate dry while the worms crawl around for 20 min to help remove OP50-1 from the cuticle and the intestine.</li>
	<li>Add 250 &#181;L of M9 to the dried plate and use a glass pipette to transfer M9 + worms to a new unseeded NGM plate. Again, let that plate dry and allow the worms to crawl around for 20 min.</li>
	<li>Add 250 &#181;L of M9 to the plate and transfer 100 &#181;L of the M9 + worms to a clean watch glass.</li>
	<li>Using two sterile 26 G syringe needles, decapitate the nematodes by holding the worm down with one needle and using the other needle to cut the worm. After decapitation, the intestine (granular) and the gonad (transparent) from the body will naturally come out of the body.</li>
	<li>Cut off a piece of the exposed intestine by holding down the intestine with one needle and cutting it with the other. 
	NOTE: If possible, verify that the microbe of interest is still present in everted intestines using Normarski microscopy, FISH, or immunohistochemistry23.</li>
	<li>Transfer a single dissected intestine into a 0.5 mL PCR tube containing 10 &#181;L of sterile water. Repeat for a total of at least 5 PCR tubes containing intestines from different animals.</li>
	<li>Freeze the PCR tubes at -80 &#176;C for a minimum of 5 min. Remove the PCR tubes from the freezer and thaw out the samples.</li>
	<li>Pipette the liquid up and down a few times to separate the bacteria from the intestine.</li>
	<li>Conduct PCR using universal primer pairs against the DNA sequence of the small ribosomal subunit of bacteria, yeast, or microsporidia, depending on the suspected type of microbe. 
	NOTE: For example, a sample protocol using universal bacterial 16S primers is presented in Table 1.</li>
	<li>Purify the amplicon using a filter-based DNA cleaning and concentration kit (see Table of Materials) and perform Sanger sequencing.</li>
</ol>

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The authors declare no conflicts of interest.

This protocol describes the isolation and identification of microbes from wild-isolated Caenorhabditis nematodes using a series of cleaning procedures. Numerous microbes are associated with wild-isolated nematodes, and some of them have exciting phenotypes that can be used for future studies in host-microbe interactions and innate immunity. Many culturable microbiome and pathogenic bacteria have been isolated from wild Caenorhabditis nematodes using standard techniques for in vitro bacterial growth25,26. However, not all microbes can be cultured in vitro, and it becomes necessary to enrich them in wild nematodes. Some microbes have a resistant spore stage, such as microsporidia, and high concentrations of SDS can be used to kill most bacteria and fungi, allowing for specific enrichment of spores12. This protocol presents a method to enrich unculturable intestinal microbes that are not resistant to SDS and antibiotic treatment.
The technique presented here takes advantage of the environmental resistance seen in dauer animals due to physiological changes such as strengthening of the cuticle, suppressing pharyngeal pumping, and covering the mouth with a buccal plug27. A critical step in this protocol is the overnight incubation with various antibiotics and 0.25% SDS. This step is used to kill all external microbes while leaving internal microbes intact. While C. elegans dauers have been demonstrated to survive SDS concentrations as high at 10% for 30 min27, this protocol uses a moderate but prolonged incubation to not only kill microbes but further expose bacteria to antibiotics. Furthermore, a moderate concentration of SDS can help ensure that dauers from other Caenorhabditis species survive, as exposure of C. tropicalis to 1% SDS overnight resulted in the death of all dauer animals. If all of the dauers die, then the concentration of SDS and/or the length of exposure to SDS should be reduced. Conversely, if the F1 generation plates still have visible contamination after cleaning, the SDS concentration and incubation time should be increased.
