The authors would like to thank members of the Krachler group for critical reading of the manuscript. DHS, DV and AMK were funded by the Biotechnology and Biological Sciences Research Council (BB/L007916/1), FA was funded by a Republic of Iraq Ministry of Higher Education and Scientific Research Scholarship and NPS was funded by a CONICYT Scholarship.

1. Chemical Coupling of Proteins to Polymer Beads
<ol>
	<li>Thiol-amine directional coupling 
	Note: This protocol is suitable for coupling of cysteine containing proteins to amine-functionalized polymer beads, using Sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (Sulfo-SMPB) as cross-linking agent (Figure 1). 
	<img alt="Figure 1" src="/files/ftp_upload/53400/53400fig1.jpg" /> 
	Figure 1. Cross-linking strategy used for directional thiol-amine coupling of proteins to polymer beads. Amine-modified polystyrene beads are activated with Sulfo-SMPB. The maleimide reacts with free cysteines to directionally couple proteins to beads. <a href="https://www.jove.com/files/ftp_upload/53400/53400fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
	<ol>
		<li>Preparation of reagents:
		<ol>
			<li>Prepare PBS (100 mM sodium phosphate, 150 mM NaCl, pH 7.0) and autoclave.</li>
			<li>Prepare a 100x stock (0.5 M or 287 mg/ml in PBS) of TCEP (tris(2-carboxyethyl)phosphine) immediately before use.</li>
			<li>Prepare a 5x stock (10 mM or 4.58 mg/ml in dH2O) of Sulfo-SMPB immediately prior to use.</li>
			<li>Prepare a 10x stock (500 mM or 88 mg/ml in PBS, pH 7.0) of cysteine immediately before use.</li>
		</ol>
		</li>
		<li>Bead activation:
		<ol>
			<li>Mix the bead suspension by gently inverting and transfer the required amount of bead suspension (e.g., 12 ml) into a sterile 1.5 ml tube containing 1 ml sterile PBS, pH 7.0.</li>
			<li>Gently pipette up and down to wash the beads and pellet by centrifugation in a microcentrifuge (2 min&#160;at 16,000 x g).</li>
			<li>Carefully remove the supernatant with a pipette and discard. Resuspend the bead pellet in 1 ml of fresh sterile PBS and repeat the washing step. Resuspend the bead pellet in 0.8 ml of PBS.</li>
			<li>Add 200 ml of freshly prepared 10 mM Sulfo-SMPB, to give a final concentration of 2 mM.</li>
			<li>Incubate the bead suspension for 1 hr&#160;at 25 &#176;C on a rotating wheel.</li>
		</ol>
		</li>
		<li>Protein reduction:
		<ol>
			<li>During the incubation period of the activation step, prepare the protein for the following coupling step so it can be immediately added to the activated beads. 
			Note: Although this reduction step is not always required for GST (Glutathione S-transferase) fusion proteins, it is &#160;recommended to ensure a high coupling efficiency.
			<ol>
				<li>Check the protein concentration, and adjust it to the final concentration required for the coupling reaction. Note: 6 mM protein in PBS, and a volume of 1 ml are usually used.</li>
				<li>Add TCEP stock to give a final concentration of 5 mM. Incubate the solution for 30 min&#160;at RT. 
				Note: The reaction mixture can be directly used for the following coupling reaction.</li>
				<li>Retain a small amount (a few ml) of the protein solution for determining the protein concentration and calculation of coupling efficiency (see section 2).&#160;&#160;</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Protein coupling step:
		<ol>
			<li>Pellet the activated beads by centrifugation (2 min, 16,000 x g in a microcentrifuge), and wash the pellet once in 1 ml of fresh sterile PBS.</li>
			<li>Resuspend the pellet in the prepared protein solution (e.g., 1 ml), to give the desired protein concentration. 
			Note: The protein concentration during the coupling step will depend on the average coupling efficiency (see section 2 for determination of the coupling efficiency) and desired coupling density (as calculated in 1.1.4.3). The efficiency is approximately 85% and the desired final concentration 5 mM in the 10x bead suspension, so protein concentration during the coupling step should be approximately 6 mM.</li>
			<li>Calculate the coupling density using the following formula: 
			<img alt="Equation 1" src="/files/ftp_upload/53400/53400eq1.jpg" /> where 
			&#961;c &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; coupling density [number of protein molecules/nm2], 
			protein conc&#160;&#160; protein concentration [mg/ml], 
			protein Mw&#160;&#160;&#160;&#160; protein molecular weight [Da], 
			bead conc&#160;&#160;&#160;&#160;&#160;&#160; bead concentration [number of beads/L], 
			d&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; bead diameter [nm2] 
			&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Avogadro&#8217;s number</li>
			<li>Use the coupling density to calculate the average ligand spacing: 
			<img alt="Equation 2" src="/files/ftp_upload/53400/53400eq2.jpg" /></li>
			<li>Incubate the protein-bead suspension for 2 hr&#160;at 25 &#176;C on a rotating wheel. 
