This work was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases.

1. Plasmid Construction
<ol>
	<li>After cloning a gene that encodes a protein of interest into an appropriate expression vector, introduce amber mutations at desired locations using a site-directed mutagenesis kit. 
	NOTE: Structural information and surface exposure predictions can serve as useful guides to determine the positions of mutations. Because the efficiency of amber suppression and cross-linking can vary considerably, amber mutations should be introduced at multiple positions. Furthermore, because Bpa is an analog of tyrosine and phenylalanine, try to introduce amber mutations at positions that normally encode large hydrophobic amino acids to minimize perturbations of protein structure and function. Finally, when choosing an expression vector keep in mind that overproducing a protein of interest at a very high level might limit the efficiency of Bpa incorporation.</li>
</ol>
2. Culture Preparation
<ol>
	<li>Use electroporation to transform an appropriate E. coli strain with a plasmid that encodes the amber suppression system (pDULE-pBpa; see refrence4) and a compatible plasmid that encodes the protein of interest with an amber mutation.</li>
	<li>Plate cells on LB agar containing appropriate antibiotics (use 5 &#181;g/ml tetracycline to select for pDULE-pBpa). Incubate the plates O/N at 37&#160;&#176;C.</li>
	<li>On the following day, start O/N cultures from single colonies in 3-5 ml M9 medium14 containing 0.2% glycerol, all of the L-amino acids except methionine and cysteine (40 &#181;g/ml) and antibiotics. Incubate at 37&#160;&#176;C.</li>
	<li>On the next day, centrifuge O/N cultures at 2,500 x g for 5 min at RT to pellet cells. Resuspend the cells in 5 ml fresh M9 medium and repeat the centrifugation. 
	NOTE: This wash step is critical to maintain plasmids that encode ampicillin resistance because small amounts of b lactamase that are present in the medium from O/N cultures degrade freshly added ampicillin before significant cell growth occurs.</li>
	<li>Resuspend the cells in 2 ml fresh medium and measure cell concentration in a spectrophotometer (OD550).</li>
	<li>Add cells to a 250 ml Erlenmeyer flask containing 50 ml M9 medium and antibiotics at OD550=0.03. Incubate the cultures at 37 &#176;C in a shaking water bath.</li>
</ol>
3. Bpa Incorporation and Preparation for Photocrosslinking
NOTE: The protocol described below is for a sample time course that has three time points (0, 1, and 5 min). Adjust the volumes accordingly if a different number of time points is desired. An outline of the procedure is illustrated in Figure&#160;1.
<ol>
	<li>While the cultures are growing, prepare a fresh 1 M (1,000x) solution of Bpa in 1 M NaOH (27 mg Bpa in 100 &#956;l 1 M NaOH). Use a dark walled tube to keep the solution in the dark.</li>
	<li>When the cultures reach early to mid-log phase(OD550=0.2), add 50 &#956;l 1 M Bpa to each culture. Add Bpa one drop at a time while swirling the flask to avoid precipitation. Next, add any inducers that are needed to drive expression of the amber mutant. Continue to incubate the cultures at 37 &#176;C for an additional 30 min.</li>
	<li>During the 30 min induction period label six disposable 15 ml centrifuge tubes with the time point (0 min, 1 min, 5 min) and either &#34;+ UV&#34; or &#34;- UV&#34;. Place the tubes on ice.</li>
	<li>Fill a second ice bucket with ice and place a 6-well tissue culture plate on top of the ice.</li>
	<li>Thaw high specific activity (1,000 mCi/mmol)&#160;35S-methionine and 35S-cysteine (or a premade mixture of the two compounds) for the pulse labeling and prepare (or thaw) a solution of nonradioactive L-cysteine and L-methionine (100 mM each) for the chase.</li>
	<li>Turn on the UV lamp five min&#160;prior to radiolabeling. 
