Research in the Meredith lab was supported by startup funds provided by the Eberly College of Science (Pennsylvania State University). We thank Dr. Tatiana Laremore for expert technical advice and access to equipment at the Penn State Proteomics and Mass Spectrometry Core Facility, University Park, PA, where mass spectrometric analyses were performed.

1. Cell Growth and Lysis
<ol>
	<li>Grow bacteria in 15 mL tryptic soy broth (TSB) or similar rich media to late exponential phase (OD600 of 1.0 - 1.5). Harvest cells by centrifugation, wash once with Tris-buffered saline/EDTA (TBSE) and continue with protocol or freeze until use. 
	NOTE: TBSE: 20 mM Tris-hydrochloride (HCl), pH 8.0, 130 mM sodium chloride (NaCl), and 5 mM ethylenediaminetetraacetic acid (EDTA)). Lipoprotein expression and modification can be influenced by growth conditions (e.g. acidity, salinity, growth media) and growth phase15. Cell growth can be scaled as desired, but 15-mL of cells is recommended for a single preparation of lipoproteins as excess biomass can decrease lipoprotein yield. One 15-mL preparation generally yields enough sample for optimal loading of ~2-4 lanes on a standard SDS-PAGE mini gel.</li>
	<li>Resuspend cells in 800 &#956;L TBSE with 1 mM phenylmethyl sulfonyl fluoride (PMSF) and 0.5 mg/mL lysozyme. Transfer solution to a 2.0-mL threaded microcentrifuge tube with screw cap and O-ring and incubate for 20 min at 37 &#176;C. 
	NOTE: Certain species are not susceptible to lysis by lysozyme. An appropriate lytic enzyme should be substituted to aid in lysis.</li>
	<li>Add ~800 &#956;L 0.1 mm zirconia/silica beads to the tube and disrupt cells by shaking at maximum speed (7,000 rpm) on a homogenizer for 5 cycles of 30 s each, with 2 min rest on ice between each cycle.</li>
	<li>Centrifuge sample at 3000 x g for 5 min at 4 &#176;C (to pellet beads and unbroken cells). Transfer the supernatant to a new 2.0-mL microcentrifuge tube and keep on ice.</li>
	<li>Add 200 &#956;L TBSE to the remaining pellet and return to the homogenizer for an additional cycle. Centrifuge (as above) and combine supernatant with the previous supernatant (should be ~800-1000 &#956;L total volume).</li>
</ol>
2. Enrichment of Lipoproteins by TX-114 Phase Partitioning
<ol>
	<li>Supplement the supernatant with TX-114 surfactant to a final concentration of 2% (vol/vol)by adding an equal volume of 4% (vol/vol) the surfactant in ice-cold TBSE and incubate on ice for 1 h, mixing by inversion every ~15 min. 
	NOTE: When chilled, the supernatant and surfactant will be miscible.</li>
	<li>Transfer the tube to a 37 &#176;C water bath and incubate for 10 min to induce phase separation. Centrifuge the sample at 10,000 x g for 10 min at room temperature to maintain bi-phasic separation.</li>
	<li>Gently pipette off the upper aqueous phase and discard. Add ice-cold TBSE to the lower surfactant phase to refill the tube to its original volume and invert to mix. Incubate on ice for 10 min.</li>
	<li>Transfer the tube to a 37 &#176;C water bath and incubate for 10 min to induce phase separation, then centrifuge at 10,000 x g for 10 min at room temperature.</li>
	<li>Repeat steps 2.3-2.4 once more for a total of 3 separations. Remove the upper aqueous phase and discard.</li>
	<li>Remove pellet of precipitated proteins that formed during the course of extractions (visible at the bottom of the tube), by adding 1 volume of ice-cold TBSE to the surfactant phase. Centrifuge at 4 &#176;C at 16,000 x g for 2 min to pellet insoluble protein. 
	NOTE: Sample should remain a single phase. If phase separation occurs, re-chill sample and centrifuge again. The pellet is generally composed of non-lipoprotein contaminants, but can be retained for further analysis.</li>
