Name: non-invasive model of neuropathogenic escherichia coli infection in the neonatal rat
Uploaded: 2014-10-29T13:00:00
Duration: 8 min 27 s
Description: Watch this Scientific Journal Video about Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat at JoVE.com

This work was supported by research grants G0400268 and MR/K018396/1 from the Medical Research Council, and by Action Medical Research. Further support was provided by the National Institute for Health Research University College London Hospitals Biomedical Research Centre.

All animal experiments performed in this study conformed to national and European legislation and were approved by the Ethical Committee of the UCL School of Pharmacy and the UK Home Office (HO). All animal work was conducted under HO project licenses PPL 80/2243 and PPL 70/7773.
1. Rat Preparation
<ol>
	<li>Carry out all in vivo experiments using pathogen-free Wistar neonatal rats.</li>
	<li>Retain all rat litters (12 neonates per colony) in individual cages with their lactating mothers (9 - 11 week old females), under optimal conditions (19 - 21 &#176;C, 45 - 55% humidity, 15 - 20 air changes/hr and 12 hr light/dark cycle).</li>
</ol>
2. Bacterial Cell Preparation
<ol>
	<li>Store E. coli K1 stocks in 20% (v/v) glycerol at -80 &#176;C. For each experiment, plate out stock bacteria onto Mueller-Hinton (MH) agar plates, and incubate at 37 &#176;C O/N.</li>
	<li>The day before feeding, inoculate 10 ml of MH broth with a single E. coli K1 colony and culture O/N at 37 &#176;C, 200 rpm. As a control for medium contamination, prepare 10 ml of uninoculated MH broth and incubate at 37 &#176;C at 200 rpm.</li>
	<li>Transfer 100 &#181;l of O/N bacterial suspension into 9.9 ml of MH broth (1:100 v/v), and incubate at 37 &#176;C, 200 rpm, until mid-exponential-phase is reached, corresponding to an optical density of 0.6 measured at a wavelength of 600 nm (OD600).</li>
</ol>
3. Feeding of Neonatal Rats with E. coli K1
<ol>
	<li>Gently hold the animal vertically in one hand from the scruff of the neck, allowing the mouth to open. Slowly insert the sterile pipette tip into the mouth of the animal, and pipette 20 &#181;l of the inoculum (~37 &#176;C) into the mouth of the animal over a 30 sec time period. Return the animal to its mother immediately after feeding.</li>
	<li>Verify that 4 - 8 x 106 bacteria have been fed to each pup by serial dilution of the inoculum in PBS and spotting onto MH agar plates.</li>
</ol>
4. Assessment of Colonization by E. coli K1
<ol>
	<li>Gently hold the animal in one hand from the scruff of the neck. Moisten a sterile cotton-tipped swab in sterile PBS, and then gently swab the perianal area. Return the animal to its mother immediately after swabbing.</li>
	<li>Place the swab tip into a tube containing 300 &#181;l of sterile PBS and store on ice.</li>
	<li>Determine the numbers of viable bacteria by serial dilution in PBS and plating onto MacConkey agar plates.</li>
	<li>Determine viable E. coli K1 by bacteriophage susceptibility testing. Pick an individual colony using a sterile microbiological loop, dipped into 200 &#956;l sterile PBS, then subject to vortex mixing.</li>
	<li>Using a sterile microbiological loop, sub-culture onto MH agar in a straight line. Allow the plate to dry for 30 sec, and then pipette 10 &#181;l of bacteriophage K1E (109 plaque forming units/ml (PFU/ml)) onto the center of the line. Incubate the plate O/N at 37 &#176;C.</li>
	<li>Next day, examine the plate for bacteriophage-mediated lysis.</li>
</ol>
5. Assessment of Disease Severity
<ol>
	<li>Assess the disease severity of each animal 4 - 5 times daily using the seven-point scoring system outlined in Table 1.</li>
	<li>If an animal scores &#8805;3 out of 7, immediately cull the animal to minimize suffering. Record the animal as dead.</li>