	NOTE: The lamp should be adjustable so that it can be positioned ~3-4 cm from the samples in the multiwell plate.</li>
	<li>Add ice (~2 ml) to each tube labeled &#34;- UV&#34; and to two of the wells in the multiwell plate. 
	NOTE: Ice should be added to a third well immediately before the 5 min&#160;time point. It is essential to place ice directly into the tubes and wells to stop intracellular reactions immediately at each time point and to thereby obtain accurate &#34;snapshots&#34; of a specific biochemical pathway.</li>
	<li>At the end of the 30 min&#160;induction period transfer 25 ml of the culture to a prewarmed 125 ml disposable Erlenmeyer flask. Place the flask in the 37 &#176;C shaking water bath.</li>
</ol>
4. Pulse-chase Labeling and UV Irradiation
NOTE: The pulse-chase labeling and UV irradiation protocol has a lot of steps that must be performed in a timely manner and that require considerable organization and advance preparation. All of the reagents and tubes should be readily accessible. A protocol for an experiment that has 0, 1,&#160;and 5 min&#160;time points is presented below.
<ol>
	<li>At time 0:00 (min:sec) transfer 25 ml of culture into 125 ml flask.</li>
	<li>At time 0:30 add 30 &#181;Ci/ml radioactive amino acids; swirl quickly to mix. Place the flask into the 37&#160;&#176;C shaking water bath.</li>
	<li>At time 1:00 add 250 &#181;l of the 100 mM methionine-cysteine solution; swirl quickly to mix.
	<ol>
		<li>Immediately pipette 4 ml of the culture into one of the wells of the chilled multiwell plate that contains ice. Pipette a second 4 ml aliquot into the 15 ml tube labeled &#34;0 min - UV&#34;. Return the flask to the water bath.</li>
	</ol>
	</li>
	<li>At time 2:00 pipette 4 ml of the culture into the second well of the chilled multiwell plate that contains ice. Pipette another 4 ml aliquot into the 15 ml tube labeled &#34;1 min - UV&#34;.&#160;Return the flask to the water bath.</li>
	<li>At time 6:00 pipette 4 ml of the culture into the third well of the chilled multiwell plate that contains ice. Pipette another 4 ml aliquot into the 15 ml tube labeled &#34;5 min - UV&#34;.</li>
	<li>Place the ice bucket with the multiwell plate under the preheated UV lamp and irradiate each sample individually for 4 min. 
	NOTE: In experiments in which time points are spaced at least 4 min&#160;apart each sample should be irradiated as soon as it is pipetted into the multiwell plate.</li>
	<li>Transfer the cells into the chilled 15 ml tubes labeled &#34;0 min +UV&#34;, 1 min +UV&#34;, etc.</li>
	<li>Centrifuge cells at 2,500 x g for 10 min&#160;at 4&#160;&#176;C.</li>
	<li>Resuspend each sample in 300 &#181;l M9 medium or PBS and add 33 &#181;l 100% cold trichloroacetic acid (TCA) to precipitate proteins. Centrifuge the TCA precipitates in a microfuge at top speed for 10 min&#160;and remove the supernatant. At this point samples can be stored at - 20&#160;&#176;C.</li>
</ol>
5. Immunoprecipitation and Crosslinking Product Detection
<ol>
	<li>Add 50 &#181;l solubilization buffer (15% glycerol, 200 mM Trisbase, 15 mM EDTA, 4% SDS, 2 mM PMSF) to each sample and heat with agitation at 95&#160;&#176;C for 5 min&#160;to solubilize precipitated protein.</li>
	<li>Add 1 ml radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1.0% Igepal CA-630, 0.5% deoxycholate, 0.1% SDS) and perform standard immunoprecipitations15 using one-fifth to one-half of each sample. 
	NOTE: Igepal CA-630 (octylphenyl-polyethylene glycol) is used in place of NP-40, which is no longer commercially available. The two compounds are chemically indistinguishable.</li>
	<li>Resolve immunoprecipitated proteins by SDS-PAGE. Stain, destain,&#160;and dry gels.</li>
	<li>Expose gels to a phosphor screen O/N and detect radioactive proteins (including crosslinking products) with a phosphorimager.</li>
</ol>