	<li>Immediately transfer the supernatant to a fresh 2.0-mL microcentrifuge tube containing 1250 &#956;L of 100% acetone. Pipette up and down thoroughly to wash the sample from the tip as it will be viscous. Mix by inversion and incubate overnight at -20 &#176;C to precipitate protein.</li>
	<li>Centrifuge the sample at 16,000 x g for 20 min at room temperature to pellet lipoproteins with attention to the orientation of the tube. Lipoproteins will form a thin, white film along the outside wall of the tube.</li>
	<li>Wash pellet twice with 100% acetone. Decant acetone and allow sample to air dry.</li>
	<li>Add 20-40 &#956;L of 10 mM Tris-HCl, pH 8.0 or a standard Laemmli SDS-PAGE sample buffer16 and thoroughly resuspend by pipetting up and down against the wall with the precipitated lipoproteins. Scrape the side of the tube with the pipette tip to dislodge lipoproteins. 
	NOTE: Lipoproteins will not dissolve in Tris buffer, but rather form a suspension.
	<ol>
		<li>Store lipoproteins at -20 &#176;C until use.</li>
	</ol>
	</li>
</ol>
3. SDS-PAGE, Electroblotting, and Staining with Ponceau S
<ol>
	<li>Separate lipoproteins by SDS-PAGE using standard methods16. 
	NOTE: The appropriate gel acrylamide percentage will vary depending on its intended use and size of lipoproteins. Here, E. faecalis lipoproteins were separated over a 10% Tris-glycine gel, while a 16.5% Tris-tricine gel was used for the smaller E. coli lipoprotein Lpp17.</li>
	<li>Transfer the lipoproteins to a nitrocellulose transfer membrane using a standard electroblotting procedure. 
	NOTE: A semi-dry transfer using Bjerrum Schafer-Nielsen buffer plus 0.1% SDS was performed at 25 V, 1.3 mA for 15 min18. Alternately, polyvinylidene difluoride (PVDF) membrane could be used, however as it is more hydrophobic than nitrocellulose, it may reduce efficient elution of hydrophobic lipopeptides from the membrane at later steps.</li>
	<li>Transfer the nitrocellulose membrane to a container and cover with Ponceau S solution (0.2% (w/v) Ponceau S in 5% acetic acid). Rock gently for 5 min or until red-pink bands are visible.</li>
	<li>Pour off Ponceau S solution and carefully rinse the nitrocellulose membrane with dH2O to remove excess stain. 
	NOTE: Ponceau-stained bands will destain rapidly. If bands disappear, repeat staining process.</li>
	<li>With a clean razor blade, excise the desired band and transfer to a microcentrifuge tube. Wash three times with 1 mL of dH2O to completely destain the band. 
	NOTE: The protocol can be paused here. Store the excised section covered with water at -20 &#176;C until use.</li>
	<li>Transfer the section to a clean surface and with a clean razor blade, dice the nitrocellulose strip into small pieces of approximately 1 mm x 1 mm. Collect the pieces into a 0.5-mL low protein binding microcentrifuge tube.</li>
	<li>Wash the pieces twice with 0.5&#160;mL of freshly-prepared 50 mM ammonium bicarbonate (NH4HCO3), pH 7.8 in high-performance liquid chromatography (HPLC) grade water.</li>
</ol>
4. Tryptic Digestion, Lipopeptide Extraction from Nitrocellulose Membrane, Deposition onto MALDI Target, and Data Acquisition
<ol>
	<li>Resuspend nitrocellulose pieces in 20 &#956;L of a 20 &#956;g/mL solution of trypsin in 50 mM NH4HCO3, pH 7.8 in HPLC grade water. Vortex to mix, then spin briefly to ensure that all pieces are fully covered by the trypsin solution. Cover the tube lid using paraffin film to prevent evaporation and incubate digest overnight at 37 &#176;C.</li>
	<li>Spin the sample at 16,000 x g for 30 s and remove the liquid by pipetting. 
	NOTE: This removes trypsinized hydrophilic peptides that can be examined later by MALDI-TOF MS for protein identification.</li>
	<li>Add 50 &#956;L of 0.5% trifluoroacetic acid (TFA) in HPLC grade water. Vortex to mix, then spin the sample briefly to ensure all pieces are covered by the solution. Incubate for 10 min at room temperature. Remove the liquid by pipetting. 