</ol>
6. Euthanization of Neonatal Rats and Blood Collection
<ol>
	<li>Gently hold the animal in one hand, exposing the head and neck. Disinfect&#160;the neck of the animal by wiping with an alcohol swab. Disinfect&#160;a large pair of scissors using 70% ethanol, before rinsing in sterile PBS to remove trace ethanol.</li>
	<li>Gently hold the animal directly above a Petri dish. Decapitate the neonate using sharp surgical scissors, ensuring that the blood drips into the Petri dish. Avoid contact with the head to prevent contamination of blood with skin flora.</li>
	<li>Immediately collect the blood using a sterile pipette and mix with heparin sodium salt at a concentration of 20 - 50 units/ml in a 0.5 ml microcentrifuge tube.</li>
	<li>Determine the numbers of viable bacteria by serial dilution in PBS and plating onto MacConkey agar plates. Determine the numbers of viable E. coli K1 by testing the susceptibility of viable bacteria to bacteriophage K1E.</li>
</ol>
7. Dissection
<ol>
	<li>Following decapitation of the neonatal rat, use aseptic conditions to excise and collect tissues of interest. Wash all instruments (shown in Figure 1) in 70% ethanol and sterile PBS.</li>
	<li>Clean the dissection board and wet the abdomen completely with 70% ethanol. Position the corpse on its back and fix to the dissection board by (i) pinning the left hind leg to the operation table with a sterile needle, (ii) stretching the corpse and pinning the right foreleg to the operation table, (iii) pinning the right hind leg and left foreleg to the operation table.</li>
	<li>Pull the skin along the left hand side of the lower abdomen using forceps, then cut along the left hand side of the corpse from the lower abdomen to the sternum using small dissection scissors.</li>
	<li>Extend the excision across the sternum, and then down the right hand side of the corpse from the sternum to the lower abdomen using forceps and small dissection scissors, ensuring that none of the underlying structures are damaged.</li>
	<li>Finally, gently pull down the skin flap from the sternum to the lower abdomen using iris curved dissection forceps to expose the peritoneum.</li>
	<li>Gently raise the peritoneum with forceps and cut vertically to expose the internal organs, ensuring that none of the underlying organs are damaged.</li>
</ol>
8. Collecting the GI Tract
<ol>
	<li>Identify the stomach, small intestine, cecum, colon and mesenteric lymphatic system.</li>
	<li>Remove the stomach by gently transecting at either side, then place into a bijou containing 1.6 ml of sterile PBS using sterile forceps.</li>
	<li>Transect the colon at the rectum. Carefully pull the entire intestinal mass from the corpse using dissection forceps and place in a sterile Petri dish. Ensure that this is done gently so that the intestinal structure and mesenteric lymphatic structures do not separate.</li>
</ol>
9. Separation of the GI Tract and the Mesenteric Lymphatic System (Figure 2)
<ol>
	<li>Pour 30 ml of sterile PBS into the Petri dish, ensuring the GI tract is completely submerged.</li>
	<li>Identify the central mass of the mesenteric lymphatic system, and pinch using fine dissection forceps. Identify the most proximal part of the small intestine, and pinch using fine dissection forceps.</li>
	<li>Slowly pull the central mass of mesenteric lymphatic system and the distal small intestine in opposite directions until the two tissues are completely separated from each other. Perform this process with care to ensure that the small intestines are not stretched, as this prevents reproducible collection of proximal, middle and distal regions. Place the mesenteric lymphatic system in 300 - 500 &#956;l sterile PBS and place on ice.</li>
</ol>
10. Sectioning of the GI Tract
<ol>
	<li>Transect the GI tract at the cecum to separate the small intestine from the colon.</li>