<ol>
	<li>Wang, L., Xie, J., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+genetic+code.">Expanding the genetic code.</a> Annu. Rev. Biophys. Biomol. Struct. 35, 225-249 (2006).</li><li>von Mering, 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=Comparative+assessment+of+large-scale+data+sets+of+protein-protein+interactions.">Comparative assessment of large-scale data sets of protein-protein interactions.</a> Nature. 417, 399-403 (2002).</li><li>Chin, J. W., Martin, A. B., King, D. S., Wang, L., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Addition+of+a+photocrosslinking+amino+acid+to+the+genetic+code+of+Escherichia+coli.">Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli.</a> PNAS. 99, 11020-11024 (2002).</li><li>Farrell, I. S., Toroney, R., Hazen, J. L., Mehr, R. A., Chin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photo-cross-linking+interacting+proteins+with+a+genetically+encoded+benzophenone.">Photo-cross-linking interacting proteins with a genetically encoded benzophenone.</a> Nat. Methods. 2 (5), 377-384 (2005).</li><li>Wittelsberger, A., Mierke, D. F., Rosenblatt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+ligand-receptor+interfaces:+approaching+the+resolution+limit+of+benzophenone-based+photaffinity+scanning.">Mapping ligand-receptor interfaces: approaching the resolution limit of benzophenone-based photaffinity scanning.</a> Chem. Biol. Drug Des. 71, 380-383 (2008).</li><li>Ieva, R., Tian, P., Peterson, J. H., Bernstein, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+and+spatially+restricted+interactions+of+assembly+factors+with+an+autotransporter+beta+domain.">Sequential and spatially restricted interactions of assembly factors with an autotransporter beta domain.</a> PNAS. 108, 383-391 (2011).</li><li>Wang, F., Robbins, S., Guo, J., Shen, W., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+incorporation+of+unnatural+amino+acids+into+proteins+in+Mycobacterium+tuberculosis.">Genetic incorporation of unnatural amino acids into proteins in Mycobacterium tuberculosis.</a> PLoS One. 5, (2010).</li><li>Deiters, A., 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=Adding+amino+acids+with+novel+reactivity+to+the+genetic+code+of+Saccharyomycescerevisiae.">Adding amino acids with novel reactivity to the genetic code of Saccharyomycescerevisiae.</a> J. Am. Chem. Soc. 125, 11782-11783 (2003).</li><li>Young, T. S., Ahmad, I., Brock, A., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+genetic+repertoire+of+the+methylotrophic+yeast+Pichiapastoris.">Expanding the genetic repertoire of the methylotrophic yeast Pichiapastoris.</a> Biochemistry. 48 (12), 2643-2653 (2009).</li><li>Hino, N., 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=Protein+photo-cross-linking+in+mammalian+cells+by+site-specific+incorporation+of+a+photoreactive+amino+acid.">Protein photo-cross-linking in mammalian cells by site-specific incorporation of a photoreactive amino acid.</a> Nat. Methods. 2 (3), 201-206 (2005).</li><li>Liu, W., Brock, A., Chen, S., Chen, S., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+incorporation+of+unnatural+amino+acids+into+proteins+in+mammalian+cells.">Genetic incorporation of unnatural amino acids into proteins in mammalian cells.</a> Nat. Methods. 4 (3), 239-244 (2007).</li><li>Leyton, D. L., Rossiter, A. E., Henderson, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+self+sufficiency+to+dependence:+mechanisms+and+factors+important+for+autotransporter+biogenesis.">From self sufficiency to dependence: mechanisms and factors important for autotransporter biogenesis.</a> Nat. Rev. Microbiol. 10 (3), 213-225 (2012).</li><li>Ieva, R., Bernstein, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+an+autotransporter+passenger+domain+with+BamA+during+its+translocation+across+the+bacterial+outer+membrane.">Interaction of an autotransporter passenger domain with BamA during its translocation across the bacterial outer membrane.</a> PNAS. 106, 19120-19125 (2009).</li><li>Pavlova, O., Peterson, J. H., Ieva, R., Bernstein, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+link+between+barrel+assembly+and+the+initiation+of+autotransporter+secretion.">Mechanistic link between barrel assembly and the initiation of autotransporter secretion.</a> PNAS. 110, (2013).</li><li>Harlow, D., Lane, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Using antibodies: a laboratory manual. , (1999).</li><li>Barnard, T. J., Dautin, N., Lukacik, N., Bernstein, P., Buchanan, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autotransporter+structure+reveals+intra-barrel+cleavage+followed+by+conformational+changes.">Autotransporter structure reveals intra-barrel cleavage followed by conformational changes.</a> Nat. Struct. Mol. Biol. 14 (12), 1214-1220 (2007).</li><li>Akiyama, Y., Ito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SecY+protein,+a+membrane-embedded+secretion+factor+of+E.+coli,+is+cleaved+by+the+ompT+protease+in+vitro.">SecY protein, a membrane-embedded secretion factor of E. coli, is cleaved by the ompT protease in vitro.</a> Biochem. Biophys. Res. Commun. 167 (2), 711-715 (1990).</li></ol>