			Note: Some proteins may not be stable at RT. In these cases, the reaction can be carried out at 4 &#176;C O/N.</li>
			<li>Deactivate remaining activated groups on the beads by adding cysteine stock to a final concentration of 50 mM and incubate the suspension for 30 min&#160;at 25 &#176;C on a rotating wheel. Pellet the beads by centrifugation (2 min, 16,000 x g in a microcentrifuge).</li>
			<li>Keep the supernatant for determining the protein concentration and calculation of coupling efficiency (see section 2).</li>
			<li>Wash the bead pellet twice with 1 ml PBS and resuspend in 1 ml fresh PBS to give the final product. 
			Note: The above protocol will typically give 1 ml of coupled protein, at a final concentration of 5 mM protein, which can be used as a 10x stock for subsequent experiments (see section 3).</li>
			<li>To proceed to section 3 of the protocol, work with 100 ml/ml of bead stock, or at a final concentration of 500 nM protein. Note: A good starting point for this procedure will be 2&#215;1012 beads/ml, resulting in an average coupling density of 3&#215;10-4 proteins/nm2 or an average spacing of 57 nm on a bead of 2 mm diameter.&#160;&#160;&#160;&#160;&#160;&#160;</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Thiol-carboxy directional coupling 
	Note: This protocol is suitable for coupling cysteine containing proteins to carboxyl-functionalized polystyrene beads. The carboxyl moiety is first activated using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/ N-hydroxysuccinimide (NHS), amine modified and then cross-linked using Sulfo-SMPB (Figure 2). 
	<img alt="Figure 2" src="/files/ftp_upload/53400/53400fig2.jpg" /> 
	Figure 2. Cross-linking strategy used for directional thiol-carboxyl coupling of proteins to polymer beads. Carboxylated polystyrene beads are first activated with EDC and modified with NHS to form a semi-stable NHS-ester. Subsequent amine-coupling of ethylenediamine results in a free amine group, which reacts with Sulfo-SMPB. Maleimide activated beads can then react with free cysteines to directionally couple proteins to beads. <a href="https://www.jove.com/files/ftp_upload/53400/53400fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
	<ol>
		<li>Preparation of reagents:
		<ol>
			<li>Prepare PBS (100 mM sodium phosphate, 150 mM NaCl, pH 7.0) and autoclave.</li>
			<li>Prepare a 100x stock (0.5 M or 287 mg/ml in PBS,) of TCEP immediately before use.</li>
			<li>Prepare a 10x stock (20 mM, or 4 mg/ml in PBS) of EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) immediately before use.</li>
			<li>Prepare a 10x stock (50 mM, or 6 mg/ml in PBS) of NHS (N-hydroxysuccinimide) immediately prior to use.</li>
			<li>Prepare a 5x stock (10 mM or 4.58 mg/ml in dH2O) of Sulfo-SMPB immediately prior to use.</li>
			<li>Prepare a 10x stock (500 mM or 88 mg/ml in PBS, pH 7.0) of cysteine immediately before use.</li>
		</ol>
		</li>
		<li>Bead activation:
		<ol>
			<li>Mix bead suspension by gently inverting and transfer the required amount of bead suspension (e.g., 12 ml) into a sterile 1.5 ml tube containing 1 ml sterile PBS, pH 7.0.</li>
			<li>Gently pipette up and down to wash the beads and pellet by centrifugation in a microcentrifuge (2 min&#160;at 16,000 x g).</li>
			<li>Carefully remove the supernatant with a pipette and discard.</li>
			<li>Resuspend the bead pellet in 1 ml of fresh sterile PBS and repeat the washing step.</li>
			<li>Resuspend the bead pellet in 0.8 ml of PBS.</li>
			<li>Add 100 ml of 10x EDC stock (2 mM final concentration) and immediately 100 ml of 10x NHS stock solution (5 mM final concentration) to the bead suspension.</li>
			<li>Incubate the bead suspension for 30 min&#160;at 25 &#176;C on a rotating wheel.</li>
			<li>Wash beads once in 1 ml of PBS and resuspend in 0.8 ml fresh sterile PBS.</li>
			<li>Add 200 ml ethylenediamine, and incubate the bead suspension for 1 hr&#160;at 25 &#176;C on a rotating wheel.</li>
			<li>Wash beads once with 1 ml PBS and resuspend the beads in 0.8 ml of fresh PBS.</li>
			<li>Proceed from section 1.1.2.4 (bead activation with Sulfo-SMPB) as described under section 1.1.2 and follow the rest of the protocol described in section 1.1 (Thiol-amine directional coupling). Perform protein preparation and protein coupling steps in a manner identical to those described in section 1.1. 