	<li>Place the colon in 300 - 500 &#956;l sterile PBS and place on ice.</li>
	<li>Collect representative tissues from proximal, middle and distal regions of the small intestine. Align the small intestine from the mid-point.</li>
	<li>Then, (i) collect the last 2 cm of tissue prior to the caecum as the distal small intestine, (ii) collect the tissue from 5 - 7 cm above the mid-point as the proximal small intestine, and (iii) collect the tissue from 3 - 5 cm below the mid-point as the middle small intestine.</li>
</ol>
11. Collecting the Liver
<ol>
	<li>Identify the three lobes of the rat liver after removal of the stomach.</li>
	<li>Pull lobes away from the abdominal cavity using fine dissection forceps.</li>
	<li>Remove the liver by cutting any attaching ligaments using fine scissors. Place liver in 300 - 500 &#956;l sterile PBS and place on ice.</li>
</ol>
12. Collecting the Spleen
<ol>
	<li>Identify the spleen.</li>
	<li>Pull spleen away from the stomach and use fine scissors to cut the gastrosplenic ligament.</li>
	<li>Place spleen in 300 - 500 &#956;l sterile PBS and place on ice.</li>
</ol>
13. Collecting the Kidneys
<ol>
	<li>Identify the kidneys that will become visible at the back of the abdominal cavity upon removal of the gastrointestinal tract and other organs.</li>
	<li>Cut ureters and any attaching ligaments using fine dissection scissors and remove kidneys using fine forceps. Remove adrenal glands and any fatty material (if still attached) using fine forceps and dissection scissors.</li>
	<li>Place both kidneys in 300-500 &#956;l sterile PBS and place on ice.</li>
</ol>
14. Collecting the Brain
<ol>
	<li>After decapitation, hold the head in an upright position using curved forceps. Pinch skin on top of head longitudinally using another set of curved forceps, and make an incision using fine dissection scissors. Cut remaining skin near neck, again longitudinally with fine dissection scissors.</li>
	<li>Pull skin in an outwards direction using fine forceps on both sides to reveal the skull and remove skin flaps using fine dissection scissors. Place fine dissection scissors in spinal opening and make an incision along the skull towards the rostrum.</li>
	<li>Pull both sections of skull in an outwards direction using fine forceps. Cut to remove skull segments.</li>
	<li>Remove brain using broad forceps, using an upwards scooping motion from the spinal opening towards the rostrum. Wash the brain in PBS twice to remove any blood from decapitation procedure.</li>
</ol>
15. Tissue Processing and Homogenization
<ol>
	<li>For experiments requiring viability counts or DNA extraction, place tissues into tubes containing sterile PBS. Immediately determine the numbers of viable bacteria by serial dilution in PBS and plating onto MacConkey agar plates, or store at -20 &#176;C with 20% glycerol for short term. For DNA extraction, store tissues at -20 &#176;C.</li>
	<li>For experiments requiring RNA extraction, place tissues into appropriate volumes of RNAlater solution (10 &#956;l per 1 mg tissue), then store immediately at -20 &#176;C for short term or at -80 &#176;C long term storage.</li>
	<li>For experiments requiring imaging, place tissues in 10 ml Methacarn solution (60% methanol, 30% chloroform and 10% acetic acid), then store at RT (20 - 25 &#176;C) for up to 3 weeks.</li>
	<li>Calculate tissue weight by comparison of weight of tubes before and after addition of tissues. Homogenize tissues using a homogenizer. Wash the homogenizer once in 70% ethanol and twice in sterile PBS in between homogenization of each sample. 
	NOTE: Methacarn fixation is desirable for fixation of intestinal samples in order to preserve mucin-related mucosal defense structures; formalin fixation may be used for systemic tissues.</li>
</ol>