Another critical step is the isolation of single dauer animals after cleaning. This step is crucial as not all animals are clean after SDS and antibiotic treatment. Therefore, the animals are placed in the center of a 10 cm NGM plate with OP50-1 and allowed to crawl radially outward. Often it is best to pick more distal animals, as extended crawling through OP50-1 appears to help remove any potential surviving microbes attached to the cuticle. However, this leads to a limitation of the protocol, as it will be more challenging to enrich for a microbe of interest if it is not present in the population at a high frequency. Here, the adhering Alphaproteobacteria was present in 90%-95% of the population; therefore, most clean plates had the microbiome bacterium. However, if a microbe of interest is present at a much lower frequency in the population, it may be necessary to screen many more F1 plates.
This protocol could likely be used to isolate any number of non-culturable microbes of interest found in wild nematodes. However, the microbe must be in a tissue protected by the dauer cuticle, capable of surviving in dauer animals, and have an observable phenotype in the host. As such, this technique can be used to enrich other microbiome bacteria in the intestinal lumen besides the Alphaproteobacteria species described here, including bacteria that do not adhere. Also, the protocol was used to enrich for a facultative intracellular bacterium, Bordetella atropi, which infects the nematode Oscheius tipulae28. After enrichment, B. atropi was found to form colonies on NGM plates, showing that a microbe of interest may be discovered to be culturable in vitro once faster-growing contaminants are removed. This technique would likely work for microsproidians and viruses, including the Orsay virus, given this capacity to enrich an intracellular bacterium. However, these microbes must be capable of surviving the transition into and out of dauer.
It is important to remember that while this protocol can be performed in a Biosafety Level 1 laboratory, a sterile technique must be maintained throughout to prevent further microbial contamination. The protocol can be changed according to the researcher&#39;s needs, including the types/concentrations of antibiotics, the percentage of SDS, and/or the addition of antifungals such as nystatin. Often, the number of contaminating microbes found in a wild-isolated nematode can vary dramatically. Here, the apparent loss of non-OP50-1 E. coli growth on NGM plates was used as a readout for a clean nematode strain. But, there may be non-culturable populations of contaminating microbes present, so it is essential to conduct a metagenomics method such as 16S rRNA amplicon sequencing to see the extent of contamination26. Once the worm strain is cleaned, it can be frozen and stored away for future studies. Overall, this protocol allows researchers to enrich unculturable microbes in wild nematodes, allowing them to study effects on host fitness, characterize phenotypes of colonization or infection, and take advantage of genetic tools to understand the mechanisms underlying host-microbe interactions.

