Name: separation of the cell envelope for gram-negative bacteria into inner and outer membrane fractions with technical adjustments for acinetobacter baumannii
Uploaded: 2020-04-10T13:00:00
Description: Watch this Scientific Journal Video about Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii at J

This work was funded by P20GM10344 and R01AI139248 awarded to Z. D. Dalebroux.

1. General Reagents and Media Preparation for Membrane Extraction
<ol>
	<li>Bacterial growth media: Prepare and sterilize 1 L of broth media in a thoroughly cleaned and autoclaved 2 L flask.</li>
	<li>General resuspension buffer (1 M Tris Buffer pH 7.5; 50 mL): Dissolve 6.05 g of Tris base in 30 mL of H2O. Adjust pH to 7.5 with 5 M HCl. Adjust final volume to 50 mL with ultrapure H2O.</li>
	<li>Master stock of divalent cation chelation solution (0.5 M EDTA pH 8; 100 mL): Add 18.6 g of disodium eth...

<ol>
	<li>Silhavy, T. J., Kahne, D., Walker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bacterial+cell+envelope.">The bacterial cell envelope.</a> Cold Spring Harbor Perspectives in Biology. 2, 000414 (2010).</li><li>Egan, A. J., Vollmer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physiology+of+bacterial+cell+division.">The physiology of bacterial cell division.</a> Annals of the New York Academy of Sciences. 1277, 8-28 (2013).</li><li>Pazos, M., Peters, K., Vollmer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+peptidoglycan+growth+by+dynamic+and+variable+multi-protein+complexes.">Robust peptidoglycan growth by dynamic and variable multi-protein complexes.</a> Current Opinion in Microbiology. 36, 55-61 (2017).</li><li>Raetz, C. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fortifying+the+barrier:+the+impact+of+lipid+A+remodelling+on+bacterial+pathogenesis.">Fortifying the barrier: the impact of lipid A remodelling on bacterial pathogenesis.</a> Nature Reviews Microbiology. 11, 467-481 (2013).</li><li>Simpson, B. W., Trent, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pushing+the+envelope:+LPS+modifications+and+their+consequences.">Pushing the envelope: LPS modifications and their consequences.</a> Nature Reviews Microbiology. 17, 403-416 (2019).</li><li>Ebbensgaard, A., Mordhorst, H., Aarestrup, F. M., Hansen, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Outer+Membrane+Proteins+and+Lipopolysaccharides+for+the+Sensitivity+of+Escherichia+coli+to+Antimicrobial+Peptides.">The Role of Outer Membrane Proteins and Lipopolysaccharides for the Sensitivity of Escherichia coli to Antimicrobial Peptides.</a> Frontiers in Microbiology. 9, 2153 (2018).</li><li>Liu, D., Reeves, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+K12+regains+its+O+antigen.">Escherichia coli K12 regains its O antigen.</a> Microbiology. 140 (1), 49-57 (1994).</li><li>Kalynych, S., Morona, R., Cygler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+understanding+the+assembly+process+of+bacterial+O-antigen.">Progress in understanding the assembly process of bacterial O-antigen.</a> FEMS Clinical Microbiology Reviews. 38, 1048-1065 (2014).</li><li>Osborn, M. J., Gander, J. E., Parisi, E., Carson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+assembly+of+the+outer+membrane+of+Salmonella+typhimurium.+Isolation+and+characterization+of+cytoplasmic+and+outer+membrane.">Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane.</a> Journal of Biological Chemistry. 247, 3962-3972 (1972).</li><li>Osborn, M. J., Munson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+the+inner+(cytoplasmic)+and+outer+membranes+of+Gram-negative+bacteria.">Separation of the inner (cytoplasmic) and outer membranes of Gram-negative bacteria.</a> Methods in Enzymology. 31, 642-653 (1974).</li><li>Cian, M. B., Giordano, N. P., Masilamani, R., Minor, K. E., Dalebroux, Z. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+enterica+serovar+Typhimurium+use+PbgA/YejM+to+regulate+lipopolysaccharide+assembly+during+bacteremia.">Salmonella enterica serovar Typhimurium use PbgA/YejM to regulate lipopolysaccharide assembly during bacteremia.</a> Infection and Immunity. , (2019).</li><li>Masilamani, R., Cian, M. B., Dalebroux, Z. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+Tol-Pal+Reduces+Outer+Membrane+Glycerophospholipid+Levels+for+Envelope+Homeostasis+and+Survival+during+Bacteremia.">Salmonella Tol-Pal Reduces Outer Membrane Glycerophospholipid Levels for Envelope Homeostasis and Survival during Bacteremia.</a> Infection and Immunity. 86, (2018).</li><li>Nikaido, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outer+Membrane+of+Salmonella-Typhimurium+Transmembrane+Diffusion+of+Some+Hydrophobic+Substances.">Outer Membrane of Salmonella-Typhimurium Transmembrane Diffusion of Some Hydrophobic Substances.</a> Biochimica Et Biophysica Acta. 433, 118-132 (1976).</li><li>Dalebroux, Z. D., Matamouros, S., Whittington, D., Bishop, R. E., Miller, S. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+analysis+of+the+outer+membrane+and+other+layers+of+the+cell+envelope+of+Acinetobacter+sp.">Chemical analysis of the outer membrane and other layers of the cell envelope of Acinetobacter sp.</a> Journal of Bacteriology. 116, 410-417 (1973).</li><li>Geisinger, E., Huo, W., Hernandez-Bird, J., Isberg, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acinetobacter+baumannii:+Envelope+Determinants+That+Control+Drug+Resistance,+Virulence,+and+Surface+Variability.">Acinetobacter baumannii: Envelope Determinants That Control Drug Resistance, Virulence, and Surface Variability.</a> Annual Review of Microbiology. 73, 481-506 (2019).</li><li>Kamischke, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Acinetobacter+baumannii+Mla+system+and+glycerophospholipid+transport+to+the+outer+membrane.">The Acinetobacter baumannii Mla system and glycerophospholipid transport to the outer membrane.</a> Elife. 8, (2019).</li></ol>

