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 ethylene tetraacetate&#183;2H2O to 80 mL of H2O. Stir vigorously and adjust pH to 8.0 with NaOH. Adjust final volume to 100 mL with ultrapure H2O. 
	NOTE: The disodium salt of EDTA will not dissolve until the pH of the solution is adjusted to ~8.0 by the addition of NaOH.</li>
	<li>Osmotic buffer A (0.5 M sucrose, 10 mM Tris pH 7.5; 1 L): Weigh 171.15 g of sucrose and transfer to a 1 L cylinder. Add 10 mL of 1 M Tris pH 7.5. Adjust to a final volume of 1 L with ultrapure H2O. Store at 4 &#176;C.</li>
	<li>Lysozyme (10 mg/mL; 5 mL): Weigh 50 mg of (chicken egg-white) lysozyme and dissolve in 5 mL ultrapure H2O. Store at 4 &#176;C.</li>
	<li>Diluted divalent cation chelation solution (1.5 mM EDTA; 500 mL): Add 1.5 mL of 0.5 M EDTA (Step 1.3) to 497.5 mL ultrapure H2O. Store at 4 &#176;C.</li>
	<li>Osmotic buffer B (0.2 M sucrose, 10 mM Tris pH 7.5; 2 L): Weigh 136.8 g of sucrose and transfer to a 2 L cylinder. Add 20 mL of 1 M Tris pH 7.5. Adjust final volume to 2 L with ultrapure H2O. Store at 4 &#176;C.</li>
	<li>Nuclease co-factor (1 M MgCl2; 10 mL): Dissolve 2.03 g of MgCl2&#183;6H2O in 8 mL of ultrapure H2O. Adjust volume to 10 mL. Store at room temperature.</li>
	<li>Nuclease solution cocktail including RNase and DNase enzymes: See Table of Materials. Store at -20 &#176;C.</li>
	<li>Protease inhibitor cocktail: See Table of Materials. Store at 4 &#176;C.</li>
	<li>Low-density isopycnic sucrose gradient solution (20% w/v sucrose, 1 mM EDTA, 1 mM Tris pH 7.5 Solution; 100 mL): Weigh 20 g of sucrose and transfer to a 200 mL cylinder. Add 100 &#181;L of 1 M Tris Buffer pH 7.5 and 200 &#181;L of 0.5 M EDTA pH 8. Adjust final volume to 100 mL with ultrapure H2O. Store at room temperature.</li>
	<li>Medium-density isopycnic sucrose gradient solution (53% w/v sucrose, 1 mM EDTA, 1mM Tris pH 7.5 Solution; 100 mL): Weigh 53 g of sucrose and transfer to a 200 mL graduated cylinder. Add 100 &#181;L of 1 M Tris Buffer pH 7.5 and 200 &#181;L of 0.5 M EDTA pH 8. Adjust final volume to 100 mL with ultrapure H2O. Store at room temperature. 
	NOTE: Prepare this solution in a graduated cylinder to ensure accuracy due to the high percentage of sucrose. Add a magnetic stir bar and stir until the sucrose is completely dissolved in solution. This process may take several hours.</li>
	<li>High-density isopycnic sucrose gradient solution (73% w/v sucrose, 1 mM EDTA, 1 mM Tris pH 7.5 Solution; 100 mL): Weigh 73 g of sucrose and transfer to a 200 mL graduated cylinder. Add 100 &#181;L of 1 M Tris Buffer pH 7.5 and 200 &#181;L of 0.5 M EDTA pH 8. Adjust final volume to 100 mL with ultrapure H2O. Store at room temperature. 
	NOTE: Prepare this solution in a graduated cylinder to ensure accuracy due to the high percentage of sucrose. Add a magnetic stir bar and stir until the sucrose is completely dissolved. This process may take several hours.</li>
	<li>Isolated-membrane-storage buffer (10 mM Tris Buffer pH 7.5; 1 L): Add 1 mL of 1 M Tris Buffer pH 7.5 to a 1 L flask and adjust the final volume to 1 L with ultrapure H2O.</li>
	<li>&#946;-Nicotinamide adenine dinucleotide (NADH) (10 mg/mL solution): Resuspend 10 mg of NADH in ultrapure H2O. Prepare fresh stocks, weekly. Store at -20 &#176;C.</li>
	<li>Phenol solution equilibrated with 10 mM Tris HCl, pH 8.0, 1 mM EDTA: See Table of Materials. Store the solution at 4 &#176;C.</li>
	<li>Lipopolysaccharide gel stain kit: See Table of Materials.</li>
	<li>Bradford reagent: See Table of Materials. 
	NOTE: All solutions and media should be prepared within the week of performing the assay to ensure consistent results.</li>
</ol>
2. Preparation of Bacteria for Membrane Extraction
<ol>
	<li>Streak the bacteria from frozen glycerol stocks onto fresh agar plates. Store the plates at 4 &#176;C once colonies develop. Inoculate a single colony into a 5 mL tube filled with broth media and culture the bacteria as desired overnight.</li>
	<li>Back-dilute the overnight bacterial culture into 1 L of preferred broth media and culture the bacteria until the desired optical density is achieved. 
	NOTE: Inoculating a single bacterial colony into 1 L of broth media is recommended for mutant genotypes that are prone to suppressing growth phenotypes, but some Gram-negative bacteria simply grow slower than others. If it is not possible to achieve a sufficient culture density by single-colony inoculation, back diluting an overnight culture into 1 L of media is one strategy to synchronize growth. Bacterial-membrane composition varies depending upon the growth phase of the culture (logarithmic vs stationary phase)13. Growth curves measuring the change in optical density for the bacterial cultures as a function of time should be performed with all strains to correlate culture density with growth phase.</li>
	<li>Set the flasks containing the broth cultures on ice. Read the optical density at 600 nm (OD600) and calculate the volume of culture that is equivalent to between 6.0 and 8.0 x 1011 bacterial colony forming units (cfu). For S. Typhimurium, this corresponds to 1 L of culture at an OD600 of between 0.6-0.8, since an OD600 of 1.0 is equal to roughly 1.0x109 cfu/mL. Add this volume to a centrifuge tube and ensure that the remaining cultures stay on ice until they are to be used.</li>
	<li>Pellet the bacteria by centrifugation at 4 &#176;C at 7,000-10,000 x g in a fixed angle high-speed centrifuge for 10 min. 