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The authors declare that they have no competing financial interests.

The animal model described here builds on previous work that aimed to reproduce the salient features of naturally occurring infections in humans. Neonatal rats were initially employed to study infant meningitis due to H. influenzae type b as the species satisfied the key criteria for a robust model of infection. Thus, the portal of entry of the relevant pathogen should reflect that of the natural human infection and reproducibly give rise to similar pathology of sufficient duration to allow for therapeutic intervention. The techniques used should not limit the applicability of the procedure and should not contribute to disease outcome20. The model of H. influenzae meningitis in infant rats developed by Moxon and colleagues satisfies these criteria20; the natural infection occurs after colonization of the mucosal membranes of the upper respiratory tract and this important feature was replicated in the rat pups by non-traumatic instillation of the bacteria onto the membranes of the nasal passages. Importantly, the age-dependent nature of the infection was replicated in the model.
The same group were also the first to develop a non-invasive model of E. coli K1 NBM in the neonatal rat22. Pathogen-free Sprague-Dawley pups were colonized by feeding 108 to 1010 bacteria through an oral gastric tube; the inoculum was therefore considerably higher than that employed by us. Colonization with the three K1 strains examined, C94 (O7:K1:H-), EC3 (O1:K1:H-) and LH (O75:K1:H3), occurred in a relatively high proportion (48-74%) of K1-fed animals, but incidences of bacteremia, meningitis and mortality were variable and significantly lower than rates of colonization. The clonal nature of E. coli K1 experimental infection was established later23 and it is now apparent that only O18:K1 and, to a lesser extent, O7:K1 serotypes are able to consistently cause systemic infection. For this reason, these investigations of the pathogenesis of neuropathogenic E. coli K1 were based on the use of the virulence-enhanced O18:K1 strain A192PP. A comparison of E. coli K1 feeding of neonatal rats through a gastric tube, as used by Glode and colleagues22, and a droplet feeding method as employed by Achtman&#8217;s group23 revealed excessive numbers of deaths using the former method, almost certainly due to damage to mucosal surfaces by the gastric tube. As rates of colonization are comparable with these two methods, it is recommended to use the less invasive method of feeding the bacteria using a pipette with a sterile tip, as described in this communication.
E. coli strain A192PP used in our studies is O18:K1. It is a more virulent derivative of the clinical strain E. coli A192 that was originally recovered from a patient with septicemia27. The increased virulence of the strain was obtained by serial passage through neonatal rats26. The strain elicits an age-dependent disease severity, with 100% bacteremia and mortality when administered to 2 day old animals28. In contrast, 9 day old animals are completely resistant to disease. K1-specific lytic bacteriophage can be used to differentiate E. coli K1 from other E. coli strains29. In this study, susceptibility of viable bacteria to bacteriophage K1E should be used to (i) check the purity of the E. coli K1 suspensions prepared to be fed to the animals, and (ii) to differentiate E. coli K1 from other coliforms in order to calculate viability in perianal swabs, blood and tissue samples. If the colony is E. coli K1, it will be susceptible to bacteriophage K1E lysis, and bacterial growth will be inhibited at the site of bacteriophage inoculation. If the colony is not E. coli K1, it will be resistant to KIE bacteriophage lysis, and there should be an area of bacterial growth at the site of bacteriophage inoculation. It should be borne in mind that animal models cannot reflect all features of the naturally occurring disease. The current model can be modified to examine the virulence characteristics of neuropathogenic bacteria other than E. coli A192PP and variations in the size of the colonizing inoculum can be accommodated. Future applications of the technique could include the evaluation of much-needed drugs to treat the condition and to uncover details of the host response to colonization and tissue invasion.
The method described here is simple but effective. Single litters of 10 - 12 pups were employed as test or control groups and this within-litter approach ensures a high degree of reproducibility and statistical validity. It is imperative that the pups are returned to their natural mothers as soon as possible after any procedure and litters should not therefore comprise animals undergoing different interventions. It is important that the fed inoculum be warmed otherwise the pups will reject the offered culture. The pups rapidly develop a complex microbiota and within two days of birth the GI tract is colonized with a wide range of bacteria from the phyla established as the most abundant microbes in the infant and adult gut. Pups that have not been fed E. coli A192PP do not carry E. coli K1 in the GI tract17 and so determination of rates of colonization is relatively straightforward. However, the neuS-based qPCR method for detection of colonizing E. coli K1 is far more sensitive that traditional culture methods and is strongly recommended17.