This method will continue to aid researchers in understanding the role of the cell envelope in bacterial physiology and pathogenesis. Following the sequential ultracentrifugation steps a purified total, inner, and OM fraction can be obtained. These membranes can be assayed in isolation to test hypotheses related to membrane protein localization and function, transport and trafficking across the periplasm, and the composition of the individual bilayers under various environmental conditions. Biological studies exploring t...

Gram-negative bacteria produce two membranes that are separated by a periplasmic space and a peptidoglycan cell wall1. The inner membrane (IM) encases the cytosol and is a symmetric bilayer of phospholipids. Peptidoglycan protects against turgor pressure and provides the bacterium with a cell shape, and is attached to the outer membrane (OM) by lipoproteins2,3. The OM surrounds the periplasm and is predominantly asymmetric. The inner leaflet consists of phospholipids and the outer leaflet consists of glycolipids known as lipooligosaccharides (LOS) or lipopolysaccharides (LPS)4,5. The lipid asymmetry and the biochemistry of the LOS/LPS molecules in the outer leaflet confer barrier properties to the cell surface that protect the bacterium against hazards in its environment6,7.
LPS molecules are comprised of three constituents: the lipid A disaccharolipid, the core oligosaccharide, and the O-polysaccharide or O-antigen. Lipid A is a multiply acylated disaccharolipid. Core-oligosaccharides consist of 10&#8211;15 sugars known as rough LPS or R-LPS. The core is subdivided into the inner region, composed of 2-keto-3-deoxy-D-manno-octulosonic acid (kdo) and one or more heptose residues, and an outer region that consists generally of hexoses (glucose or galactose) and heptoses, or acetamido sugars5. The outer core region is more variable in its components and structure than the inner core. In Salmonella spp., only one core structure has been described; however, in Escherichia coli there are five different core structures (designated K-12, R1, R2, R3, and R4)8. E. coli K-12 DH5&#945;, which we use in this procedure carries a mutation that results in production R-LPS9. The R-LPS molecules lack the O-antigen moiety and have a similar molecular weight to LOS molecules.
The addition of O-antigen to R-LPS turns this molecule into smooth LPS, or S-LPS. The O-antigens are built from short 3-4 carbohydrate subunits and consist of multiple modalities with varying chain lengths10. Some LPS-producing bacteria, like Salmonella enterica serovar Typhimurium (S. Typhimurium), display a trimodal distribution of LPS molecules on their surface10,11. Very-long chain O-antigens can contain over one hundred subunits and weigh over one hundred kilodaltons. The O-antigens provide surface properties to the bacterium that are necessary to resist antibiotics, evade predation by bacteriophages, and cause disease.
Species of Campylobacter, Bordetella, Acinetobacter, Haemophilus, Neisseria and others generate LOS molecules instead of LPS molecules on their surface12. LOS molecules consist of lipid A and core oligosaccharides but lack the O-antigen. These types of Gram-negative bacteria modify their core oligosaccharides with additional sugars and combinations of sugars to alter surface properties12. Both LOS and LPS-producing microbes derivatize the phosphates on lipid A and core molecules with cationic moieties7. These additions include phosphoethanolamine, galactosamine and aminoarabinose substitutions, which function by neutralizing anionic surface charge and thereby protecting against cationic antimicrobial peptides. Gram-negative bacteria also modify the core oligosaccharide structure with variable non-stoichiometric substitutions of sugars, or extra kdo molecules, and alter the number of acyl chains on lipid-A disaccharolipids7.
The ability to isolate the IM from the OM of Gram-negative bacteria has been instrumental for understanding the role of the cell envelope in antimicrobial resistance and disease pathogenesis11,12. Derivations of this approach have been used to deduce mechanisms of assembly, maintenance, and remodeling of the protein, phospholipid, and glycolipid constituents for the OM.
Our lab routinely performs bacterial lipidomic analyses to study protein-mediated lipid regulation and lipid function in a variety of Gram-negative species. The volumes used in the protocol reflect the routine use of this procedure to analyze non-radiolabeled phospholipids by thin layer chromatography and liquid chromatography tandem mass spectrometry13,14.
The protocol begins by exposing a chilled suspension of Gram-negative bacteria to a high osmolar solution of sucrose and adding lysozyme to dissociate the OM from the underlying peptidoglycan layer (Figure 1)12. EDTA is then added to facilitate penetration of the lysozyme, since divalent cation sequestration disrupts the lateral electrostatic bridging interactions between adjacent LOS/LPS molecules15. The original protocol from which ours has been adapted required the formation of spheroplasts, a Gram-negative bacterial cell form that consist of a plasma membrane and cytosol, but lacks the peptidoglycan layer and an OM. It is possible that spheroplasts are produced by the adapted method; however, the technique does not rely or intend on their formation for success. Instead, the lysozyme-EDTA treated bacteria are rapidly harvested by centrifugation and re-suspended in a sucrose solution of lesser concentration before pressurized lysis. The OMs that might have been released by forming spheroplasts should in theory be harvestable from the supernatants of the treated cells, but this approach is not detailed herein. Ultimately, the treated cells are subjected to conventional homogenization and lysis, which enhances the efficiency and reproducibility of the membrane separation procedure16.
After lysis, the total membranes are collected by ultracentrifugation and applied to a discontinuous sucrose density gradient to fractionate the IMs and OMs. The classical approach uses a more continuous gradient that consists of at least five different sucrose solutions11,12. The discontinuous gradient in our protocol consists of three sucrose solutions and partitions the bilayers into two distinct fractions17. The LOS and LPS molecules within the OMs of Gram-negative bacteria drive the envelope to partition into an upper brown low-density IM fraction and a lower white high-density OM fraction (Figure 1 and Figure 2).
Acinetobacter baumannii are important multidrug resistant human pathogens that produce LOS molecules in their OM and erect a cell envelope that is difficult to separate18,19. Recent work suggests that a derivation of the protocol we present here can be used to partition the bilayers of these organisms20. Therefore, we tested our protocol on A. baumannii 17978. Initially, the procedure was inadequate. However, we modified the sucrose concentration of the middle density solution and greatly improved separation (Figure 2). An NADH dehydrogenase assay and a LOS/LPS extraction and detection procedure was used to confirm separation for A. baumannii, wild-type S. Typhimurium and two O-antigen deficient enterobacterial genotypes; namely, galE-mutant S. Typhimurium and a laboratory strain, E. coli DH5&#945; (Figure 3 and Figure 4).
The intent of this work is to supply a streamlined approach for reproducibly isolating the membranes of Gram-negative bacteria. The protocol can be used to study many types of membrane-associated molecules for these microbes.

