Name: assessment of intestinal transcytosis of neonatal escherichia coli bacteremia isolates
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Description: Watch this Scientific Journal Video about Assessment of Intestinal Transcytosis of Neonatal Escherichia coli Bacteremia Isolates at JoVE.com

This work was supported by a Sarah Morrison student grant issued by the University of Missouri-Kansas City School of Medicine to A.I.

NOTE: Perform all the manipulations of the T84 cells, bacteria, plates, and reagents in a Biosafety Level 2 (BSL-2) safety cabinet to avoid contamination. Use separate areas and incubators for all the work involving sterile T84 cells, infected T84 cells, and E. coli. The clinical E. coli isolates tested with the methods described here were obtained following the guidelines of the Institutional Review Board at our institution1,16.
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<ol>
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This method is derived from techniques used in gastroenterology and infectious disease20. In vitro models of the intestinal epithelial barrier have been used to elucidate the mechanisms by which the luminal contents interact with this relevant component of innate immunity6,8. The host-pathogen interactions of invasive neonatal E. coli have also been separately characterized through genetic analysis, studies of antimicrobi...

Escherichia coli is the most common cause of early-onset sepsis in newborns1,2,3. The mortality rate of neonatal E. coli bacteremia can reach 40%, and meningitis is a possible complication that is associated with severe neurodevelopmental disabilities2. The ingestion of maternal E. coli strains by the newborn can produce neonatal bacteremia; this process has been replicated in animal models2,4. Once ingested, pathogenic bacteria travel from the neonatal gut lumen across the intestinal barrier and enter the bloodstream, causing septicemia. Neonatal invasive E. coli strains that produce bacteremia vary in their ability to invade intestinal epithelial cells1,5. However, their ability to transcytose the intestinal epithelium after invasion has not been completely characterized.
This intestinal transcytosis model is a useful in vitro method to emulate bacterial passage across the intestinal epithelium. The overall goal of the methods presented in this manuscript is to compare the ability of neonatal E. coli isolates to transcytose the intestinal epithelium. The model described here utilizes T84 cells, which are immortalized human intestinal adenocarcinoma cells6,7. T84 cells are grown to confluence on a semipermeable membrane with two separate compartments. The rationale for using this technique is that, as happens in vivo, these intestinal cells polarize and develop mature tight junctions6,8. The side in contact with the membrane becomes the basal side. The opposite side of the cells becomes the apical side, resembling the intestinal lumen where ingested pathogens adhere and invade. The transwell membrane is permeable to bacteria, but the polarized intestinal cells form tight junctions, which impair bacterial paracellular movement9. Thus, this method provides the advantage of a controlled in vitro environment utilizing a human cell line to study the process of bacterial transcytosis, including the transcellular route. While other methods exist to investigate the transcytosis of bacteria across the intestinal epithelium, the transwell method presented here provides greater ease and accessibility. Alternative techniques, such as those utilizing ex vivo samples set up in Ussing chamber systems, are available. However, they utilize tissue specimens that may not be easily accessible, particularly if the research intends to study human physiology10. Intestinal organoids represent another example of an in vitro alternative for studying host-bacteria interactions11. While organoid monolayers can also be used in the transwell system to study bacterial transcytosis, they require the isolation and growth of stem cells and the use of specific growth factors to induce differentiation12. Thus, their use is more time-consuming and associated with greater costs as compared to the transwell method described in this manuscript.
The assessment of bacterial passage across the intestinal epithelium using this in vitro transwell system has been successfully performed for various pathogens. These studies have shown the utility of the transwell system using T84 cells to characterize the transcytosis of bacteria across the polarized intestinal epithelium13,14,15. However, the application of this transwell method to compare the transcytosis ability of bacteremia-producing neonatal E. coli strains has not been described in detail. This manuscript provides other researchers with a standard transwell protocol that is reliable and easy to use and does not require resources that are too expensive.