Systemic bacterial infections are a major threat to the well-being and survival of the newborn; preterm infants are particularly vulnerable. Neonatal bacterial meningitis (NBM), frequently associated with bacterial sepsis, continues to be a significant source of mortality and morbidity during the first few weeks of life and the problem is exacerbated by the continuing evolution of resistance to frontline antibacterial drugs1,2. A case of NBM is a medical emergency that carries a high medical, social and economic burden3; consequently, there is an urgent need for new therapeutics and, particularly, novel prophylactic strategies to reduce the burden of infection. Some features of NBM are unusual: in the developed world, Escherichia coli and Group B streptococci are responsible for the large majority of cases and the capacity of these strains to elicit NBM is almost always associated with the presence of a protective polysaccharide capsule that enables the pathogen to evade immune recognition processes4. A very high proportion (80 - 85%) of neuroinvasive E. coli express the K1 capsule5,6, an &#945;-2,8-linked polysialic acid polymer that is structurally identical to host modulators of neuronal plasticity7.
The evaluation of new therapeutics and prophylactics for NBM and associated bacteremia and sepsis would clearly benefit from a robust animal model of infection that mimics the key features of the disease in the human neonate, in particular the strong age-dependency and the natural pathway of infection. A wide range of models for Gram-positive and Gram-negative bacterial meningitis are available8,9 and these have considerably extended our knowledge of pathogenesis, pathophysiology and treatment options in these infections. Thus, experimental infections in rats, mice, rabbits and monkeys have been used to study meningitis in both the neonate and the adult. However, many of these models employ direct intracisternal or subcutaneous injection of bacteria for initiation of infection, creating an artificial pathogenesis by bypassing natural processes of dissemination from the site of colonization. In some cases these methods of inoculation led to significant changes in pathology; for example, subcutaneous administration of E. coli K1 strains abrogated the age-dependency associated with the natural infection, producing bacteremia and invasion of the central nervous system (CNS) in both neonates and adults10. Predisposition to E. coli NBM is critically dependent on vertical transmission of the causative agent from mother to infant at or soon after birth11. Maternally-derived E. coli K1 bacteria colonize the neonatal gastrointestinal (GI) tract11-13, which is sterile at birth but rapidly acquires a complex microbiota14. In colonized neonates, E. coli K1 bacteria have the capacity to translocate from the intestinal lumen into the systemic circulation before entering the CNS across the blood-brain or blood-cerebrospinal fluid barriers9,15. The design of robust models of experimental infection should take these details into account.
Although mice have been widely used for the study of some forms of bacterial meningitis8, they are unsuitable for studies of neonatal infection: they are overwhelmed by systemic infection and do not show the strong age dependency characteristic of human infants16. Further, &#945;-defensins, key peptides of the GI tract providing protection against systemic invasion by E. coli K117, are highly expressed in Paneth cells and neutrophils in humans and rats but not in mice18. There is a remarkable degree of duplication, redundancy and heterogeneity in mouse defensin and related cryptidin genes not found in other animals19. The neonatal rat was initially used by Moxon and coworkers20 to investigate the pathogenesis of Haemophilus influenzae meningitis following intranasal inoculation, replicating the natural site of colonization of this neonatal pathogen in the human, and subsequently adapted for age-dependent E. coli K1 bacteremia and meningitis. Bortolussi et al.21 employed intraperitoneal injection of the bacterial inoculum to initiate infection but the key study of Glode and coworkers22 used oral gastric feeding to parallel the natural route of infection after GI colonization. As the gastric tube may damage mucosal surfaces, the procedure was refined to include feeding of the inoculum to the neonates23. Here, the method for GI tract colonization and procedures for tracking the infection in susceptible rat pups are described; additionally, therapeutic and preventative applications of the model are discussed.