<table>
	<tbody>
		<tr>
			<td>1 L Centrifuge bottles, PC/PPCO, super speed, with sealing cap, Nalgene</td>
			<td>VWR</td>
			<td>525-0466</td>
			<td></td>
		</tr>
		<tr>
			<td>1 L Pyrex Media Storage Bottle with High Temperature Cap</td>
			<td>VWR</td>
			<td>10416-312</td>
			<td></td>
		</tr>
		<tr>
			<td>2000 mL Erlenmeyer Flask, Narrow Mouth</td>
			<td>VWR</td>
			<td>10545-844</td>
			<td></td>
		</tr>
		<tr>
			<td>4-20% mini PROTEAN Precast Protein Gels, 12 well</td>
			<td>BIORAD</td>
			<td>4561095</td>
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		<tr>
			<td>4x Laemmli Sample Buffer 10 mL</td>
			<td>BIORAD</td>
			<td>1610747</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL sterile polypropylene centrifuge tubes</td>
			<td>VWR</td>
			<td>89049-174</td>
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			<td>7 mL Dounce Tissue Homogenizer with Two Glass Pestles</td>
			<td>VWR</td>
			<td>71000-518</td>
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			<td>70 mL polycarbonate bottle assembly</td>
			<td>Beckman Coulter Life Sciences</td>
			<td>355622</td>
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			<td>VWR</td>
			<td>A10752</td>
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			<td>B10P Benchtop pH Meter with pH Probe</td>
			<td>VWR</td>
			<td>89231-664</td>
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			<td>Barnstead GenPure xCAD Plus UV/UF - TOC (bench version)</td>
			<td>ThermoFisher Scientific</td>
			<td>50136146</td>
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			<td>Benzonase Nuclease</td>
			<td>MilliporeSigma</td>
			<td>70746-3</td>
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		<tr>
			<td>EDTA disodium salt dihydrate 99.0-101.0%, crystals, ultrapure Bioreagent Molecular biology grade, J.T. Baker</td>
			<td>VWR</td>
			<td>4040-01</td>
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			<td>EmulsiFlex-C3</td>
			<td>Avestin, Inc.</td>
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			<td>Fiberlite F13-14 x 50cy Fixed Angle Rotor</td>
			<td>ThermoFisher Scientific</td>
			<td>75006526</td>
			<td></td>
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			<td>Hydrochloric acid 6.0 N</td>
			<td>VWR</td>
			<td>BDH7204</td>
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			<td>IBI Scientific Orbital Platform Shaker</td>
			<td>Fischer Scientific</td>
			<td>15-453-211</td>
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			<td>LB Broth Miller</td>
			<td>VWR</td>
			<td>214906</td>
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			<td>Lysozyme, Egg White, Ultra Pure Grade</td>
			<td>VWR</td>
			<td>VWRV0063</td>
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			<td>Magnesium chloride hexahydrate</td>
			<td>Sigma Aldrich</td>
			<td>M2670</td>
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			<td>NADH</td>
			<td>Sigma Aldrich</td>
			<td>10107735001</td>
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			<td>Optima XPN-80 - IVD</td>
			<td>Beckman Coulter Life Sciences</td>
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			<td>Phenol Solution, Equilibrated with 10 mM Tris HCl, pH 8.0, 1 mM EDTA, BioReagent, for molecular biology</td>
			<td>Sigma - Millipore</td>
			<td>P4557</td>
			<td></td>
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		<tr>
			<td>Pierce Coomassie Plus (Bradford) Assay Kit</td>
			<td>Thermofisher</td>
			<td>23238</td>
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			<td>Pro-Q emerald 300 Lipopolysaccharide Gel Stain Kit</td>
			<td>Thermofisher</td>
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			<td>Proteacease -50, EDTA free</td>
			<td>G Biosciences</td>
			<td>786-334</td>
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			<td>Proteinase K, Molecular Biology Grade</td>
			<td>New England Biolabs</td>
			<td>P8107S</td>
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			<td>Sodium hydroxide &#8805;99.99%</td>
			<td>VWR</td>
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			<td>Sorval RC 6 Plus Centrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>36-101-0816</td>
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		<tr>
			<td>Sucrose</td>
			<td>MilliporeSigma</td>
			<td>SX1075-3</td>
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			<td>SW 41 Ti Swinging-Bucket Rotor</td>
			<td>Beckman Coulter Life Sciences</td>
			<td>331362</td>
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			<td>Tris(hydroxymethyl)aminomethane (TRIS, Trometamol) &#8805;99.9% (dried basis), ultrapure Bioreagent Molecular biology grade, J.T. Baker</td>
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			<td>JT4109-6</td>
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			<td>Type 45 Ti Fixed-Angle Titanium Rotor</td>
			<td>Beckman Coulter Life Sciences</td>
			<td>339160</td>
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			<td>Ultra-Clear Tube ,14 x 89mm</td>
			<td>Beckman Coulter Life Sciences</td>
			<td>344059</td>
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			<td>Vortex-Genie 2</td>
			<td>VWR</td>
			<td>102091-234</td>
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	</tbody>
</table>