	NOTE: Pre-cool and maintain the centrifuges at a low temperature. Maintain the samples on ice during the entire procedure.</li>
	<li>Decant and discard the supernatant carefully. 
	NOTE: The pellet can be flash frozen and/or stored at -80 &#176;C if the membrane fractions are not going to be extracted immediately. However, it is recommended to proceed directly with plasmolysis on the same day the cells are harvested, and is especially recommended for non-enterobacterial species.</li>
</ol>
3. Dissociation of the Outer Membrane and Plasmolysis
<ol>
	<li>Thaw the cell pellets on ice if previously stored at -80 &#176;C and retain the samples on ice for the remainder of the procedure. Resuspend each cell pellet within the centrifuge tube in 12.5 mL of buffer A. Add a magnetic stir bar to the suspension of cells.</li>
	<li>Add 180 &#181;L of 10 mg/mL lysozyme (final concentration of 144 &#956;g/mL) to each cell resuspension. Keep the samples on ice while stirring for 2 min.</li>
	<li>Add 12.5 mL of 1.5 mM EDTA solution to each cell resuspension and continue stirring on ice for an additional 7 min.</li>
	<li>Decant the suspension into a 50 mL conical tube and centrifuge at 9,000-11,000 x g for 10 min at 4 &#176;C.</li>
	<li>Discard supernatants into a biohazard waste container and retain the pellets on ice.</li>
	<li>Add 25 mL of buffer B to the cell pellet.</li>
	<li>Add 55 &#181;L of 1 M MgCl2, 1 &#181;L of RNase/DNase nuclease reagent (to avoid viscosity problems associated with bacteria undergoing plasmolysis prior to homogenization), and 1 &#181;L of protease inhibitor cocktail to the volume of buffer B that sits atop the cell pellet</li>
	<li>Resuspend the pellet in the buffer B mixture. Vigorously pipet and vortex until observing a homogenous solution. 
	NOTE: It is very important to have a homogenous solution before proceeding to Step 4 of this protocol. The resuspended cells should have a viscous cake-batter like appearance and consistency.</li>
	<li>Vortex each sample for 15 s. Retain suspensions on ice and proceed to Step 4.</li>
</ol>
4. Pressurized Homogenization and Lysis
NOTE: Several methods can be used for lysis. Sonication is not ideal due to the generation of heat. Osmotic lysis can be achieved but is often inefficient. Therefore, we recommend high-pressure lysis. High pressure lysis can be achieved using a variety of instruments. We suggest homogenization machines, such as the French Press or the Emusliflex. We work with many types of Gram-negative bacteria whose response to high osmolar sucrose solutions varies. The high-pressure homogenization step improves efficiency, reproducibility, and yield.
<ol>
	<li>Prechill the French Press cell at 4 &#176;C or insert the metal coil from the homogenizer machine on ice.</li>
	<li>Pour the sample into the French-pressure cell or the homogenizer-sample cylinder and bring the cell under the desired homogenization pressure (10,000 psi should be adequate when using a French Press or 20,000 psi when using a homogenizer).</li>
	<li>Adjust the outlet flow rate to approximately one drop per second while maintaining the pressure if utilizing a French Press.</li>
	<li>Collect the cell lysate in 50 mL conical tubes while keeping samples on ice.</li>
	<li>Repeat steps 4.2-4.4 three to five times to achieve complete lysis, typically indicated by gradual increase in transparency of sample. 
	NOTE: The sample chamber should be washed and equilibrated with Buffer B in between samples.</li>
	<li>Keep lysed cells on ice.</li>
</ol>
5. Total Membrane Fractionation
<ol>
	<li>Centrifuge the lysed bacterial samples at 6,169 x g for 10 min at 4 &#176;C to pellet remaining intact cell material. (e.g., unlysed bacterial cells).</li>
	<li>Distribute the remaining portion of the supernatant, which now contains the homogenized membranes into a polycarbonate bottle for ultracentrifugation. 
	CAUTION: If needed, cell samples can be balanced by diluting with buffer B.</li>
	<li>Ultracentrifuge the cell lysates at 184,500 x g for at least 1 h, at 4 &#176;C. This step can be performed overnight without affecting the quality of the membranes.</li>
	<li>Discard remaining supernatant present in the ultracentrifuge tube and retain membrane pellets on ice (Figure 1).</li>
	<li>Resuspend the membrane pellets in 1 mL of the low-density isopycnic-sucrose gradient solution using a glass-dounce homogenizer. Use a glass Pasteur pipette to transfer the sample homogenate to a 1.5 mL microcentrifuge tube and retain on ice. 
	NOTE: If only total membrane composition analysis is desired, substitute 1 mL of low-density isopycnic-sucrose gradient solution for 1 mL of isolated-membrane storage buffer. Step 5.5 is the endpoint of isolation if only total-bacterial membrane samples are desired. Store samples at -20 &#176;C until further downstream analysis is required.</li>
</ol>
6. Density Gradient Ultracentrifugation to Separate the Dual Membranes
<ol>
	<li>Gather the appropriate number of 13 mL polypropylene or ultra-clear open-top tubes specified for a swinging bucket rotor and ultracentrifuge.</li>
	<li>Hold the tube in a slightly tilted position and prepare the sucrose gradient by slowly adding sucrose solutions from higher density to lower density in the following order: 
	2 mL of 73% w/v sucrose,1 mM EDTA, 1 mM Tris pH 7.5 
	4 mL of 53% w/v sucrose, 1 mM EDTA, 1 mM Tris pH 7.5
	<ol>
		<li>Next, add the total membrane fraction (1 mL), which has been resuspended in the 20% w/v sucrose solution (step 5.5). Avoid mixing the membrane fraction with the sucrose solution that lies beneath it. Divisions should be visible between each of these layers.</li>