	CAUTION: TFA is harmful if inhaled. Handle with caution and use in the chemical fume hood. Avoid contact with skin.</li>
	<li>Repeat step 4.3 with 50 &#956;L of 10% acetonitrile in HPLC grade water. 
	CAUTION: Acetonitrile is harmful if inhaled. Handle with caution and use in the chemical fume hood. Avoid contact with skin.</li>
	<li>Repeat step 4.3 with 50 &#956;L of 20% acetonitrile in HPLC grade water. 
	NOTE: This removes any moderately hydrophobic peptides loosely bound to the nitrocellulose.</li>
	<li>To elute the tightly-bound lipopeptides, add 15 &#956;L of freshly-made 10 mg/mL &#945;-cyano-4-hydroxycinnamic acid (CHCA) matrix dissolved in chloroform-methanol (2:1, v/v) and incubate for 10 min at room temperature with intermittent vortexing.
	<ol>
		<li>Alternately, add 15 &#956;L chloroform-methanol to elute lipopeptides and mix with matrix at a later time. Other matrices, such as 2,5-dihydroxybenzoic acid (DHB), should be tested as alternatives to CHCA if unsatisfactory results are obtained for a particular lipopeptide composition. 
		CAUTION: Both chloroform and methanol are harmful if inhaled. Handle with caution and use in the chemical fume hood. Avoid contact with skin.</li>
		<li>Optional: To promote sodium adduct formation, supplement the solution of CHCA in chloroform-methanol with aqueous sodium bicarbonate (NaHCO3) to a final concentration of 1 mM.</li>
	</ol>
	</li>
	<li>Spin the sample briefly. With a pipette, carefully transfer the liquid to a new low protein binding microcentrifuge tube. This solution will contain the bulk of the N-terminal lipopeptides. 
	NOTE: The nitrocellulose may partially or fully dissolve in the chloroform-methanol solution. This will not negatively affect MS results, and may be used as an alternate method to increase lipopeptide return. PVDF membrane will not dissolve in the same manner.</li>
	<li>Deposit 1 &#956;L of the eluted lipopeptides with CHCA onto a polished steel MALDI target. 
	NOTE: The chloroform-methanol will evaporate quickly, leaving behind crystallized CHCA and lipopeptides. An optional second 1 &#956;L aliquot can be deposited onto the same spot to increase sample concentration. Chloroform-methanol may spread significantly after deposition onto the target and as such, care should be taken to avoid sample mixing on the target. It is recommended to deposit and shoot multiple spots of each sample.</li>
	<li>Immediately proceed to mass spectrometry. Scan all areas of the spot for lipopeptide signal, especially if the spot contains two layers of sample, as the second layer may push the lipopeptides to the spot&#39;s outer rim. 
	NOTE: Recommended starting laser intensity is 25%, though it may be necessary to increase intensity to acquire signal and sum multiple spectra (20 or more scans) to achieve adequate signal-to-noise ratio. Here, spectra were acquired on a MALDI-TOF-TOF instrument (see the Table of Materials) using a factory-configured instrument method for the reflector positive-ion detection over the 700-3500 m/z range. The instrument was calibrated with a bovine serum albumin (BSA) tryptic peptide mixture.</li>
</ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipoproteins+of+bacterial+pathogens.">Lipoproteins of bacterial pathogens.</a> Infect Immun. 79 (2), 548-561 (2011).</li><li>Nguyen, M. T., G&#246;tz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipoproteins+of+Gram-Positive+Bacteria:+Key+Players+in+the+Immune+Response+and+Virulence.">Lipoproteins of Gram-Positive Bacteria: Key Players in the Immune Response and Virulence.</a> Microbiol Mol Biol Rev. 80 (3), 891-903 (2016).</li><li>Nakayama, H., Kurokawa, K., Lee, B. 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The authors have nothing to disclose.