separation of the cell envelope for gram-negative bacteria into inner and outer membrane fractions with technical adjustments for acinetobacter baumannii

This method works by partitioning the envelope of Gram-negative bacteria into total, inner, and outer membrane (OM) fractions and concludes with assays to assess the purity of the bilayers. The OM has an increased overall density compared to the inner membrane, largely due to the presence of lipooligosaccharides (LOS) and lipopolysaccharides (LPS) within the outer leaflet. LOS and LPS molecules are amphipathic glycolipids that have a similar structure, which consists of a lipid-A disaccharolipid and core-oligosaccharide substituent. However, only LPS molecules are decorated with a third subunit known as the O-polysaccharide, or O-antigen. The type and amount of glycolipids present will impact an organism&#8217;s OM density. Therefore, we tested whether the membranes of bacteria with varied glycolipid content could be similarly isolated using our technique. For the LPS-producing organisms, Salmonella enterica serovar Typhimurium and Escherichia coli, the membranes were easily isolated and the LPS O-antigen moiety did not impact bilayer partitioning. Acinetobacter baumannii produces LOS molecules, which have a similar mass to O-antigen deficient LPS molecules; however, the membranes of these microbes could not initially be separated. We reasoned that the OM of A. baumannii was less dense than that of Enterobacteriaceae, so the sucrose gradient was adjusted and the membranes were isolated. The technique can therefore be adapted and modified for use with other organisms.

This technique provides an effective means to isolate the IMs and OMs for Gram-negative bacteria. An outline of the entire procedure is illustrated (Figure 1). In general, the normalization of cultures to an OD600 of 0.6-0.8 in 1 L of media, or harvesting between 6.0 and 8.0 x 1011 bacterial cells will ensure that the appropriate amount of membrane material is collected for subsequent separation.
Upon lysing the bacteria and ultracentrifuging...

Watch this Scientific Journal Video about Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii at J

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii

Gram-negative bacteria produce two spatially segregated membranes. The outer membrane is partitioned from the inner membrane by a periplasm and a peptidoglycan layer. The ability to isolate the dual bilayers of these microbes has been critical for understanding their physiology and pathogenesis.

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii

This method works by partitioning the envelope of Gram-negative bacteria into total, inner, and outer membrane (OM) fractions and concludes with assays to ...