		<li>Finally, fill the tube with the low-density isopycnic-sucrose gradient solution (approx. 6 mL). Polypropylene and ultra-clear open-top tubes should be filled as full as possible (2 or 3 mm from the tube top) for tube support.</li>
	</ol>
	</li>
	<li>Adjustment for Acinetobacter baumannii 17978
	<ol>
		<li>Adapt an adjusted sucrose gradient for use with different bacterial specimens. For A. baumannii, the following sucrose gradient afforded more complete separation of the membranes (Figure 2). 
		2 mL of 73% w/v sucrose, 1 mM EDTA, 1 mM Tris pH 7.5 
		4 mL of 45% w/v sucrose, 1 mM EDTA, 1 mM Tris pH 7.5</li>
		<li>Next, add the total membrane fraction (1 mL), which has been resuspended in the 20% w/v sucrose solution (step 5.5). Avoid mixing the membrane fraction with the sucrose solution that lies beneath it. Divisions should be visible between each of these layers.</li>
		<li>Finally, fill the tube with low-density isopycnic-sucrose gradient solution (approx. 6 mL). Polypropylene and ultra-clear open-top tubes should be filled as full as possible (2 or 3 mm from the tube top) for tube support.</li>
	</ol>
	</li>
	<li>Ultracentrifuge the samples using a swinging-bucket rotor at 288,000 x g and 4 &#176;C overnight. 
	NOTE: For the volumes used in the previous steps, we recommend centrifugation times between 16 h and 23 h.</li>
	<li>Cut the end of a P1000 pipette tip about 5 mm from the point. Remove the upper-brown IM layer using the pipette. Transfer the IM fraction into a polycarbonate bottle for ultracentrifugation.</li>
	<li>Leave about 2 mL of the sucrose solution above the 53-73% interface to ensure that the lower white OM is not cross contaminated with the IM fraction. Repeat the pipetting procedure from step 6.4 for the OM fraction (Figure 1). 
	NOTE: The membranes can also be collected by puncturing the centrifuge tubes at the bottom using a needle and collecting the membranes as fractions dropwise.</li>
	<li>Fill the remaining void of the ultracentrifuge tube with isolated-membrane storage buffer and mix by inversion or pipetting. Retain the samples on ice.</li>
	<li>Collect the now washed and isolated membranes by ultracentrifugation at 184,500 x g for 1 h and 4 &#176;C.</li>
	<li>Discard the supernatant and resuspend the membranes by dounce-homogenization. Add 500-1000 &#956;L of storage buffer. Collect samples in 2 mL microcentrifuge tubes.</li>
	<li>Store the bacterial membrane samples at -20 &#176;C.</li>
</ol>
7. Confirming Separation of the Bilayers
NOTE: Incomplete separation of the bilayers can occur due to technical error or the unique cell envelope composition of some species. To confirm separation, we advise using two independent assays to quantify the degree of cross contamination between the bilayers. The first assay detects the enzymatic activity of NADH dehydrogenase, which exists exclusively in the IM. The second assay detects the presence of LOS or LPS, which predominantly exists in the OM.
<ol>
	<li>Confirming that the OMs are isolated from the IMs
	<ol>
		<li>Measure the protein concentration in each isolated membrane fraction using a Bradford protein assay.</li>
		<li>Add the volume of sample corresponding to between 50 and 500 &#181;g of protein to an empty 2 mL microcentrifuge tube. The concentration will vary depending upon the species, but 50 &#181;g is typically sufficient for Enterobacteriaceae. Add the appropriate volume of 10 mM Tris-buffer to achieve a total volume of 990 &#956;L. Transfer the content to a cuvette for spectrophotometry.</li>
		<li>Add 10 &#956;L of a 10 mg/mL solution of NADH to the sample and measure the initial absorbance at 340 nm.</li>
		<li>Continue measuring the absorbance every 30 s for 5 min.</li>
		<li>Compile the data and graph the change in absorbance (y-axis) vs. the change in time (x-axis) for each membrane fraction (Figure 3).</li>
	</ol>
	</li>
	<li>Confirming that the IMs are isolated from the OMs
	<ol>
		<li>Add the volume of sample corresponding to between 50 and 500 &#181;g of protein to an empty 2 mL microcentrifuge tube. The concentration will vary depending upon the bacterial species, but 50 &#181;g is typically sufficient for Enterobacteriaceae. Fill to a final volume of 100 &#956;L with phosphate buffered saline, which is hereafter referred to as the aqueous phase.</li>
		<li>Add 5 &#956;L of Proteinase K (stock 800 U/mL) to the aqueous phase and incubate overnight at 59 &#176;C.</li>
		<li>Warm a 10 mL aliquot of Tris-saturated Phenol for 10 min at 68 &#176;C.</li>
		<li>Spin down the aqueous phase samples that were treated with the Proteinase K and add the &#8220;hot&#8221; Tris-saturated Phenol at a 1:1 ratio with the aqueous phase, vortex vigorously and incubate at 68 &#176;C for 10 min.</li>
		<li>Transfer the now milky-white samples from 68 &#176;C to an ice-water bath and incubate for 10 min.</li>
		<li>Centrifuge the samples at 2,100 x g for 10 min at 4 &#176;C.</li>
		<li>Transfer the upper aqueous phase to a new tube and store the tube at -20 &#176;C if the sample is not to be used immediately.</li>
		<li>Dilute samples in SDS-loading buffer and load the wells of a 4-20% Tris-glycine gradient gel.</li>
		<li>Electrophorese for 45 min or until the dye-front is at the bottom of the gel.</li>
		<li>Stain the gel using the LPS staining kit following manufacturer&#8217;s instructions.</li>
	</ol>
	</li>
</ol>