<table border="0" cellpadding="0" cellspacing="0" width="1042">
	<tbody>
		<tr>
			<td>Pathogen-free Wistar rats (12 x neonate, 1 x lactating mother)</td>
			<td>Harlan, UK</td>
			<td>www.harlan.com</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli K1 A192PP</td>
			<td>Taylor lab</td>
			<td>www.ucl.ac.uk</td>
			<td>Mushtaq et al. 2004</td>
		</tr>
		<tr>
			<td>Bacteriophage K1E</td>
			<td>Taylor lab</td>
			<td>www.ucl.ac.uk</td>
			<td>Mushtaq et al. 2004</td>
		</tr>
		<tr>
			<td>Glycerol&#160;</td>
			<td>Sigma, UK</td>
			<td>www.sigmaaldrich.com</td>
			<td>G5516</td>
		</tr>
		<tr>
			<td>Mueller-Hinton Agar</td>
			<td>Oxoid, UK</td>
			<td>www.oxoid.com</td>
			<td>CM0337</td>
		</tr>
		<tr>
			<td>Mueller-Hinton Broth</td>
			<td>Oxoid, UK</td>
			<td>www.oxoid.com</td>
			<td>CM0405</td>
		</tr>
		<tr>
			<td>MacConkey Agar</td>
			<td>Oxoid, UK</td>
			<td>www.oxoid.com</td>
			<td>CM0007</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma, UK</td>
			<td>www.sigmaaldrich.com</td>
			<td>P4417</td>
		</tr>
		<tr>
			<td>Ethanol 100%</td>
			<td>Sigma, UK</td>
			<td>www.sigmaaldrich.com</td>
			<td>E7023</td>
		</tr>
		<tr>
			<td>Heparin Sodium Salt</td>
			<td>Sigma, UK</td>
			<td>www.sigmaaldrich.com</td>
			<td>84020 
			Prepare 20-50 units/ml</td>
		</tr>
		<tr>
			<td>RNAlater Solution</td>
			<td>Sigma, UK</td>
			<td>www.sigmaaldrich.com</td>
			<td>R0901 
			10&#181;l/mg tissue</td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Sigma, UK</td>
			<td>www.sigmaaldrich.com</td>
			<td>320099</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma, UK</td>
			<td>www.sigmaaldrich.com</td>
			<td>C2432</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma, UK</td>
			<td>www.sigmaaldrich.com</td>
			<td>322415</td>
		</tr>
		<tr>
			<td>Cotton-tipped swabs</td>
			<td>Fisher Scientific, UK</td>
			<td>www.fisher.co.uk</td>
			<td>11542483</td>
		</tr>
		<tr>
			<td>Alcotip Swabs</td>
			<td>Scientific Laboratory Supplies, UK</td>
			<td>www.scientificlabs.co.uk</td>
			<td>SWA1000</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Sigma, UK</td>
			<td>www.sigmaaldrich.com</td>
			<td>P5856</td>
		</tr>
		<tr>
			<td>30mL Universal Tube</td>
			<td>AlphaLaboratories, UK</td>
			<td>www.alphalabs.co.uk</td>
			<td>CW3890</td>
		</tr>
		<tr>
			<td>0.5ml microcentrifuge tubes</td>
			<td>StarLab, UK</td>
			<td>www.starlab.co.uk</td>
			<td>I1405-1500</td>
		</tr>
		<tr>
			<td>1.5ml microcentrifuge tubes</td>
			<td>StarLab, UK</td>
			<td>www.starlab.co.uk</td>
			<td>I1415-1000</td>
		</tr>
		<tr>
			<td>0.1&#181;l calibrated loops</td>
			<td>StarLab, UK</td>
			<td>www.starlab.co.uk</td>
			<td>E1412-0112</td>
		</tr>
		<tr>
			<td>L-shaped spreaders</td>
			<td>StarLab, UK</td>
			<td>www.starlab.co.uk</td>
			<td>E1412-1005</td>
		</tr>
		<tr>
			<td>Cuvettes</td>
			<td>Fisher Scientific, UK</td>
			<td>www.fisher.co.uk</td>
			<td>10594175</td>
		</tr>
		<tr>
			<td>Forceps straight with fine points</td>
			<td>Fisher Scientific, UK</td>
			<td>www.fisher.co.uk</td>
			<td>12780036</td>
		</tr>
		<tr>
			<td>Forceps straight with blunt tips</td>
			<td>Fisher Scientific, UK</td>
			<td>www.fisher.co.uk</td>
			<td>12391369</td>
		</tr>
		<tr>
			<td>Forceps watchmaker&#39;s curved with very fine points&#160;</td>
			<td>Fisher Scientific, UK</td>
			<td>www.fisher.co.uk</td>
			<td>12740926</td>
		</tr>
		<tr>
			<td>Scissors straight with very fine points</td>
			<td>Fisher Scientific, UK</td>
			<td>www.fisher.co.uk</td>
			<td>12972055</td>
		</tr>
		<tr>
			<td>Laboratory Scissors</td>
			<td>VWR, UK</td>
			<td>www.vwr.com</td>
			<td>USBE4251</td>
		</tr>
		<tr>
			<td>25g Syringe Needles</td>
			<td>Greiner Bio-One Ltd</td>
			<td>www.greinerbioone.com</td>
			<td>N2525</td>
		</tr>
		<tr>
			<td>LAMBDA 25 UV/Vis Spectrophotometers</td>
			<td>PerkinElmer, UK</td>
			<td>www.perkinelmer.co.uk</td>
			<td>L60000BB</td>
		</tr>
		<tr>
			<td>Unitemp Incubator</td>
			<td>B&#38;T, UK</td>
			<td></td>
			<td>OP958</td>
		</tr>
		<tr>
			<td>Multitron shaking incubator</td>
			<td>INFORS HT, UK</td>
			<td>www.infors-ht.com</td>
			<td>AJ118</td>
		</tr>
		<tr>
			<td>Ultra-Turrax T-10 homogenizer</td>
			<td>IKA Werke</td>
			<td>www.ika.com</td>
			<td>0003737000</td>
		</tr>
	</tbody>
</table>