Studying how microbes regulate the chemical composition of the individual bilayers will inform our knowledge of mechanisms of antibiotic killing, antimicrobial resistance and disease pathogenesis. This technique partitions the cell envelope of gram-negative bacteria into two defined fractions without using detergent. Therefore, the lipids, proteins and sugars can be assessed in an environment that is semi-native.Some of the steps are time dependent and require focus and coordination. Initially, use one or two samples. When one's comfort level increases, more samples can be added.No more than six samples should be handled at once. This is a multi-day procedure and visual checkpoints are helpful to assess efficacy and error. Prior to performing this sometimes final steps.Streak the bacteria from frozen glycerol stocks onto fresh agar plates. Once colonies develop, store the plates at four degrees Celsius. Use a single colony to inoculate a five milliliter tube filled with broth media and culture the bacteria as desired overnight.Back dilute the overnight bacterial culture into one liter of the preferred broth media and incubate the flasks. When the desired optical density has been achieved, remove the flasks from the incubator and place them on ice. Measure the optical density of the culture at 600 nanometers and calculate the volume of culture that is equivalent to between six and eight times 10 to the eleventh bacterial colony forming units.Add this volume of culture to a centrifuge tube and ensure that the remaining cultures stay on ice until they are to be used. Pellet the bacteria by centrifugation for 10 minutes ina a fixed angle, high speed centrifuge at 7, 000 to 10, 000 times G and four degrees Celsius. Carefully decant the supernatant and discard it.Resuspend each cell pellet in 12.5 milliliters of buffer A.Add a magnetic stir bar to the cell suspension. Add 180 microliters of 10 milligrams per milliliter lysozyme to each cell resuspension. After collecting the lysozyme EDTA treated bacteria by centrifugation, quickly add the protease inhibitor, magnesium chloride and nuclease to the bacteria and rapidly resuspend the cells in buffer B.Stir for two minutes, keeping the suspensions on ice.Next, add 12.5 milliliters of 1.5 millimolar EDTA solution to each cell re-suspension and continue stirring on ice for an additional seven minutes. Decant the suspensions into 15 milliliter conical tubes. Centrifuge the suspensions at 9, 000 to 11, 000 times G for 10 minutes at four degrees Celsius.Discard the supernatants into a biohazard waste container and retain the pellets on ice. Add 25 milliliters of buffer B to each cell pellet. Then, add 55 microliters of one molar magnesium chloride, one microliter of rnase/dnase/nuclease reagent and one microliter of protease inhibitor cocktail.Resuspend the pellets in the mixture. Vigorously pipette and vortex until the solutions appear homogenous. Store the suspensions on ice.Pour the sample into the homogenizer sample cylinder and bring the cell to 20, 000 PSI. To avoid aerosolization of pathogens, condition the pressurized apparatus in buffer B and adjust the pressure levels before adding bacterial suspension. Engage the pressure cell and allude 10 milliliters of buffer B as a precaution.Collect the cell lysate in 50 milliliter conical tubes on ice while also keeping the samples on ice. To achieve complete lysis, repeat this process three to five times. To pellet the remaining, intact, cell material, centrifuge the lysed bacterial samples at 6, 169 times G and four degrees Celsius for 10 minutes.Distribute the supernatants which now contain the homogenized membranes, into polycarbonate bottles for ultracentrifugation. Ultracentrifuge the cell lysates at 184, 500 times G and four degrees Celsius for at least one hour. Discard the supernatants and retain the membrane pellets on ice.Using a