To compare the ability of neonatal invasive E. coli strains to transcytose the intestinal epithelium, the apical side of the intestinal epithelial monolayer can be infected with a known number of bacterial cells. After incubation, the medium on the basal side of the epithelium can be collected and the bacteria quantified to determine the amount of bacterial transcytosis over time. In this manuscript, the methods presented are utilized to study the transcytosis ability of neonatal E. coli clinical strains recovered from newborns hospitalized with bacteremia. The inclusion criteria for the selection of these neonatal clinical isolates for transcytosis studies have been published previously1,2,16. When this method is performed using different E. coli strains, their transcytosis abilities can be compared. Through this process, the intestinal transcytosis model provides valuable data to characterize the virulence factors of E. coli that contribute to the multistep process that culminates in the development of neonatal bacteremia.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10,000 U/ mL Penicillin/Streptomycin Mixture</td>
			<td>Fisher Scientific</td>
			<td>15-140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL sterile conical tubes</td>
			<td>MidSci</td>
			<td>C15B</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL microcentrifuge tubes</td>
			<td>Avant</td>
			<td>AVSS2000</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL sterile polypropylene conical tubes</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Aspirator</td>
			<td>Corning</td>
			<td>4930</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety Cabinets</td>
			<td>Labconco</td>
			<td>30441010028343</td>
			<td>Three of these are used in the method: one for sterile tissue work, one for infected tissue work, and one for bacterial work.</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Sorvall</td>
			<td>Legend RT</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable inoculation loops</td>
			<td>Fisherbrand</td>
			<td>22363605</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11965-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Epithelial Volt/Ohm Meter</td>
			<td>World Precision Instruments</td>
			<td>EVOM</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Fisher Scientific</td>
			<td>10437028</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#39;s F-12 Nutrient Mixture</td>
			<td>Gibco</td>
			<td>11765-047</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Sigma Aldrich, Bright Line</td>
			<td>Z359629</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator shaker</td>
			<td>New Brunswick</td>
			<td>Innova 4080</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubators</td>
			<td>Thermo Scientific</td>
			<td>51030284</td>
			<td>Three of these are used in the method: one for sterile tissue culturing, one for infected tissue culturing, and one for bacterial incubation.</td>
		</tr>
		<tr>
			<td>Lysogeny broth</td>
			<td>Difco</td>
			<td>244610</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysogeny broth agar</td>
			<td>IBI Scientific</td>
			<td>IB49101</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse TS2R Microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Unico</td>
			<td>1100RS</td>
			<td></td>
		</tr>
		<tr>
			<td>T84 Intestinal Cells</td>
			<td>American Tissue Culture Collection</td>
			<td>CCL248</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture inserts, with polyethylene trephthalate membrane, 3 &#181;m pores,&#160; 24 well format</td>
			<td>Falcon</td>
			<td>353096</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture plate, 24 wells</td>
			<td>Falcon</td>
			<td>353504</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue stain</td>
			<td>Fisher Scientific</td>
			<td>T10282</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of intestinal transcytosis of neonatal escherichia coli bacteremia isolates

Newborns ingest maternal E. coli strains that colonize their intestinal tract around the time of delivery. E. coli strains with the ability to translocate across the gut invade the newborn's bloodstream, causing life-threatening bacteremia. The methodology presented here utilizes polarized intestinal epithelial cells grown on semipermeable inserts to assess the transcytosis of neonatal E. coli bacteremia isolates in vitro. This method uses the established T84 intestinal cell line that has the ability to grow to confluence and form tight junctions and desmosomes. After reaching confluence, mature T84 monolayers develop transepithelial resistance (TEER), which can be quantified using a voltmeter. The TEER values are inversely correlated with the paracellular permeability of extracellular components, including bacteria, across the intestinal monolayer. The transcellular passage of bacteria (transcytosis), on the other hand, does not necessarily alter the TEER measurements. In this model, bacterial passage across the intestinal monolayer is quantified for up to 6 h post-infection, and repeated measurements of TEER are made to monitor the paracellular permeability. In addition, this method facilitates the use of techniques such as immunostaining to study the structural changes in tight junctions and other cell-to-cell adhesion proteins during bacterial transcytosis across the polarized epithelium. The use of this model contributes to the characterization of the mechanisms by which neonatal E. coli transcytose across the intestinal epithelium to produce bacteremia.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64241/64241fig01.jpg" /> 
Figure 1: T84 TEER over time. As the T84 cell layer matures on the insert, the electrical resistance of the monolayer increases. At a TEER of at least 1,000 &#937;&#183;cm2, the cell layer is sufficiently developed to decrease the paracellular bacterial transport and allow the measurement of primarily transcellular bacterial transit....