			Note: The typical coupling efficiency using this protocol is slightly lower (approx. 75%) compared to section 1.1, therefore adjust the initial protein concentration accordingly to achieve the same coupling density.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Determination of Coupling Efficiency
Note: To determine protein concentration, use Bradford Reagent 10 and colorimetric assays as follows:
<ol>
	<li>Gently invert Bradford Reagent to ensure homogeneity of the reagent.</li>
	<li>Using a 10 mg/ml BSA stock solution, prepare protein standards covering concentrations from 0.1 to 1.5 mg/ml BSA in buffer.</li>
	<li>Add 250 ml of Bradford Reagent to wells of a 96-well plate. Prepare enough wells for all samples, protein standards and negative controls (buffer only).</li>
	<li>Add 5 ml of protein sample (protein standards or buffer, for the negative control) to the reagent in the 96-well plate.</li>
	<li>Incubate the plate on an orbital shaker at RT for 10 min.</li>
	<li>Measure absorbance at 595 nm using a plate reader.</li>
	<li>Generate a standard curve of BSA concentration versus A595 nm and use this to determine protein concentrations in initial and supernatant samples.</li>
	<li>Calculate the concentration of coupled protein as follows: 
	<img alt="Equation 3" src="/files/ftp_upload/53400/53400eq3.jpg" /></li>
	<li>Calculate the coupling efficiency as: 
	<img alt="Equation 4" src="/files/ftp_upload/53400/53400eq4.jpg" /></li>
</ol>
3. Use of Bead-coupled Adhesins in Competition Assays
<ol>
	<li>Preparation:
	<ol>
		<li>Seed 1 ml per well of Hela cells at a concentration of 150,000 cells/ml into a 24-well plate the day before the competition assay, to allow cells to reach approximately 80% confluency prior to starting the experiment.</li>
		<li>Set up each experimental condition in triplicate. Include wells for negative controls (no bacteria added during competition assays), positive controls (control beads coupled to fusion-tag only added during competition assays) and lysis controls (for cytotoxicity experiments).</li>
		<li>Inoculate a 5 ml marine LB (MLB) culture with a fresh colony of V. parahaemolyticus and grow O/N at 30 &#176;C, shaking.</li>
		<li>Prepare sufficient bead-coupled MAM, as described in section 1. (Coupling). Allow for 100 ml of 10x bead stock per well.</li>
	</ol>
	</li>
	<li>Competition assay:
	<ol>
		<li>On the day of the competition experiment, measure the OD600 of the bacterial culture.</li>
		<li>Prepare infection media by diluting bacterial cultures into colorless DMEM without additives, pre-warmed to 37 &#176;C, containing 10% v/v bead suspension (either adhesin-coupled beads or control beads), to give an MOI of 10. Prepare 1 ml/well and 10-20% excess volume per sample. Note: For the above mentioned conditions (24-well plate, V. parahaemolyticus, MOI 10), the necessary volume of O/N culture to be added per well (ml/ml) is calculated as 3/OD600 of the culture. For example, 1 ml of infection medium will typically contain 100 ml bead suspension, a few ml of bacterial culture and be made up to 1 ml with colorless, unsupplemented DMEM.</li>
		<li>Remove old medium from wells and wash cultured Hela cells by adding 1 ml of sterile PBS pre-warmed to 37 &#176;C to each well.</li>
		<li>Remove PBS and add 1 ml of infection medium per well. Also set up controls, by adding solutions containing control beads and bacteria (positive control) or adhesin beads and no bacteria (negative controls), or DMEM containing 0.1% Triton X-100 (lysis control, only necessary for cytotoxicity measurements).</li>
		<li>Incubate the plate in a tissue culture incubator at 37 &#176;C for the desired amount of time (e.g. 4 hr&#160;for cytotoxicity measurements or 1 hr&#160;for adhesion measurements). 