glass, Dounce homogenizer, resuspend the membrane pellets in one milliliter of the low-density isopycnic sucrose gradient solution. Then, use a glass, Pasteur pipette to transfer the sample homogenate to a 1.5 milliliter microcentrifuge tube and place the tube on ice. To prepare the sucrose gradient, hold a polypropylene or Ultra-clear Open-Top tube in a slightly tilted position.Slowly add two milliliters of the higher density sucrose solution. Then add four milliliters of the lower density sucrose solution. When preparing the density gradient, add the sucrose solution slowly to avoid mixing the layers.The sucrose layers should be slightly visible and appear generally defined. Next, add the total membrane fraction which was previously resuspended in one milliliter of low density, isopycnic sucrose solution. Finally, fill the remainder of the tube by adding approximately six milliliters of the low density, isopycnic sucrose gradient solution.Ultracentrifuge the samples using a swinging bucket rotor at 288, 000 times G and four degrees Celsius for 16 to 23 hours. Cut the end off a P1000 pipette tip about five milliliters from the point. After centrifugation is complete, use the pipette to remove the upper, brown layer from the tube.Transfer this layer, which contains the inner membrane fraction, to a polycarbonate bottle for ultracentrifugation. Leave about two milliliters of the sucrose solution above the interface between the 53%sucrose and the 73%sucrose to ensure that the lower, white, outer membrane fraction does not cross contaminate the inner membrane fraction. Again using a P1000 pipette tip with the end removed, remove the outer membrane layer and transfer it to a polycarbonate bottle.Fill the remaining void of polycarbonate bottle with isolated membrane storage buffer and mix by inversion or pippetting. Retain the samples on ice. To collect the membranes, ultracentrifuge the polycarbonate bottles at 184, 500 times G and four degrees Celsius for one hour.Finally, discard the supernatants and add 500 to 1, 000 microliters of storage buffer. Resuspend the membranes by Dounce homogenization. Collect the samples in two milliliter microcentrifuge tubes.Total membrane pellets were scraped, Dounce homogenized and ultracentrifuged over sucrose density gradients to separate the inner and outer membranes. A sucrose density gradient of 20%53%73%was sufficient for partitioning some bacterial strains. But not for partitioning A.baumannii which was successfully partitioned using a 20%45%73%gradient.To assess the efficiency of the separation, membrane samples were tested for NADH dehydrogenase, an enzyme located in the bacterial inner membrane. A decrease in optical density at 340 nanometers indicated the presence of the enzyme. Optical density was measured for 50 microgram samples of inner and outer membrane fractions from wild type S.typhimurium, E.coli K12 DH5a and galE mutant S.typhimurium.A higher concentration of protein was added when assaying the purity of A.baumannii membrane fractions because an initial assay using 50 micrograms suggested that the relative levels of NADH dehydrogenase were lower for A.baumannii than for the other bacterial strains. To assess cross contamination of the inner membrane fractions with outer membrane fractions, LPS and LOS were extracted and visualized. The extracts were loaded onto a polyacrylamide gel and stained with Pro-Q Emerald 300.Acinetobacter baumannii is a top priority, multi drug resistant, human pathogen. This protocol should aid researchers in understanding the roles of lipooligosaccharides, phospholipids and lipoproteins in the resistance and virulence mechanisms of this unique and important pathogen. This method is ideal for downstream analysis of phospholipids, glycolipids carbohydrates, proteins and small molecules that typically exist within the dual membranes of gram-negative bacteria.