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Assessment of Intestinal Transcytosis of Neonatal Escherichia coli Bacteremia Isolates

Escherichia coli&#160;causes sepsis in neonates who ingest the bacteria around the time of birth. The process involved in&#160;E. coli&#8217;s&#160;ability to travel from the enteric tract to the bloodstream is poorly understood. This&#160;in vitro&#160;model assesses the ability of&#160;E. coli&#160;strains to travel through the intestinal epithelial cells.

Assessment of Intestinal Transcytosis of Neonatal Escherichia coli Bacteremia Isolates

Newborns ingest maternal E. coli strains that colonize their intestinal tract around the time of delivery. E. coli strains with the ability to translocate ...

This method allows comparing the transcytosis abilities of various equalized strains across polarized intestinal epithelial cells with mature junctions. This is a relatively easy technique that does not require extensive use of resources or time, and allows investigating an important step in the pathogenesis of neonatal E.coli invasive disease. Demonstrating the procedure will be Josh Wheatley, a research assistant in my laboratory.Working inside a biosafety cabinet, seed T84 cells into polyethylene terephthalate membrane cell culture Transwell inserts. In a 24 well plate designed to hold Transwell inserts, fill the desired number of collecting wells with one milliliter of tissue culture medium or TCM with antibiotics. Seed these inserts with one times 10 to the fifth T84 cells suspended in 500 microliters of TCM with antibiotics.Approximately 48 hours after seeding, verify with light microscopy if the monolayers have started to become confluent. Every two days after seeding, measure and record the transepithelial electrical resistance or TEER using an epithelial volt per OM meter or EVOM. Before measuring the TEER, decontaminate the electrical probe by submerging it in five milliliters of 70%ethanol for 10 to 15 minutes.Remove the probe, shake off the excess ethanol, and let it air dry inside the biosafety cabinet for 10 minutes. Test the EVOM and probe by placing the dry decontaminated probe in a sterile well containing one milliliter of TCM with antibiotics with a sterile insert inside containing 500 microliters of TCM with antibiotics. Ensure that the EVOM reading is less than 200 OM.Record this blank value to use it in the later resistance calculations.Remove the probe from the tube of TCM with antibiotics. Gently lower the probe into the first insert with the long electrode in the collecting well. Allow the electrode to touch the bottom of the collecting well, but do not push down as this may disrupt the epithelial monolayer and the short electrode inside the insert.When finished, decontaminate the probe in the ethanol. Subtract the blank resistance value from each value obtained from each insert containing T84 cells. Then multiply the resulting resistance for each insert by the area of the bottom of each insert to obtain the final TEER measurement.Once the TEER reaches 1000 OMs per square centimeter, the epithelial monolayer is mature and ready for infection assays. As the TEER matures, provide the cells with a fresh medium every one to two days. In a new 24 well plate, add one milliliter of TCM with antibiotics to one well for each seeded insert.Using sterile forceps, carefully transfer the inserts to the newly replenished wells. Replace the media in the inserts. Remove the old media from the inserts by tilting the plate, and gently remove the media with a pipette tip along the side of the insert.Add 500 microliters of TCM with antibiotics to the inserts and visualize the monolayer to verify that it remains intact. The day before the experiment, measure and record the TEER. Replace the medium with one milliliter of TCM without antibiotics in the plate well, and 500 microliters in the insert.Take a labeled 15 milliliter conical tube with five milliliters of sterile lysogeny broth or LB, and use a sterile lube to inoculate the broth with one colony from one Escherichia coli strain. Incubate the culture overnight with the cap from the tube loosened. The next day, add 250 microliters from overnight LB culture to 25 milliliters of TCM without antibiotics in a 50 milliliter conical tube.Incubate the tube for two hours. Measure the TEER across each insert. Record these as the TEERs at the time T to zero hour.Move the inserts to wells in a new plate and change the media. Fill the new collecting wells with 500 microliters and inserts with 400 microliters of TCM without hand antibiotics. Keep the inserts inside the tissue culture incubator until the time of infection.After two hours, remove the morning bacterial cultures from the shaker and centrifuge. Resuspend the pellet in TCM without antibiotics. Then use a spectrophotometer to adjust the optical density or OD to 0.7 or 0.9 and further dilute to a concentration of 1 million colony forming units or CFU per milliliter.Infect each insert with 100 microliters of the OD adjusted inoculum. Note the time and record it as time zero hour. Place the inoculum bacterial suspension to quantity CFU per milliliter using the track dilution method plating 10 microliter aliquot on a square LB auger plate.Every 30 minutes following the inoculation, fill new wells with 500 microliters of TCM without antibiotics, and transfer the inserts to these new wells. Collect the media from the used collecting well for each insert into a separate labeled tube and place the tube on ice. Return the Transwell plate to the incubator between time points.For each insert, combine the collected media from different time points and vortex briefly. Plate the collected media on LB auger plates using the track dilution method to quantify the number of bacteria transcytose in the first two hours of the experiment. Collect the media at four and six hours.Additionally, at six hours, plate the media from the control wells. At six hours, measure and record the TEER. After overnight incubation, count the bacterial colonies manually on the track dilution LB plates.During the proliferation of the sterile T84 cells, the TEERs across the T84 monolayers continuously rise over time, surpassing 1000 OMs per square centimeter approximately after seven days from seeding. Transcytosis of neonatal E.coli isolates over time is shown here. The comparisons of the mean transcytosis values demonstrated significant differences between the neonatal E.coli isolates one versus two at two hours post-infection.In contrast, the nonpathogenic E.coli strain DH5 alpha underwent minimal transcytosis. TEER results before and after infection with two neonatal E.coli clinical isolates with different abilities to transcytose intestinal epithelial cells are shown here. The rate and the amount of transcytosis vary among strains but the TEER significantly increases after infection with both pathogenic strains compared to the non-infected inserts.Performing TEER measurements in a consistent manner is crucial for obtaining reproducible results with this method. Following strict sterile techniques to avoid contamination is also very important.