The genetic model organism C. elegans is an excellent in vivo system to study host-microbe interactions1,2. They have relatively simple physiology compared to other animals, yet, much of their cell biology is fundamentally similar to mammals making them a good model for biological research1,3,4. Additionally, they are microscopic, easy to maintain, and remain transparent throughout their short lifespan. These properties enable rapid studies into the mechanisms governing host-microbe interactions and visualization of in vivo infection and colonization of the genetically pliable hosts5,6. Finally, C. elegans rapidly&#160;responds to bacterial, fungal, and viral infections, making them an excellent model to study host-microbe interactions and the gut microbiome7,8,9.
An increase in the sampling of wild C. elegans and other nematodes has allowed for research into the ecology of free-living nematodes and natural genetic variation10,11. Concurrently, sampling has also increased the discovery of naturally occurring biological pathogens and microbes that interact with C. elegans12,13,14,15, leading to the establishment of many host-microbe model systems that study interactions with&#160;viruses, bacteria, microsporidia, oomycetes, or fungi16,17,18,19,20. Typically, wild C. elegans is found in rotting stems and fruits, often in more temperate climates, and mostly they are self-reproducing21. When these samples are brought into the lab, wild nematodes are isolated into clonal populations, carrying an array of associated microbiota. When discovering new microbes of interest in Caenorhabditis nematodes, animals are often directly screened for infection or colonization by microscopy using easily visualized phenotypes. For example, viral infection can be visualized as a disintegration of intestinal structures, and microsporidian stages can be seen inside host cells as spores or meronts14,22. When a microbe of interest is discovered for future investigation, it must be separated from the other contaminating microbes found in the wild nematodes so that it can be studied in isolation. In many cases, the microbe of interest cannot be cultured in vitro, making it essential to enrich the microbe in the host nematode.
For example, this protocol describes a wild isolate of C. tropicalis containing a bacterium that colonizes within the intestinal lumen of nematodes, adhering to the intestinal epithelial cells in a directional manner. Phenotypically, the bacterium grows perpendicular along the intestinal lumen&#39;s inner sides, giving it a bristle-like appearance, visualized on a standard Normarski microscope in all stages of the animal, including the dauer stage. The nematode growth medium (NGM) plate on which this wild C. tropicalis strain was grown contained visible contamination with other microbes. This protocol was developed to reduce additional contaminating microbial growth on the plates for studying this unknown adhering bacterium. The nematodes were forced into the dauer stage to protect the bacteria in the lumen, and then cleaned using a series of washes. Afterward, the unknown bacterial species was identified by dissection of the intestines and PCR amplification of the 16S ribosomal DNA for sequencing.
Overall, this protocol can potentially enrich any microbe of interest associated with a wild-caught nematode. Afterward, researchers will identify the target microbe, visualize in vivo infection or colonization phenotypes via microscopy, and study effects on host fitness or other aspects of host-microbe interactions. The isolation and investigation of novel microbial species that interact with Caenorhabditis nematodes can uncover the genetic mechanisms of host immunity and novel paradigms of host-microbe interactions relevant to microbial pathogenesis and microbiome studies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Fisher Scientific</td>
			<td>BP1356</td>
			<td></td>
		</tr>
		<tr>
			<td>10% SDS</td>
			<td>Invitrogen</td>
			<td>AM9822</td>
			<td></td>
		</tr>
		<tr>
			<td>BD PrecisionGlide Needle - 26 G</td>
			<td>Fisher Scientific</td>
			<td>305115</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbenicillin</td>
			<td>Millipore-Sigma</td>
			<td>C1389-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cefotaxime</td>
			<td>Millipore-Sigma</td>
			<td>C7039-500mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Millipore-Sigma</td>
			<td>C0378-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Clean and Concentrator Kit</td>
			<td>Zymo Research</td>
			<td>11-303C</td>
			<td></td>
		</tr>
		<tr>
			<td>DreamTaq Polymerase</td>
			<td>Fisher Scientific</td>
			<td>EP0711</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Millipore-Sigma</td>
			<td>G1264-250mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Millipore-Sigma</td>
			<td>K1876-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Fisher Scientific</td>
			<td>P-286</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>S-671</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Fisher Scientific</td>
			<td>A-661</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>Millipore-Sigma</td>
			<td>S6501-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracyclin</td>
			<td>Millipore-Sigma</td>
			<td>T7660-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP-151</td>
			<td></td>
		</tr>
		<tr>
			<td>Watch glasses</td>
			<td>VWR</td>
			<td>470144-850</td>
			<td></td>
		</tr>
	</tbody>
</table>