The authors declare that they have no competing financial interests.

This paper describes a method that combines site-specific photocrosslinking with pulse-chase labeling to examine the dynamics of interactions between a protein of interest and other components of a living cell. The method is especially useful to study multistep processes in which the protein interacts sequentially with other factors. Unlike traditional chemical cross-linking approaches, site-specific photocrosslinking provides detailed information about the status of individual segments or even individual residues of a p...

It is often essential to identify and characterize interactions between a protein of interest and other cellular components to define its role in a biological pathway, to understand its assembly, or to elucidate its mechanism of action. A wide variety of approaches are commonly used to study protein-protein interactions, but all have their limitations. The simplest unbiased method to identify proteins that bind to a protein of interest is copurification (or coimmunoprecipitation) but this approach requires that a protein complex remain intact during the purification procedure. Weak or transient protein interactions can be stabilized through chemical cross-linking, but this method typically requires the use of compounds that link proteins through primary amines that are widely scattered in the protein sequence. Complicated cross-linking patterns that are difficult to interpret can be generated, especially when proteins of interest are components of multiprotein complexes. Furthermore, because the spacer arms of chemical cross-linkers generally exceed 10 &#197; in length, covalent bonds can be formed with proteins that are located in proximity to but do not interact directly with the protein of interest. Methods such as affinity chromatography and two-hybrid screens are also often useful to identify protein-protein interactions. The former approach, however, requires reproducing conditions that promote physiologically significant interactions and cannot be used to detect weak interactions. The latter approach requires that binding interactions can be replicated in the host organism and are maintained when a protein of interest and its binding partners are placed in the context of a fusion protein. Two-hybrid methods also tend to generate false-positive results2. Once a protein-protein interaction has been established, considerable work is often required to map the site of interaction. Perhaps the most significant drawback of traditional approaches is that they do not provide any information about the temporal sequence of intermolecular interactions or the kinetics of binding.
This paper describes a simple method that overcomes some of the limitations of other approaches that are used to study protein-protein interactions. Initially a single amber mutation is introduced into the gene that encodes a protein of interest. The amber mutant is then coexpressed with an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii that have been engineered to incorporate the photoactivatable amino acid analog p-benzoylphenylalanine (Bpa) only at amber codons3. Irradiation of cells with long wavelength ultraviolet (UV) light (~365 nm) then facilitates formation of a covalent bond between Bpa and proteins that are within ~3-8 &#197;4,5. In some experimental contexts, cross-links can also be formed between activated Bpa and nonprotein components of cells such as lipids and nucleic acids6. Molecules that are terminated at the amber codon should generally be easily distinguishable from the full-length protein on SDS-PAGE and cannot be cross-linked to other proteins. By subjecting cells to pulse-chase radiolabeling prior to cross-linking and then immunoprecipitating or affinity purifying cross-linking products, the sequence and duration of protein-protein interactions can be established. The ability to generate temporal information about intermolecular interactions is especially valuable to study the progression of a protein of interest through any ordered multistep assembly, trafficking or signaling pathway and to identify pathway intermediates. Like chemical cross-linking, site-specific cross-linking detects protein-protein interactions that occur under physiological conditions, but the introduction of a cross-linker at a single position facilitates the analysis of interactions between individual segments of a protein and other factors. This unique feature of site-specific cross-linking is especially advantageous when the protein of interest has multiple segments or domains that have the potential to interact with distinct binding partners. Furthermore, site-specific cross-linking can be used in combination with methods such as mass spectrometry to map the residues that mediate protein-protein interactions with precision. Although this method is optimized for use in E. coli, the incorporation of photactivatable amino acid analogs into proteins by amber suppression has been reported in Mycobacterium, yeast and mammalian cells7-11. Thus, in principle our method can be used in other systems.
We have used this method extensively to identify intermolecular interactions that facilitate the biogenesis of EspP, an E. coli O157:H7 virulence factor. EspP is a member of a superfamily of proteins known as &#34;autotransporters&#34; that contain a large N-terminal extracellular domain (&#34;passenger domain&#34;) and aC-terminal domain (&#34;b domain&#34;) that folds into a cylindrical b barrel structure and anchors the protein to the outer membrane (OM)12. The mechanism by which the passenger domain is translocated across the OM has been a long-standing mystery. Like all bacterial cell surface proteins, EspP must be transported across the inner membrane, shuttled across the periplasm (the space between the inner and outer membranes), and targeted to the OM in a series of ordered events. Following its translocation across the OM, the EspP passenger domain is also separated from the b domain by a proteolytic cleavage and released from the cell surface. By combining site-specific cross-linking with pulse-chase radiolabeling, we have shown that both domains of the protein initially interact with the molecular chaperone Skp in the periplasm6. Subsequently, discrete segments of the b domain interact in a stereospecific fashion with components of a heterooligomer called the Bam complex, which catalyzes the integration of b barrel proteins into the bacterial OM by an unknown mechanism. Perhaps most interestingly, we have found that the passenger domain is in contact with one of the Bam complex subunits (BamA) as it traverses the OM13. Our results have enabled us to construct a detailed model for autotransporter assembly14 and have implicated the Bam complex in the transport of the passenger domain across the OM.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QuikChange II Site-Directed Mutagenesis Kit</td>
			<td style="width:71px;">Agilent</td>
			<td align="right" style="width:119px;">200521</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BPA (H-p-Bz-Phe-OH)</td>
			<td style="width:71px;">Bachem</td>
			<td style="width:119px;">F-2800</td>
			<td></td>
		</tr>
		<tr height="93">
			<td height="93" style="height:93px;width:216px;">TRAN35S-LABEL, Metabolic Labeling Reagent (35S-L-methionine and 35S-L-cysteine, &#62;1,000 Ci/mmol)</td>
			<td style="width:71px;">MP Biomedicals</td>
			<td align="right" style="width:119px;">51006</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">Spectroline SB-100P Super-High-Intensity UV Lamp, 365 nm</td>
			<td style="width:71px;">Spectronics Corporation</td>
			<td style="width:119px;">SB-110P</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">Spectroline Replacement Bulb 100S</td>
			<td style="width:71px;">Spectronics Corporation</td>
			<td style="width:119px;">11-992-15</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Falcon Tissue Culture Plate, 6-well</td>
			<td style="width:71px;">Becton Dickinson Labware</td>
			<td align="right" style="width:119px;">353046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable 125 ml Erlenmeyer flask</td>
			<td style="width:71px;">Corning</td>
			<td align="right" style="width:119px;">430421</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Innova 3100 shaking water bath</td>
			<td style="width:71px;">New Brunswick Scientific</td>
			<td style="width:119px;">n.a.</td>
			<td></td>
		</tr>
	</tbody>
</table>