<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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymology,+genetics,+and+regulation+of+membrane+phospholipid+synthesis+in+Escherichia+coli.">Enzymology, genetics, and regulation of membrane phospholipid synthesis in Escherichia coli.</a> Clinical Microbiology Reviews. 42, 614-659 (1978).</li><li>Whitfield, C., 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=Biosynthesis+and+export+of+bacterial+lipopolysaccharides.">Biosynthesis and export of bacterial lipopolysaccharides.</a> Annual Review of Biochemistry. 83, 99-128 (2014).</li><li>Needham, B. D., 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=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. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PhoPQ+regulates+acidic+glycerophospholipid+content+of+the+Salmonella+Typhimurium+outer+membrane.">PhoPQ regulates acidic glycerophospholipid content of the Salmonella Typhimurium outer membrane.</a> Proceedings of the National Academy of Sciences of the United States of America. 111, 1963-1968 (2014).</li><li>Castanie-Cornet, M. P., Cam, K., Jacq, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RcsF+is+an+outer+membrane+lipoprotein+involved+in+the+RcsCDB+phosphorelay+signaling+pathway+in+Escherichia+coli.">RcsF is an outer membrane lipoprotein involved in the RcsCDB phosphorelay signaling pathway in Escherichia coli.</a> Journal of Bacteriology. 188, 4264-4270 (2006).</li><li>Thorne, K. J., Thornley, M. J., Glauert, A. 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>