		Note: Both bacterial adhesion and cytotoxicity on host cells can be used as read-outs for the efficacy of inhibition. If the time points of cytotoxicity and adhesion measurements coincide, both assays may be performed using samples from the same well, since cytotoxicity is determined using the culture supernatant and attachment assays use samples derived from the remaining cell layer.&#160;</li>
	</ol>
	</li>
	<li>Cytotoxicity measurements:
	<ol>
		<li>At indicated time points (e.g., 4 hr&#160;post infection), remove three times 200 ml from each 24-well and transfer to a 96-well plate.</li>
		<li>Spin 96-well plates at 1,500 x g, 5 min&#160;and transfer 100 ml from each well into a fresh 96-well plate. Add 100 ml of the media used during infection experiments to fresh wells of the 96-well plate in triplicate (these will be used as blanks).</li>
		<li>Carry out the lactate dehydrogenase (LDH) release assay using a LDH cytotoxicity detection kit and following the manufacturer&#8217;s instructions.
		<ol>
			<li>Briefly, calculate the amount of reagent needed in increments of 25 (e.g., if 62 samples are to be measured, make up enough reagent mix for 75 etc.).</li>
			<li>For example, for 100 samples, mix 11.25 ml of reagent A with 250 &#181;l of reagent B. Invert, do not vortex to avoid foaming.</li>
			<li>Put the mixture in a reservoir to be able to pipet with a multi-channel pipette. &#160;Add 100 &#181;l of reagent mix to each sample.</li>
			<li>Incubate plate at RT and read the absorbance at 490 nm on a plate reader at 10, 20, 30 min.</li>
			<li>Analyze the data set for which the absorbance of the lysis control sample is high but still within the linear range of the plate reader (typically, 2-3 absorbance units).</li>
			<li>Express results as % cytotoxicity, using the following formula for conversion: 
			<img alt="Equation 5" src="/files/ftp_upload/53400/53400eq5.jpg" /></li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Measurement of bacterial adhesion:
	<ol>
		<li>At indicated time points (e.g., 1 hr&#160;post infection), remove media from the cell layer.</li>
		<li>Thoroughly wash the cell layer with sterile, pre-warmed PBS (at least 3-4 washes of 1 ml PBS each) to remove any un-attached cells.</li>
		<li>Lyse host cells by adding 1 ml of a sterile 1 % v/v Triton X-100 solution in PBS per well. Incubate the plate at 37 &#176;C for 5 min.</li>
		<li>Pipette each sample up and down several times before transferring the contents of each well to separate 1.5 ml tubes. Prepare 10-fold serial dilutions of samples into sterile PBS (e.g., use 100 ml of sample and 900 ml of PBS).</li>
		<li>Plate 100 ml of each sample on MLB agar and spread using a cell spreader. Optimize which dilutions to plate depending on the bacterial strains and time point. Note: For the described experimental setup, 105 or 106 fold dilutions give a suitable number of CFUs.</li>
		<li>Incubate plates at 37 &#176;C O/N and enumerate bacteria by colony counting.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Lim, J., Stones, D. H., Hawley, C. A., Watson, C. A., Krachler, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multivalent+Adhesion+Molecule+7+clusters+act+as+signaling+platform+for+host+cellular+GTPase+activation+and+facilitate+epithelial+barrier+dysfunction.">Multivalent Adhesion Molecule 7 clusters act as signaling platform for host cellular GTPase activation and facilitate epithelial barrier dysfunction.</a> PLoS Pathog. 10 (9), e1004421 (2014).</li><li>Krachler, A. M., Ham, H., Orth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outer+membrane+adhesion+factor+multivalent+adhesion+molecule+7+initiates+host+cell+binding+during+infection+by+gram-negative+pathogens.">Outer membrane adhesion factor multivalent adhesion molecule 7 initiates host cell binding during infection by gram-negative pathogens.</a> Proc Natl Acad Sci USA. 108 (28), 11614-11619 (2011).</li><li>Krachler, A. M., Orth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterization+of+the+interaction+between+bacterial+adhesin+multivalent+adhesion+molecule+7+(MAM7)+protein+and+its+host+cell+ligands.">Functional characterization of the interaction between bacterial adhesin multivalent adhesion molecule 7 (MAM7) protein and its host cell ligands.</a> J Biol Chem. 286 (45), 38939-38947 (2011).</li><li>Joh, D., Speziale, P., Gurusiddappa, S., Manor, J., Hook, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+specificities+of+the+staphylococcal+and+streptococcal+fibronectin-binding+microbial+surface+components+recognizing+adhesive+matrix+molecules.">Multiple specificities of the staphylococcal and streptococcal fibronectin-binding microbial surface components recognizing adhesive matrix molecules.</a> Eur J Biochem. 258 (2), 897-905 (1998).