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Research

JoVE Journal

Immunology and Infection

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) <a href="http://www.usaid.gov">(www.usaid.gov)</a>. The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.
The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at &tilde;20 &Aring; resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH <a href="http://biowulf.nih.gov">(http://biowulf.nih.gov)</a>. This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the causative agent of Legionnaires' pneumonia, gains a pathogenic advantage over in vitro cultured bacteria when first harvested from protozoan cells prior to infection of mammalian macrophages. This suggests that important virulence factors may not be properly expressed in vitro. We have developed a tractable system for priming L. pneumophila through its natural protozoan host Acanthamoeba castellanii prior to mammalian cell infection. The contribution of any virulence factor can be examined by comparing intracellular growth of a mutant strain to wild-type bacteria after protozoan priming. GFP-expressing wild-type and mutant L. pneumophila strains are used to infect protozoan monolayers in a priming step and allowed to reach late stages of intracellular growth. Fluorescent bacteria are then harvested from these infected cells and normalized by spectrophotometry to generate comparable numbers of bacteria for a subsequent infection into mammalian macrophages. For quantification, live bacteria are monitored after infection using fluorescence microscopy, flow cytometry, and by colony plating. This technique highlights and relies on the contribution of host cell-dependent gene expression by mimicking the environment that would be encountered in a natural acquisition route. This approach can be modified to accommodate any bacterium that uses an intermediary host as a means for gaining a pathogenic advantage.

This technique provides a method to harvest, normalize and quantify intracellular growth of bacterial pathogens that are pre-cultivated in natural protozoan host cells prior to infections of mammalian cells. This method can be modified to accommodate a wide variety of host cells for the priming stage as well as target cell types.

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to the frequent failure of traditional antibiotic treatments. Alternative therapeutics are urgently needed to tackle MDR bacteria.
An innovative non-antibiotic approach, antimicrobial blue light (aBL), has shown promising effectiveness against MDR infections. The mechanism of action of aBL is not yet well understood. It is commonly hypothesized that naturally occurring endogenous photosensitizing chromophores in bacteria (e.g., iron-free porphyrins, flavins, etc.) are excited by aBL, which in turn produces cytotoxic reactive oxygen species (ROS) through a photochemical process.
Unlike another light-based antimicrobial approach, antimicrobial photodynamic therapy (aPDT), aBL therapy does not require the involvement of an exogenous photosensitizer. All it needs to take effect is the irradiation of blue light; therefore, it is simple and inexpensive. The aBL receptors are the endogenous cellular photosensitizers in bacteria, rather than the DNA. Thus, aBL is believed to be much less genotoxic to host cells than ultraviolet-C (UVC) irradiation, which directly causes DNA damage in host cells.
In this paper, we present a protocol to assess the effectiveness of aBL therapy for MDR Acinetobacter baumannii infections in a mouse model of burn injury. By using an engineered bioluminescent strain, we were able to noninvasively monitor the extent of infection in real time in living animals. This technique is also an effective tool for monitoring the spatial distribution of infections in animals.