selective cleaning of wild caenorhabditis nematodes to enrich for intestinal microbiome bacteria

Caenorhabditis elegans (C. elegans)&#160;has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the context of the intestines. Recently, ecological sampling of wild Caenorhabditis nematodes has discovered a diverse array of associated microbes, including bacteria, viruses, fungi, and microsporidia. Many of these microbes have interesting colonization or infection phenotypes that warrant further study, but they are often unculturable. This protocol presents a method to enrich the desired intestinal microbes in C. elegans and related nematodes and reduce the presence of the many contaminating microbes adhering to the cuticle. This protocol involves forcing animals into the dauer stage of development and using a series of antibiotic and detergent washes to remove external contamination. As dauer animals have physiological changes that protect nematodes from harsh environmental conditions, any intestinal microbes will be protected from these conditions. But, for enrichment to work, the microbe of interest must be maintained when animals develop into dauers. When the animals leave the dauer stage, they are singly propagated into individual lines. F1 populations are then selected for desired microbes or infection phenotypes and against visible contamination. These methods will allow researchers to enrich unculturable microbes in the intestinal lumen, which make up part of the natural microbiome of C. elegans and intracellular intestinal pathogens. These microbes can then be studied for colonization or infection phenotypes and their effects on the host fitness.

A wild C. tropicalis strain (JU1848) was isolated from rotten palm tree fruits in the Nouragues Forest of French Guiana (Figure 1A)24. This strain was found to have thin microbes that colonize the lumen of the intestine in a directional manner (Figure 1B). This microbe was easily transferred to C. elegans strain N2 via co-culture with strain JU1848, where it colonized the lumen of the intestine similarly.
Propagation of JU1848 on standard NGM plates seeded with E. coli OP50-1 over multiple generations continually resulted in visible contamination, seen as various dark, mucoid colonies on and off the OP50-1 lawn (Figure 2A). A plate of wild JU1848 was starved to force animals into dauer and cleaned as described. Single dauer animals that survived cleaning were plated onto individual 6 cm NGM plates seeded with OP50-1 and allowed to grow for 4 days at 20 &#176;C. Multiple plates of F1 progeny were observed without visible microbial contamination (Figure 2B). The F1 progeny were verified to still&#160;contain adhering bacteria in the lumen of the intestine (see below).
Clean JU1848 animals were washed and decapitated to isolate intestinal pieces as described in the protocol (steps 6.1-6.12). Adhering bacteria in the lumen of the dissected intestine was verified via Nomarski microscopy (Figure 3). The microbe in the lumen of JU1848 was suspected to be a bacterium, so the dissected intestines were used as a template for PCR using universal bacterial 16S primers, 27F, and 1492R. From a total of six individual dissected intestines, the PCR products were sequenced via Sanger, and clean chromatographs showed that all the six sequences were identical. Based on these sequences, this bacterium was identified as a new species in the class Alphaproteobacteria but could not be classified into a known order or genus (Supplementary File 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62937/62937fig01.jpg" /> 
Figure 1: Adhering bacteria colonizing the lumen of a wild C. tropicalis. (A) Field sample image of rotten Euterpe sp. (Family: Arecaceae) palm tree fruits in the Nouragues forest of French Guiana.&#160;(B) Nomarski image of strain JU1848 seen with thousands of long, thin bacteria that form a brush-like appearance in the lumen (lu) of the host intestine (in). The bacterial (ba) layers coating the intestine are indicated with brackets ([). <a href="https://www.jove.com/files/ftp_upload/62937/62937fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62937/62937fig02.jpg" /> 
Figure 2: Contaminating microbial growth is lost after nematode cleaning. (A) Wild strain JU1848 propagates noticeable microbial growth on standard 6 cm NGM plates seeded with E. coli OP50-1 bacteria. (B) After cleaning, a plate of F1 progeny from a single dauer shows no visible microbial contamination after 4 days of incubation at 20 &#176;C. <a href="https://www.jove.com/files/ftp_upload/62937/62937fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62937/62937fig03.jpg" /> 
Figure 3: Adhering bacteria are seen in the lumen of the dissected intestine. Nomarski image of a clean JU1848 animal that was decapitated so that the intestines spill out. The colonizing bacteria (ba) are indicated with a bracket ([) and are seen in the lumen (lu) of a piece of the intestine (in) that is outside of the nematode body (nb). <a href="https://www.jove.com/files/ftp_upload/62937/62937fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Concentration</td>
			<td>Amount</td>
		</tr>
		<tr>
			<td>27F primer&#160; (5&#8217;-AGAGTTTGATCMTGGCTCAG-3&#8217;)</td>
			<td>20 mM</td>
			<td>2.5 &#181;L</td>
		</tr>
		<tr>
			<td>1492R primer (5&#8217;-GGTTACCTTGTTACGACTT-3&#8217;)</td>
			<td>20 mM</td>
			<td>2.5 &#181;L</td>
		</tr>
		<tr>
			<td>Dissected intestine in water</td>
			<td>N/A</td>
			<td>3 &#181;L</td>
		</tr>
		<tr>
			<td>10x PCR buffer</td>
			<td>10x</td>
			<td>5 &#181;L</td>
		</tr>
		<tr>
			<td>dNTP</td>
			<td>10 mM</td>
			<td>1 &#181;L</td>
		</tr>
		<tr>
			<td>Taq Polymerase</td>
			<td>5 U/&#181;L</td>
			<td>0.5 &#181;L</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>N/A</td>
			<td>35.5 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 1: Sample PCR protocol using universal bacterial primers and dissected intestine.
Supplementary File 1: MUSCLE alignment of bacterial 16S rDNA sequences derived from PCR of six dissected JU1848 intestines. <a href="https://www.jove.com/files/ftp_upload/62937/Supplementary File 1.docx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria at JoVE.com

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Wild Caenorhabditis nematodes are associated with many microbes, often in the gut lumen or infecting the intestine. This protocol details a method to enrich unculturable microbes colonizing the intestine, taking advantage of the resistance of the dauer cuticle.