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Research

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Immunology and Infection

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine via a series of lymphatic vessels and nodes that converge at the superior mesenteric lymph duct. Cannulation of the mesenteric lymph duct thus enables the collection of mesenteric lymph flowing from the intestine. Mesenteric lymph consists of a cellular fraction of immune cells (99% lymphocytes), aqueous fraction (fluid, peptides and proteins such as cytokines and gut hormones) and lipoprotein fraction (lipids, lipophilic molecules and apo-proteins). The mesenteric lymph duct cannulation model can therefore be used to measure the concentration and rate of transport of a range of factors from the intestine via the lymphatic system. Changes to these factors in response to different challenges (e.g., diets, antigens, drugs) and in disease (e.g., inflammatory bowel disease, HIV, diabetes) can also be determined. An area of expanding interest is the role of lymphatic transport in the absorption of orally administered lipophilic drugs and prodrugs that associate with intestinal lipid absorption pathways. Here we describe, in detail, a mesenteric lymph duct cannulated rat model which enables evaluation of the rate and extent of lipid and drug transport via the lymphatic system for several hours following intestinal delivery. The method is easily adaptable to the measurement of other parameters in lymph. We provide detailed descriptions of the difficulties that may be encountered when establishing this complex surgical method, as well as representative data from failed and successful experiments to provide instruction on how to confirm experimental success and interpret the data obtained.

Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

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.

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

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

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 ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

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.

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

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.