monitoring the assembly of a secreted bacterial virulence factor using site-specific crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

We used site-specific photocrosslinking to identify proteins that interact with EspP during its journey to the OM. In the experiments that are shown here, phenylalanine codons at residues 1113 and 1214 were replaced with amber codons. Both positions are located in the bdomain, which integrates into the OM and serves as a membrane anchor. The crystal structure of the EspP&#946; domain16 shows that the two residues are on the periplasmic side of the b barrel but are separated by ~120&#176; (see ...

Watch this Scientific Journal Video about Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking at JoVE.com

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

monitoring-assembly-secreted-bacterial-virulence-factor-using-site

Research

JoVE Journal

Immunology and Infection

6.1K Views.  National Institutes of Health. This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution “snapshots” of an ordered assembly pathway in a living cell.

Video: Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species1-5, as well as the wealth of information available regarding the insect's immune system6-8, and the pending genome sequence9 make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.
The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter10. The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking11 and oral toxicity assays12. Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula.
The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides7; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR13. The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes6. To analyze these responses, injected insects can be dissected and visualized by microscopy13, 14.

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

The method described here utilizes direct injection of entomopathogenic bacteria into the hemocoel of Manduca sexta insect larvae. M. sexta is a commercially available and well-studied insect. Thus, this method represents a simple approach to analyzing host-bacterial interactions from the perspective of one or both partners.

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

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 ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

Here, a method using a permeable membrane insert-based infection system to study the effects of Streptolysin S, a secreted toxin produced by Group A Streptococcus, on keratinocytes is described. This system can be readily applied to the study of other secreted bacterial proteins on various host cell types during infection.

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

Assembly and Purification of Prototype Foamy Virus Intasomes

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an integrase (IN) enzyme that catalyzes the covalent joining of viral to host DNA, which is known as strand transfer. Integration may be modeled in vitro with recombinant retroviral IN and DNA oligomers mimicking the ends of the viral genome. In order to more closely recapitulate the integration reaction that occurs in vivo, integration complexes are assembled from recombinant IN and synthetic oligomers by dialysis in a reduced salt concentration buffer. The integration complex, called an intasome, may be purified by size exclusion chromatography. In the case of prototype foamy virus (PFV), the intasome is a tetramer of IN and two DNA oligomers and is readily separated from monomeric IN and free oligomer DNA. The integration efficiency of PFV intasomes may be assayed under a variety of experimental conditions to better understand the dynamics and mechanics of retroviral integration.

Recombinant retroviral integrase and DNA oligomers mimicking viral DNA ends can form an enzymatically active complex known as an intasome. Intasomes may be used for biochemical, structural, and kinetic studies. This protocol details how to assemble and purify prototype foamy virus intasomes.

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an ...