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of massage on skeletal muscles, the molecular mechanisms behind are poorly understood. We have recently developed a simple device to apply local cyclical compression (LCC), which can generate intramuscular pressure waves with varying amplitudes. Using this device, we have demonstrated that LCC modulates inflammatory responses of macrophages in situ and alleviates immobilization-induced muscle atrophy. Here, we describe protocols for the optimization and application of LCC as a massage-like intervention against immobilization-induced inflammation and atrophy of skeletal muscles of mouse hindlimbs. The protocol that we have developed can be useful for investigating the mechanism underlying beneficial effects of physical exercise and massage. Our experimental system provides a prototype of the analytical approach to elucidate the mechanical regulation of muscle homeostasis, although further development needs to be made for more comprehensive studies.

Here we describe the protocols for applying defined mechanical loads to mouse calves and for monitoring the concomitant intramuscular pressure changes. The experimental systems that we have developed can be useful for investigating the mechanism behind the beneficial effects of physical exercise and massage.

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...

Separation of Rat Epidermis and Dermis with Thermolysin to Detect Site-Specific Inflammatory mRNA and Protein

Easy-to-use and inexpensive techniques are needed to determine the site-specific production of inflammatory mediators and neurotrophins during skin injury, inflammation, and/or sensitization. The goal of this study is to describe an epidermal-dermal separation protocol using thermolysin, a proteinase that is active at 4 &deg;C. To illustrate this procedure, Sprague Dawley rats are anesthetized, and right hind paws are injected with carrageenan. Six and twelve hours after injection, rats with inflammation and na&iuml;ve rats are euthanized, and a piece of hind paw, glabrous skin is placed in cold Dulbecco's Modified Eagle Medium. The epidermis is then separated at the basement membrane from the dermis by thermolysin in PBS with calcium chloride. Next, the dermis is secured by microdissection forceps, and the epidermis is gently teased away. Toluidine blue staining of tissue sections show that the epidermis is separated cleanly from the dermis at the basement membrane. All keratinocyte cell layers remain intact, and the epidermal rete ridges along with indentations from dermal papillae are clearly observed. Qualitative and real-time RT-PCR is used to determine nerve growth factor and interleukin-6 expression levels. Western blotting and immunohistochemistry are finally performed to detect amounts of nerve growth factor. This report illustrates that cold thermolysin digestion is an effective method to separate epidermis from dermis for evaluation of mRNA and protein alterations during inflammation.

Presented here is a protocol for the separation of epidermis from dermis to evaluate inflammatory mediator production. Following inflammation, rat hind paw epidermis is separated from the dermis by thermolysin at 4 &#176;C. The epidermis is then used for mRNA analysis by RT-PCR and protein evaluation by western blot and immunohistochemistry.

Easy-to-use and inexpensive techniques are needed to determine the site-specific production of inflammatory mediators and neurotrophins during skin ...

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a protocol for studying the pathogenesis of E. coli O1:K1:H7, a serotype responsible for high mortality rates in neonates. Our method utilizes intravital imaging of neonatal pups at different time points during the progression of infection. This imaging, paralleled by measurement of bacteria in the blood, inflammatory profiling, and tissue histopathology, signifies a rigorous approach to understanding infection dynamics during sepsis. In the current report, we model two infectious inoculums for comparison of bacterial burdens and severity of disease. We find that subscapular infection leads to disseminated infection by 10 h post-infection. By 24 h, infection of luminescent E. coli was abundant in the blood, lungs, and other peripheral tissues. Expression of inflammatory cytokines in the lungs is significant at 24 h, and this is followed by cellular infiltration and evidence of tissue damage that increases with infectious dose. Intravital imaging does have some limitations. This includes a luminescent signal threshold and some complications that can arise with neonates during anesthesia. Despite some limitations, we find that our infection model offers an insight for understanding longitudinal infection dynamics during neonatal murine sepsis, that has not been thoroughly examined to date. We expect this model can also be adapted to study other critical bacterial infections during early life.

Infection of neonatal mice with bioluminescent E. coli O1:K1:H7 results in a septic infection with significant pulmonary inflammation and lung pathology. Here, we describe procedures to model and further study neonatal sepsis using longitudinal intravital imaging in parallel with enumeration of systemic bacterial burdens, inflammatory profiling, and lung histopathology.

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a ...