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Caenorhabditis elegans (C. elegans)&#160;has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the ...

selective-cleaning-wild-caenorhabditis-nematodes-to-enrich-for

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

2.7K Views.  San Diego State University. Wild Caenorhabditis nematodes are associated with many microbes, often in the gut lumen or infecting the intestine. This protocol details a method to enrich unculturable microbes colonizing the intestine, taking advantage of the resistance of the dauer cuticle.

Video: Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans has emerged as a new genetic model to study host-pathogen interactions. Here we describe a protocol to infect C. elegans with Salmonella typhimurium coupled with the double-strand RNAi interference technique to examine the role of host genes in defense against Salmonella infection.

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

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Selective Harvesting of Marginating-pulmonary Leukocytes

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Evidence suggests higher cytotoxicity of natural killer cells, and a distinct pro- and anti-inflammatory profile of the MP-leukocyte population compared to circulating or splenic immunocytes. The method presented herein enables selective harvesting of MP-leukocytes by forced perfusion of the lungs in either mice or rats. In contrast to other methods used to extract lung-leukocytes, such as tissue grinding and biological degradation, this method exclusively yields leukocytes from the lung capillaries, uncontaminated with parenchymal, interstitial, and broncho-alveolar cells. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, without inducing physiological responses due to tissue processing. This unique MP leukocyte population is strategically located to identify and react towards abnormal circulating cells, as all circulating malignant cells and infected cells are detained while passing through the lung capillaries, physically interacting with endothelial cells and resident leukocytes,. Thus, selective harvesting of MP-leukocytes and their study under various conditions may advance our understanding of their biological and clinical significance, specifically with respect to controlling circulating aberrant cells and lung-related diseases.

Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of infected cells, including organelle ultrastructure, cytoskeletal network organization, as well as cellular processes such as Stress Granule (SG) formation. The characterization of how pathogens subvert host processes is an important and integral part of the field of pathogenesis. While variable phenotypes may be readily visible, the precise analysis of the qualitative and quantitative differences in the cellular structures induced by pathogen challenge is essential for defining statistically significant differences between experimental and control samples. SG formation is an evolutionarily conserved stress response that leads to antiviral responses and has long been investigated using viral infections1. SG formation also affects signaling cascades and may have other still unknown consequences2. The characterization of this stress response to pathogens other than viruses, such as bacterial pathogens, is currently an emerging area of research3. For now, quantitative and qualitative analysis of SG formation is not yet routinely used, even in the viral systems. Here we describe a simple method for inducing and characterizing SG formation in uninfected cells and in cells infected with a cytosolic bacterial pathogen, which affects the formation of SGs in response to various exogenous stresses. Analysis of SG formation and composition is achieved by using a number of different SG markers and the spot detector plug-in of ICY, an open source image analysis tool.

We describe a method for the qualitative and quantitative analysis of stress granule formation in mammalian cells after the cells are challenged with bacteria and a number of different stresses. This protocol can be applied to investigate the cellular stress granule response in a wide range of host-bacterial interactions.

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of ...

Assaying for Inorganic Polyphosphate in Bacteria

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many bacteria. There are a variety of methods for quantifying polyP in biological materials, many of which are either labor-intensive or insensitive, limiting their usefulness. We present here a streamlined method for polyP quantification in bacteria, using a silica membrane column extraction optimized for rapid processing of multiple samples, digestion of polyP with the polyP-specific exopolyphosphatase ScPPX, and detection of the resulting free phosphate with a sensitive ascorbic acid-based colorimetric assay. This procedure is straightforward, inexpensive, and allows reliable polyP quantification in diverse bacterial species. We present representative polyP quantification from the Gram-negative bacterium (Escherichia coli), the Gram-positive lactic acid bacterium (Lactobacillus reuteri), and the mycobacterial species (Mycobacterium smegmatis). We also include a simple protocol for nickel affinity purification of mg quantities of ScPPX, which is not currently commercially available.