The protocol herein describes two distinct stages of lipoprotein characterization: enrichment by TX-114 phase partitioning and structural determination by MALDI-TOF MS. During TX-114 extraction, additional centrifugation removes contaminating proteins that precipitate during this process, followed by acetone precipitation to yield highly enriched lipoproteins. By limiting the scale of each preparation to 15-mL worth of cells, several samples can be easily processed in parallel, and if desired, pooled at the end of the pr...

Bacterial lipoproteins are characterized by a conserved N-terminal lipid-modified cysteine that anchors the globular protein domain to the cell membrane surface. They are universally distributed in bacteria, constituting 2 to 5% of all cellular genes within a typical genome1. Lipoproteins play critical roles in a wide variety of cellular processes, including nutrient uptake, signal transduction, assembly of protein complexes, and in maintaining cell envelope structural integrity2. In pathogenic bacteria, lipoproteins serve as virulence factors3,4. During an infection, recognition of N-terminal lipopeptides by Toll-like receptor (TLR) 2 incites an innate immune response to remove invading pathogens. Depending on the N-terminal acylation state, lipoproteins are generally recognized by alternate TLR2 heterodimeric complexes. TLR2-TLR1 recognizes N-acylated lipopeptides, while TLR2-TLR6 binds free lipopeptide &#945;-amino termini. Once bound, the signaling pathways converge to induce secretion of proinflammatory cytokines3,4.
It was previously thought that lipoproteins from Gram-positive bacteria were diacylated and those from Gram-negative bacteria were triacylated, differing in the absence or presence of an amide-linked fatty acid on the conserved N-terminal cysteine residue. This assumption was supported by the lack of sequence orthologs in Gram-positive genomes to Lnt, the Gram-negative N-acyl transferase that forms triacylated lipoproteins5. However, recent studies have revealed lipoprotein triacylation in Gram-positive Firmicutes that lack lnt, as well as three novel N-terminal lipoprotein structures, termed the peptidyl, lyso, and N-acetyl forms6,7,8. These findings raise questions about possible yet-to-be-discovered lipoprotein forms, alongside fundamental questions about how these novel lipoproteins are made and what physiological purpose or advantage the various forms impart. Furthermore, they clearly demonstrate the current inability of genomics to predict lipoprotein structure. Indeed, we recently identified a novel class of lipoprotein N-acyl transferases, called Lit, from Enterococcus faecalis and Bacillus cereus that makes lyso-form lipoproteins9. This indicates the need to experimentally verify lipoprotein structure, which can be challenging due to their extremely hydrophobic nature and limited methods available to characterize their molecular structure.
To facilitate studies on lipoprotein induction of the host immune response, as well as N-terminal structural determination, we have adapted several previously-described protocols in order to purify bacterial lipoproteins and prepare the N-terminal tryptic lipopeptides for analysis by MALDI-TOF MS6,10,11,12. Lipoproteins are enriched using an established Triton X-114 (henceforth referred to as surfactant or TX-114) phase partitioning method, with optimization to remove contaminating non-lipoproteins and increase lipoprotein yield. These lipoproteins are suitable for direct use in TLR assays or for further purification by SDS-PAGE. For MALDI-TOF MS, transfer of the lipoproteins to nitrocellulose membrane provides a scaffold for efficient in situ trypsin digestion, washing, and subsequent elution from the membrane surface, resulting in highly purified N-terminal lipopeptides. Nitrocellulose has been shown to facilitate sample handling and improve sequence coverage for highly hydrophobic peptides from integral membrane proteins13,14, as well as lipoproteins9,10. The method has the additional advantage of fractionating peptides based on polarity, so that intermediate wash solutions can be analyzed for high confidence protein identification simultaneously with N-terminal structural determination in a single experiment. This protocol uniquely features intentional sodium adduct formation to promote parent ion fragmentation towards dehydroalanyl ions during MS/MS, aiding in structural assignment of the N-acylation state. The N-terminus is both the most variable and key feature related to TLR recognition of lipoproteins. Taken together, this protocol has allowed intensive and reproducible studies on lipoproteins, with the individual stages of purification and structural determination by MALDI-TOF MS easily adapted depending on the overall goal of the experiment.

<table>
	<tbody>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.01 mm Zirconia/silica beads</td>
			<td>BioSpec Products</td>
			<td>110791012</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>EMD</td>
			<td>AX0073-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>EMD</td>
			<td>AX0116-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>EMD</td>
			<td>AX0142-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Fluka Analytical</td>
			<td>09830</td>
			<td></td>
		</tr>
		<tr>
			<td>BioTrace NT Nitrocellulose</td>
			<td>PALL Life Sciences</td>
			<td>66485</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA) digest standard</td>
			<td>Protea</td>
			<td>PS-204-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Acros Organics</td>
			<td>423550025</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Fisher Scientific</td>
			<td>BP118</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC Grade water</td>
			<td>EMD</td>
			<td>WX0008-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Fisher Scientific</td>
			<td>BP535-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethyl sulfonyl fluoride (PMSF)</td>
			<td>Amresco</td>
			<td>0754</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce trypsin protease</td>
			<td>Thermo Scientific</td>
			<td>90057</td>
			<td></td>
		</tr>
		<tr>
			<td>Ponceau S</td>
			<td>Acros Organics</td>
			<td>161470100</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind Tube 0.5mL</td>
			<td>Eppendorf</td>
			<td>022431064</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>792519</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Macron Fine Chemicals</td>
			<td>7581-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid (TFA)</td>
			<td>Sigma-Aldrich</td>
			<td>299537</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-hydrochloride (HCl)</td>
			<td>Fisher Scientific</td>
			<td>BP152</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-114</td>
			<td>Sigma-Aldrich</td>
			<td>93422</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic soy broth (TSB)</td>
			<td>BD</td>
			<td>211822</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-cyano-4-hydroxycinnamic acid (MS Grade)</td>
			<td>Sigma-Aldrich</td>
			<td>C8982</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MagNALyser</td>
			<td>Roche</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot Turbo Transfer System</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MALDI-TOF target</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultraflextreme MALDI-TOF-TOF</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td>Equipped with a 355 nm frequency-tripled Nd:YAG smartbeam-II laser</td>
		</tr>
	</tbody>
</table>