No conflicts of interest declared.

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.

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		<tr>
			<td>1 L Centrifuge bottles, PC/PPCO, super speed, with sealing cap, Nalgene</td>
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			<td>1 L Pyrex Media Storage Bottle with High Temperature Cap</td>
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			<td>4x Laemmli Sample Buffer 10 mL</td>
			<td>BIORAD</td>
			<td>1610747</td>
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			<td>50 mL sterile polypropylene centrifuge tubes</td>
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			<td>EDTA disodium salt dihydrate 99.0-101.0%, crystals, ultrapure Bioreagent Molecular biology grade, J.T. Baker</td>
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			<td>Fiberlite F13-14 x 50cy Fixed Angle Rotor</td>
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			<td>Hydrochloric acid 6.0 N</td>
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			<td>Lysozyme, Egg White, Ultra Pure Grade</td>
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			<td>VWRV0063</td>
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			<td>Magnesium chloride hexahydrate</td>
			<td>Sigma Aldrich</td>
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			<td>NADH</td>
			<td>Sigma Aldrich</td>
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			<td>Optima XPN-80 - IVD</td>
			<td>Beckman Coulter Life Sciences</td>
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			<td>Pharmco Products&#160;PURE ALCOHOL 200 PROOF GL 4/CS&#160;</td>
			<td>Fischer Scientific</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>
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			<td>Thermofisher</td>
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			<td>G Biosciences</td>
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		<tr>
			<td>Proteinase K, Molecular Biology Grade</td>
			<td>New England Biolabs</td>
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			<td>Sodium hydroxide &#8805;99.99%</td>
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			<td>ThermoFisher Scientific</td>
			<td>36-101-0816</td>
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			<td>Sucrose</td>
			<td>MilliporeSigma</td>
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			<td>SW 41 Ti Swinging-Bucket Rotor</td>
			<td>Beckman Coulter Life Sciences</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>
			<td>VWR</td>
			<td>JT4109-6</td>
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			<td>Type 45 Ti Fixed-Angle Titanium Rotor</td>
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			<td>Beckman Coulter Life Sciences</td>
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			<td>Vortex-Genie 2</td>
			<td>VWR</td>
			<td>102091-234</td>
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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 ...

separation-cell-envelope-for-gram-negative-bacteria-into-inner-outer

Research

JoVE Journal

Immunology and Infection

12.9K Views.  University of Oklahoma Health Sciences Center. 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.

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

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 ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

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 ...