</li><li>Bingham, R. J., 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=Crystal+structures+of+fibronectin-binding+sites+from+Staphylococcus+aureus+FnBPA+in+complex+with+fibronectin+domains.">Crystal structures of fibronectin-binding sites from Staphylococcus aureus FnBPA in complex with fibronectin domains.</a> Proc Natl Acad Sci U.S.A. 105 (34), 12254-12258 (2008).</li><li>Ensenberger, M. G., 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=Specific+interactions+between+F1+adhesin+of+Streptococcus+pyogenes+and+N-terminal+modules+of+fibronectin.">Specific interactions between F1 adhesin of Streptococcus pyogenes and N-terminal modules of fibronectin.</a> J Biol Chem. 276 (38), 35606-35613 (2001).</li><li>Hawley, C. A., Watson, C. A., Orth, K., Krachler, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+MAM7+peptide-based+inhibitor+of+Staphylococcus+aureus+adhesion+does+not+interfere+with+in+vitro+host+cell+function.">A MAM7 peptide-based inhibitor of Staphylococcus aureus adhesion does not interfere with in vitro host cell function.</a> PLoS One. 8 (11), e81216 (2013).</li><li>Krachler, A. M., Ham, H., Orth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turnabout+is+fair+play:+use+of+the+bacterial+Multivalent+Adhesion+Molecule+7+as+an+antimicrobial+agent.">Turnabout is fair play: use of the bacterial Multivalent Adhesion Molecule 7 as an antimicrobial agent.</a> Virulence. 3 (1), 68-71 (2012).</li><li>Krachler, A. M., Mende, K., Murray, C., Orth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+characterization+of+multivalent+adhesion+molecule+7-based+inhibition+of+multidrug-resistant+bacteria+isolated+from+wounded+military+personnel.">In vitro characterization of multivalent adhesion molecule 7-based inhibition of multidrug-resistant bacteria isolated from wounded military personnel.</a> Virulence. 3 (4), 389-399 (2012).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal Biochem. 72, 248-254 (1976).</li></ol>

The authors have nothing to disclose.

Herein, we describe two protocols, which can be used to couple thiol-containing proteins to amine- or carboxylate-modified polystyrene beads, respectively. Due to ease of the procedure, thiol-amine coupling is preferable, but depending on the desired bead specification (diameter, fluorescence properties), use of amine-functionalized beads may not be possible and we have therefore included a protocol which will convert the carboxylate- into an amine moiety to give the researcher the greatest possible flexibility in choice of scaffold. Although both thiol-amine and thiol-carboxyl coupling work for any cysteine containing protein, directional coupling (i.e., immobilization of the protein per its N-terminus, mimicking surface display) requires a protein without cysteine residues, that is produced as a GST fusion protein, or that contains a single terminal cysteine residue introduced by site directed mutagenesis. If multiple reactive cysteines are contained within the protein, this will lead to random immobilization which may impede protein function. Many bacterial adhesins do not contain cysteines naturally. For others, these may be removed by site directed mutagenesis, although this would require extensive assays to ensure native structure and function are retained in the mutant. For GST fusion proteins, purified GST-tag coupled to beads can be used as a suitable negative control. Using uncoupled beads as a control should be avoided, as these often have a higher tendency to clump together or adhere to cells non-specifically. A simplified version of protocol 1.2., using only EDC, can be used to couple proteins to carboxyl-functionalized polymer beads, however in this case coupling takes place via primary amines in the protein and therefore does not guarantee directional coupling.
TCEP as a reducing agent must not be replaced with other commercially available and commonly used reducing agents, such as dithiothreitol (DTT) or 2-mercaptoethanol (BME), as the thiols contained within them will compete with protein coupling in the thiol-maleimide coupling step. PBS may be replaced with other buffers, but with the following considerations: Buffers may not contain primary amines (so Tris-containing buffers are not suitable). Use of buffers containing very low (&#60; 10mM) salt concentrations leads to bead clumping and should also be avoided. Protein purity is also an important factor to consider during this procedure, and to achieve high quality data, pure proteins should be used. We routinely purify proteins in multiple steps, including at least an affinity purification and gel filtration step, but in some cases ion exchange chromatography is done as a third step. As a result the purity of proteins used for coupling is usually 90% or higher, as judged by SDS-PAGE.