We describe a simple method for rapid quantification of inorganic polyphosphate in diverse bacteria, including Gram-negative, Gram-positive, and mycobacterial species.

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many ...

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota and the vertebrate gut. The spatial landscape of the gut microbiota is tightly controlled by physical features - intestinal mucus, crypts, and folds - and is affected by host-controlled properties such as pH, oxygen availability, and immune factors. These properties limit the ability of commensal microbes and pathogens alike to colonize the gut stably. At the micron-scale, microbial organization determines the close-range interactions between different microbes as well as the interactions between microbes and their host. These interactions then affect large-scale organ function and host health.
This protocol enables the visualization of the gut microbiota spatial organization from distances between cells to organ-wide scales. The method is based on fixing gut tissues while preserving intestinal structure and mucus properties. The fixed samples are then embedded, sectioned, and stained to highlight specific bacterial species through fluorescence in situ hybridization (FISH). Host features, such as mucus and host cell components, are labeled with fluorescently labeled lectins. Finally, the stained sections are imaged using a confocal microscope utilizing tile-scan imaging at high magnification to bridge the micron to centimeter length scales. This type of imaging can be applied to intestinal sections from animal models and biopsies from human tissues to determine the biogeography of the microbiota in the gut in health and disease.

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

This streamlined protocol details a workflow to detect and image bacteria in complex tissue samples, from fixing the tissue to staining microbes with fluorescent in situ hybridization.

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota ...

Chemical Cartography Approaches to Study Trypanosomatid Infection

Pathogen tropism and disease tropism refer to the tissue locations selectively colonized or damaged by pathogens, leading to localized disease symptoms. Human-infective trypanosomatid parasites include Trypanosoma cruzi, the causative agent of Chagas disease; Trypanosoma brucei, the&#160;causative agent of sleeping sickness; and Leishmania species, causative agents of leishmaniasis. Jointly, they affect 20 million people across the globe. These parasites show specific tropism: heart, esophagus, colon for T. cruzi, adipose tissue, pancreas, skin, circulatory system and central nervous system for T. brucei, skin for dermotropic Leishmania strains, and liver, spleen, and bone marrow for viscerotropic Leishmania strains. A spatial perspective is therefore essential to understand trypanosomatid disease pathogenesis. Chemical cartography generates 3D visualizations of small molecule abundance generated via liquid chromatography-mass spectrometry, in comparison to microbiological and immunological parameters. This protocol demonstrates how chemical cartography can be applied to study pathogenic processes during trypanosomatid infection, beginning from systematic tissue sampling and metabolite extraction, followed by liquid chromatography-tandem mass spectrometry data acquisition, and concluding with the generation of 3D maps of metabolite distribution. This method can be used for multiple research questions, such as nutrient requirements for tissue colonization by T. cruzi, T. brucei, or Leishmania, immunometabolism at sites of infection, and the relationship between local tissue metabolic perturbation and clinical disease symptoms, leading to comprehensive insight into trypanosomatid disease pathogenesis.

This protocol describes the steps to generate a 3D model of metabolite distribution during trypanosomatid infection, including sample collection, metabolite extraction, an overview of liquid chromatography-tandem mass spectrometry data acquisition, 3D model generation, and finally, data visualization.

Pathogen tropism and disease tropism refer to the tissue locations selectively colonized or damaged by pathogens, leading to localized disease symptoms. ...

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Summary

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Chapters in this video

Application of a Mouse Ligated Peyer’s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

Biomimetic Materials to Characterize Bacteria-host Interactions

Selective Harvesting of Marginating-pulmonary Leukocytes

Selective Harvesting of Marginating-hepatic Leukocytes

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Assaying for Inorganic Polyphosphate in Bacteria

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

Chemical Cartography Approaches to Study Trypanosomatid Infection