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.

The E. coli K1 systemic infection model described here replicates many of the features of the natural infection in humans. The bacteria are ingested, colonize the GI tract, translocate into the blood compartment via the mesenteric lymph nodes before establishing organ-specific disease with associated inflammation of the brain24. Importantly, the model displays strong age dependency; as shown in Figure 3, two-day-old (P2) rat pups are highly susceptible to invasive disease but over a seven-day period the animals become progressively more refractory to infection, but not to GI tract colonization17. After transit from the site of GI colonization to the blood compartment, the bacteria can be visualized in blood samples by fluorescence microscopy (Figure 4) before entering the CNS predominantly at the choroid plexus25. In some animals, there is extensive invasion of other major organs such as lung, spleen and kidney25.
Bacterial numbers in tissues can vary substantially between individual pups25 but when the bioburden is scored as either present or absent there is a high degree of reproducibility with regard to organ invasion. With a litter of 12 pups as a single test cohort, power calculations using G* Power Software determined that this sample size equates to a 98.6% probability of finding an effect based on survival using six animals from the cohort and &#62;99% probability if all twelve are taken into consideration. The model is therefore suitable for evaluation of novel agents specifically tailored for the treatment of neonatal bacterial infections and has been used in the procedure to evaluate the therapeutic potential of the capsule depolymerase EndoE that selectively removes the K1 capsule from the bacterial surface24-26. It may also be used to investigate host-bacteria interactions that impact on the pathogenesis of E. coli neuropathogens; within this context it has been employed for studies of E. coli A192PP colonization and dissemination. It has been demonstrated that E. coli A192PP cells persist in the GI tract of P2, P5 and P9 pups in large numbers; temporal aspects of colonization in these three groups were very similar (Figure 5) and reflects the capacity of the bacteria to replicate and maintain population density within the gut.
The virulence of the clinical isolate A192 was enhanced by serial passage in neonatal rats in order to ensure little or no redundancy of animal use. E. coli A192 colonized P2 neonatal rats with 100% efficiency, elicited bacteremia in 35% of animals and produced a lethal effect in 25%27. The passaged derivative A192PP colonizes the GI tract, produces bacteremia and causes lethality in all P2 pups. Thus, the model can be employed to investigate the virulence of different K1 strains with respect to their capacity to invade the CNS and other organ systems from the site of colonization. In this context, Pluschke and co-workers23 used a neonatal rat infection model to determine the capacity of 95 E. coli K1 strains of human origin to cause bacteremia after gut colonization; they observed wide variations in the efficiency of both colonization and invasive capability, underpinning the clonal nature of E. coli K1 neuropathogens.
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/52018/52018fig1highres.jpg" width="600" /> 
Figure 1. Materials for tissue collection: (A) weighing scale, (B) pre-weighed tubes containing necessary media, (C) operation table, ruler and needles to pin down the animal, (D) 70% (v/v) ethanol and PBS to sterilize the tissue collection equipment, (E) tissue collection kit including a large pair of scissors for decapitation and scissors and tweezers and blades of various size and shapes, (F) ice to preserve the tissues, (G) 70% (v/v) ethanol for sterilizing the operation table and surrounds. <a href="https://www.jove.com/files/ftp_upload/52018/52018fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="6in" src="/files/ftp_upload/52018/52018fig2highres.jpg" width="600" /> 