It is recommended to determine protein concentrations both in the initial reaction mixture as well as of the supernatant after reaction completion. This will help to determine the apparent concentration of bead-coupled protein, and thus coupling density. Determination of both values will also allow calculation of the coupling efficiency. This can be taken into account when preparing the initial protein solution in subsequent reactions, to achieve the desired final concentration and coupling density. Bradford reagent is particularly suited for determination of protein concentration before and after the coupling reaction, as none of the substances in the reaction interferes with the dye complex formation at the concentrations used. If lower protein concentrations are to be used, this method may have to be replaced by a more sensitive detection method, however attention has to be paid to the fact it has to be compatible with the substances contained within the coupling reaction. It is also recommended to use freshly prepared reagents and handle powdered reagents with care (e.g., store in a sealed container and use silica beads, to avoid the reagents drawing moisture) since the quality of reagents will influence the coupling efficiency. If the coupling efficiency is lower than expected, possible remedies include increasing the initial bead and protein concentrations. If higher concentrations are being used, the concentration of coupling reagents has to be increased proportionally to ensure sufficient molar excess. Modifying bead/protein concentrations is usually a better step towards optimization rather than increasing reaction times. Since the protocols for bead coupling are lengthy, we commonly prepare a large batch of material. Aliquots of the suspension can be snap-frozen in liquid nitrogen and stored at -20 &#176;C for several months. Thawed aliquots should not be refrozen and should be kept at 4 &#176;C and used within 1-2 days. However, this will vary with the nature and stability of the protein used as should be tested on a small batch initially.
Bead-coupled adhesins can be used for many applications, as discussed below. This protocol described an assay that is commonly used to measure inhibition of bacterial binding and pathogen-mediated cytotoxicity on host cells. The assay is commonly performed to measure the capacity of bead-coupled MAMs to competitively inhibit infection of Hela epithelial cells with the sea-food borne pathogen Vibrio parahaemolyticus, using either a decrease in bacterial attachment to host cells or reduced cytotoxicity as a read-out. In both cases, preparation and competition assays follow the same protocol. Depending on the readout, different strains of V. parahaemolyticus are being used: the cytotoxic strain POR1 is used for cytotoxicity measurements, while the non-cytotoxic strain POR2 is used for measuring bacterial adhesion, since cell death and cell detachment compromises the procedure for quantifying attached bacteria.
Initially, competition experiments were set up as a step-wise protocol, where host cells were first pre-incubated with beads prior to the addition of bacteria. For V. parahaemolyticus and the bead specifications used (2 &#956;m beads coupled to MAM7), both beads and bacteria can be added at the same time without changes in the resulting cytotoxicity. I.e., in this experimental setup, beads outcompete bacteria for host cell binding. Depending on the bacterial species and bead geometry used, there may be good reasons for maintaining the bead adhesion and bacterial infection as two separate steps. For example, to infect cells with non-motile bacteria, plates are commonly centrifuged after addition of the infection media. However, centrifugation of plates containing bead suspensions should be avoided, since this leads to a highly uneven distribution of beads on the cell layer. If smaller particle sizes are being used, beads will take longer to settle on the cell surface, in which case sufficient time should be allowed for bead attachment prior to the infection. When bacterial adhesion is used as a read out, samples should be taken at time points where host cells are not significantly damaged by the infecting strain, as cell detachment and lysis can compromise the quantification of attached bacteria.
Instead of enumerating bacterial adhesion by dilution plating, samples may alternatively be processed for imaging (Figure 5). In this case, tissue culture cells should be seeded onto glass cover slips, rather than directly into wells. Additionally, fluorescent beads and bacteria expressing a fluorescent protein may be used, along with infection-specific host cell markers. For example, competition experiments are commonly imaged using fluorescent red rhodamine-phalloidin to stain the host cells&#8217; actin cytoskeleton and assess morphological changes resulting from infection, together with fluorescent blue beads and fluorescent green (GFP expressing or SYTO18 stained) bacteria.
A range of bead-coupled adhesins, including Staphylococcus aureus FnBPA, Streptococcus pyogenes F1 FUD and Vibrio parahaemolyticus MAM, have been used as biomimetic materials to study adhesion, adhesion inhibition and the contribution of adhesion to pathogen-mediated cytotoxicity 1, 7, 8. One of the advantages of using this approach is the ease of visualization of attachment events, since the polymer beads used as scaffolds are available in a wide range of colors (e.g., blue, fluorescent red, blue, green, orange). Thus, direct protein labeling, which may interfere with function, can be avoided. Additionally, surface coupling mimics the multivalent display of adhesins on the bacterial surface, thus reflecting a more physiologically relevant conformation compared to soluble proteins.