enrichment of bacterial lipoproteins and preparation of n-terminal lipopeptides for structural determination by mass spectrometry

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their significance to both cell physiology and immunology, much remains to be discovered about novel lipoprotein forms, how they are synthesized, and the effect of the various forms on host immunity. To enable thorough studies on lipoproteins, this protocol describes a method for bacterial lipoprotein enrichment and preparation of N-terminal tryptic lipopeptides for structural determination by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Expanding on an established Triton X-114 phase partitioning method for lipoprotein extraction and enrichment from the bacterial cell membrane, the protocol includes additional steps to remove non-lipoprotein contaminants, increasing lipoprotein yield and purity. Since lipoproteins are commonly used in Toll-like receptor (TLR) assays, it is critical to first characterize the N-terminal structure by MALDI-TOF MS. Herein, a method is presented to isolate concentrated hydrophobic peptides enriched in N-terminal lipopeptides suitable for direct analysis by MALDI-TOF MS/MS. Lipoproteins that have been separated by Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) are transferred to a nitrocellulose membrane, digested in situ with trypsin, sequentially washed to remove polar tryptic peptides, and finally eluted with chloroform-methanol. When coupled with MS of the more polar trypsinized peptides from wash solutions, this method provides the ability to both identify the lipoprotein and characterize its N-terminus in a single experiment. Intentional sodium adduct formation can also be employed as a tool to promote more structurally informative fragmentation spectra. Ultimately, enrichment of lipoproteins and determination of their N-terminal structures will permit more extensive studies on this ubiquitous class of bacterial proteins.

A schematic of the protocol is provided in Figure 1. The lipoprotein-enriched fraction extracted from Enterococcus faecalis ATCC 19433 by TX-114 is shown in Figure 2. For comparison, the banding pattern of the precipitated protein fraction is also shown. Proteins from this fraction were confirmed by MALDI-MS to be highly abundant contaminating proteins other than lipoproteins (Table 1). The mass spectra ...

Watch this Scientific Journal Video about Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry at JoVE.com

Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

The enrichment of bacterial lipoproteins using a non-ionic surfactant phase partitioning method is described for direct use in TLR assays or other applications. Further steps are detailed to prepare N-terminal tryptic lipopeptides for structural characterization by mass spectrometry.

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their ...

enrichment-bacterial-lipoproteins-preparation-n-terminal-lipopeptides

Research

JoVE Journal

Immunology and Infection

8.5K Views.  Pennsylvania State University. The enrichment of bacterial lipoproteins using a non-ionic surfactant phase partitioning method is described for direct use in TLR assays or other applications. Further steps are detailed to prepare N-terminal tryptic lipopeptides for structural characterization by mass spectrometry.

Video: Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein complexes and assemblies. In fact, single particle electron microscopy1 and two-dimensional (2D) electron crystallography2 have become relatively routine methodologies and a large number of structures have been solved using these methods. At the same time, image processing and three-dimensional (3D) reconstruction of helical objects has rapidly developed, especially, the iterative helical real-space reconstruction (IHRSR) method3, which uses single particle analysis tools in conjunction with helical symmetry. Many biological entities function in filamentous or helical forms, including actin filaments4, microtubules5, amyloid fibers6, tobacco mosaic viruses7, and bacteria flagella8, and, because a 3D density map of a helical entity can be attained from a single projection image, compared to the many images required for 3D reconstruction of a non-helical object, with the IHRSR method, structural analysis of such flexible and disordered helical assemblies is now attainable.
In this video article, we provide detailed protocols for obtaining a 3D density map of a helical protein assembly (HIV-1 capsid9 is our example), including protocols for cryo-EM specimen preparation, low dose data collection by cryo-EM, indexing of helical diffraction patterns, and image processing and 3D reconstruction using IHRSR. Compared to other techniques, cryo-EM offers optimal specimen preservation under near native conditions. Samples are embedded in a thin layer of vitreous ice, by rapid freezing, and imaged in electron microscopes at liquid nitrogen temperature, under low dose conditions to minimize the radiation damage. Sample images are obtained under near native conditions at the expense of low signal and low contrast in the recorded micrographs. Fortunately, the process of helical reconstruction has largely been automated, with the exception of indexing the helical diffraction pattern. Here, we describe an approach to index helical structure and determine helical symmetries (helical parameters) from digitized micrographs, an essential step for 3D helical reconstruction. Briefly, we obtain an initial 3D density map by applying the IHRSR method. This initial map is then iteratively refined by introducing constraints for the alignment parameters of each segment, thus controlling their degrees of freedom. Further improvement is achieved by correcting for the contrast transfer function (CTF) of the electron microscope (amplitude and phase correction) and by optimizing the helical symmetry of the assembly.