Figure 2. Separation of the GI tract and the mesenteric lymphatic system. (A) Grip the central mass of the mesenteric lymphatic system with fine dissection forceps. (B) Grip the proximal part of the small intestine, and pull in opposite directions. (C) The GI tract and mesenteric lymphatic system will fully separate. <a href="https://www.jove.com/files/ftp_upload/52018/52018fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/52018/52018fig3highres.jpg" width="500" /> 
Figure 3. Survival of neonatal rat pups aged from two days (P2) to nine days (P9) following oral administration of E. coli A192PP, illustrating the strong age dependency of systemic infection. Each group represents 24 neonates.
<img alt="Figure 4" fo:content-width="6in" src="/files/ftp_upload/52018/52018fig4highres.jpg" width="600" /> 
Figure 4. Fluorescence images of E. coli A192PP cells in a blood smear from a P2 pup infected following oral administration of bacteria. The lipopolysaccharide O antigen at the bacterial surface was stained with rabbit anti-O18 polyclonal antibody and Alexa546-conjugated goat-anti-rabbit second antibody. The K1 capsule was visualized with EndoE-GFP reagent. Virtually all bacteria detected in blood samples displayed the protective K1 capsule. Images were captured by Dr Andrea Zelmer. <a href="https://www.jove.com/files/ftp_upload/52018/52018fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/52018/52018fig5highres.jpg" width="500" /> 
Figure 5. E. coli A192PP intestinal colonization following administration of the bacterial inoculum. DNA was extracted from whole intestine and E. coli K1 colony forming units/g (CFU/g) tissue determined by quantitative polymerase chain reaction (qPCR) targeting the polysialyltransferase (neuS) gene as described elsewhere17. LOD: limit of detection.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Feature</td>
			<td>Healthy</td>
			<td>Unhealthy</td>
		</tr>
		<tr>
			<td>Color of the skin</td>
			<td>Pink</td>
			<td>Pale/Yellow</td>
		</tr>
		<tr>
			<td>Agility (righting reflex)</td>
			<td>Pup immediately reverses on backward placement</td>
			<td>Difficulty in reversing backward placement (&#62; 3 sec) or cannot achieve</td>
		</tr>
		<tr>
			<td>Gentle pressure on the abdomen</td>
			<td>No sound</td>
			<td>Sound of agitation</td>
		</tr>
		<tr>
			<td>Stomach/milk line</td>
			<td>Visible and white</td>
			<td>Not visible</td>
		</tr>
		<tr>
			<td>Temperature</td>
			<td>Warm</td>
			<td>Relatively cold*</td>
		</tr>
		<tr>
			<td>Weight</td>
			<td>Gain of 1.5-2 g per day</td>
			<td>No weight gain or weight loss</td>
		</tr>
		<tr>
			<td>Behavior when placed in cage</td>
			<td>Moves towards mother and starts feeding</td>
			<td>Cannot move towards mother and shows difficulty feeding</td>
		</tr>
	</tbody>
</table>
Table 1. Seven-point scoring system: The first three scores listed are usually the initial signs observed. *Neonates with systemic infection experience elevated body temperature (&#62; 2 &deg;C). However, due to the lack of agility of the animals to reach their mother to maintain body temperature, unhealthy animals may become separated from the litter and feel cold to the bare hand.

Watch this Scientific Journal Video about Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat at JoVE.com

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.

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 ...

non-invasive-model-neuropathogenic-escherichia-coli-infection

Research

JoVE Journal

Immunology and Infection

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

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. ...

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 ...

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. ...