Compared to studies using intact bacteria or bacterial mutants, the bead approach circumvents problems associated with bacterial growth. For example, longer-term (e.g., O/N) studies of bacterial adhesion to host cells using intact bacteria are often compromised by phenomena accompanying bacterial growth &#8211; acidification of the growth medium and nutrient depletion negatively affect host cells, and bacterial replication eventually compromises imaging quality.
More recently, the use of bead-coupled adhesins has been extended to include their use as tools for affinity purifications of host cellular factors involved in signaling processes downstream of bacterial attachment. V. parahaemolyticus MAM7, via binding to phosphatidic acids in the host cell membrane, triggers RhoA activation and actin rearrangements 1, 3. MAM-coupled beads are being used to purify and identify proteins involved in the signaling platforms assembled as a consequence of MAM-host cell binding. Since beads can easily be separated from the supernatant by a short centrifugation step, and the protein of interest is covalently coupled, this is a good method to achieve separation from contaminant proteins and enrich relevant protein complexes, which can be used for downstream applications such as proteomics or Western Blotting.

Dissecting the interactions between bacterial adhesins and host surface receptors at the host-pathogen interface is an essential step towards our understanding of the underlying mechanisms driving bacterial adhesion. Ultimately, this will help us to identify strategies to interfere with these processes during infections. In conducting such studies, we often face a dilemma: Biochemical and biophysical analysis of the molecular mechanisms of adhesin binding requires their separation from other cell wall components, which may interfere with adhesion events. On the other hand, the use of recombinant soluble proteins over-simplifies the adhesion event, by disregarding protein anchoring in the bacterial cell wall and multivalency of binding achieved through bacterial surface display. Equally, from the host cell&#8217;s perspective, encountering and binding to single adhesin molecules does not always have the same impact in terms of plasma membrane organization, membrane fluidity and receptor clustering1 and this, ultimately, makes it difficult to evaluate the impact of adhesion on host cellular signaling and the outcome of bacteria-host interactions. 
Recently a method was devised, whereby recombinant purified adhesins or adhesin fragments are directionally and covalently coupled to polymer particles similar in size to bacteria, thus mimicking bacterial surface display. This approach has been used on a range of different adhesins, from Gram-negative Multivalent Adhesion Molecules (MAMs)2, 3, to Gram-positive adhesins including Staphylococcus aureus fibronectin binding protein (FnBPA)4, 5 and Streptococcus pyogenes F1 6, 7. This method has allowed the dissection of adhesin fragments important for host cell binding, identification of host surface receptors and determination of their behavior on the host cell surface, while taking both binding affinity and avidity into consideration 1, 8. Additionally, this approach has been used to investigate the efficacy of immobilized adhesins and derivatives as inhibitors of host-pathogen interactions 8, 9. It has been demonstrated that surface coupled derivatives of the Vibrio parahaemolyticus Multivalent Adhesion Molecule (MAM) 7 can be used to attenuate a range of bacterial infections in vitro, including those caused by multidrug-resistant pathogens such as Acinetobacter baumannii and methicillin-resistant S. aureus (MRSA) 7, 9. 
Herein, we describe different chemistries which can be used to immobilize adhesins to commercially available functionalized polystyrene particles. Due to the wide array of available surface functionalities, labels and particle sizes, these are a useful scaffold for the production of bacteriomimetic materials to investigate adhesin-host interactions. We further describe methods for the initial characterization of the coupling reaction, and for the calculation of important properties of the resulting materials. Finally, the use of bead-coupled adhesins as competitive inhibitors of in vitro bacterial infections is discussed as an example of their application, as well as the future scope and limitations of this approach.