This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at all on selective media that are usually used to cultivate bacteria. For this reason, many of these pathogens were discovered only recently or following important outbreaks. These pathogens are often associated with amoebae, which serve as host-cell and allow the survival and growth of the bacteria. We intend here to provide a demonstration of two techniques that allow isolation and characterization of intracellular pathogens present in clinical or environmental samples: the amoebal coculture and the amoebal enrichment. Amoebal coculture allows recovery of intracellular bacteria by inoculating the investigated sample onto an amoebal lawn that can be infected and lysed by the intracellular bacteria present in the sample. Amoebal enrichment allows recovery of amoebae present in a clinical or environmental sample. This can lead to discovery of new amoebal species but also of new intracellular bacteria growing specifically in these amoebae. Together, these two techniques help to discover new intracellular bacteria able to grow in amoebae. Because of their ability to infect amoebae and resist phagocytosis, these intracellular bacteria might also escape phagocytosis by macrophages and thus, be pathogenic for higher eukaryotes.

Amoebal coculture is a cell culture system using adherent amoebae to selectively grow intracellular pathogens able to resist phagocytic cells such as amoebae and macrophages. It thus represents a key tool to discover new infectious agents. Amoebal enrichment allows discovery of new amoebal species and of their specific intracellular bacteria.

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at ...

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 ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

Electroporation of Functional Bacterial Effectors into Mammalian Cells

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Identification of Nucleolar Factors During HIV-1 Replication Through Rev Immunoprecipitation and Mass Spectrometry

The HIV-1 infectious cycle requires viral protein interactions with host factors to facilitate viral replication, packaging, and release. The infectious cycle further requires the formation of viral/host protein complexes with HIV-1 RNA to regulate the splicing and enable nucleocytoplasmic transport. The HIV-1 Rev protein accomplishes the nuclear export of HIV-1 mRNAs through multimerization with intronic cis-acting targets - the Rev response element (RRE). A nucleolar localization signal (NoLS) exists within the COOH-terminus of the Rev arginine-rich motif (ARM), allowing the accumulation of Rev/RRE complexes in the nucleolus. Nucleolar factors are speculated to support the HIV-1 infectious cycle through various other functions in addition to mediating mRNA-independent nuclear export and splicing. We describe an immunoprecipitation method of wild-type (WT) Rev in comparison to Rev nucleolar mutations (deletion and single-point Rev-NoLS mutations) in the presence of HIV-1 replication for mass spectrometry. Nucleolar factors implicated in the nucleocytoplasmic transport (nucleophosmin B23 and nucleolin C23), as well as cellular splicing factors, lose interaction with Rev in the presence of Rev-NoLS mutations. Various other nucleolar factors, such as snoRNA C/D box 58, are identified to lose interaction with Rev mutations, yet their function in the HIV-1 replication cycle remain unknown. The results presented here demonstrate the use of this approach for the identification of viral/host nucleolar factors that maintain the HIV-1 infectious cycle. The concepts used in this approach are applicable to other viral and disease models requiring the characterization of understudied pathways.

Here we describe Rev immunoprecipitation in the presence of HIV-1 replication for mass spectrometry. The methods described can be used for the identification of nucleolar factors involved in the HIV-1 infectious cycle and are applicable to other disease models for the characterization of understudied pathways.

The HIV-1 infectious cycle requires viral protein interactions with host factors to facilitate viral replication, packaging, and release. The infectious ...

Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

Summary

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Chapters in this video

Invasion of Human Cells by a Bacterial Pathogen

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

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

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Electroporation of Functional Bacterial Effectors into Mammalian Cells

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Identification of Nucleolar Factors During HIV-1 Replication Through Rev Immunoprecipitation and Mass Spectrometry