<table><tbody><tr><td>Reagents</td><td></td><td></td><td></td></tr><tr><td>Tris(2-carboxyethyl)phosphine (TCEP)</td><td>Sigma</td><td>646547</td><td>Danger; corrosive&lt;br/&gt; can be bought as solution or freshly prepared</td></tr><tr><td>Sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (Sulfo-SMPB)</td><td>Fisher</td><td>PN22317</td><td>Danger; toxic</td></tr><tr><td>L-cysteine</td><td>Sigma</td><td>30089</td><td>Danger; toxic</td></tr><tr><td>Latex beads, amine-modified polystyrene, fluorescent blue - aqueous suspension, 2.0&amp;nbsp;&amp;mu;m mean particle size</td><td>Sigma</td><td>L0280</td><td>harmful;&lt;br/&gt; a range of alternative particle sizes and colors is available</td></tr><tr><td>1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)</td><td>Thermo scientific</td><td>22980</td><td></td></tr><tr><td>N-hydroxysuccinimide (NHS)</td><td>Thermo scientific</td><td>24500</td><td></td></tr><tr><td>Carboxylate-modified polystyrene beads</td><td>Sigma</td><td>CLB9</td><td>harmful;&lt;br/&gt; a range of alternative particle sizes and colors is available</td></tr><tr><td>Ethylenediamine</td><td>Sigma</td><td>E26266</td><td>Danger; flammable, corrosive, toxic, sensitizing</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Promega</td><td>W3841</td><td></td></tr><tr><td>Bradford reagent</td><td>Sigma</td><td>B6916</td><td>Danger; corrosive and toxic</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium - high glucose - With 4,500 mg/L glucose and sodium bicarbonate, without&amp;nbsp;L-glutamine, sodium pyruvate, and phenol red, liquid, sterile-filtered, suitable for cell culture</td><td>Sigma</td><td>D1145</td><td>for infection experiments</td></tr><tr><td>DMEM</td><td>Sigma</td><td>D6429</td><td>for maintenance of cultured host cells</td></tr><tr><td>Fetal Bovine Serum, heat inactivated</td><td>Sigma</td><td>F9665</td><td>supplement for tissue culture medium</td></tr><tr><td>Penicillin-Streptomycin -&lt;br/&gt; Solution stabilized, with 10,000 units penicillin and 10&amp;nbsp;mg streptomycin/mL, sterile-filtered, BioReagent, suitable for cell culture</td><td>Sigma</td><td>F9665</td><td>supplement for tissue culture medium</td></tr><tr><td>PBS</td><td>Sigma</td><td>D8537</td><td></td></tr><tr><td>LDH Cytotoxicity detection kit</td><td>Takara</td><td>MK401</td><td></td></tr><tr><td>Plasticware</td><td></td><td></td><td></td></tr><tr><td>reagent reservoirs (25ml)</td><td>SLS</td><td>F267660</td><td></td></tr><tr><td>24-well plates</td><td>Greiner</td><td>662160</td><td></td></tr><tr><td>96-well plates</td><td>Greiner</td><td>655182</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>haemocytometer</td><td></td><td></td><td></td></tr><tr><td>microcentrifuge</td><td></td><td></td><td></td></tr><tr><td>plate reader</td><td></td><td></td><td></td></tr><tr><td>tissue culture facilities</td><td></td><td></td><td></td></tr><tr><td>multichannel pipette</td><td></td><td></td><td></td></tr></tbody></table>

biomimetic materials to characterize bacteria-host interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

The V. parahaemolyticus adhesin MAM7 contains seven tandem mammalian cell entry (MCE) domains involved in recognition of host surface receptors. We used polystyrene beads coupled to recombinant, purified fragments encompassing either all seven tandem MCE domains (MAM7) or only the first MCE domain (MAM1) to test the ability of these materials to compete with V. parahaemolyticus for host cell binding and the resulting efficacy of these materials as adhesion inhibitors. Hela cells were infected with V. parahaemolyticus strain POR1, and cytotoxicity resulting from in vitro infection was evaluated after 4 hr (Figure 3). Treatment of Hela cells with 0.1% Triton X-100 (positive lysis control) resulted in complete cell lysis, uninfected cells displayed very low levels of cytotoxicity. In vitro infection of untreated cells with POR1 resulted in very high levels of cell lysis, and this was inhibited by MAM7-coupled beads but not MAM1-coupled or GST-coupled control beads (Figure 3).
<img alt="Figure 3" src="/files/ftp_upload/53400/53400fig3.jpg" /> 
Figure 3. Characterization of Vibrio parahaemolyticus induced cytotoxicity in epithelial cells during competition assays. Cells were treated with V. parahaemolyticus (V.p), indicated with (+), or no bacteria (-) in the presence of different competing entities (comp.) as indicated. These included either no comp. (-), MAM7 beads (MAM7), MAM1 beads (MAM1) or GST control beads (GST). As controls, cells were treated either with Triton X-100 (triton, lysis control), or left uninfected (uninf). LDH release after 4 hr was measured as described in section 3, and results normalized to Triton controls (100 %) and blanks (0 %) as described above. Results are means &#177; s.e.m from triplicate experiments. <a href="https://www.jove.com/files/ftp_upload/53400/53400fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Enumeration of V. parahaemolyticus attached to either untreated Hela cells, or cells incubated with MAM7-, MAM1-, or GST control beads, revealed that MAM7-beads but not MAM1- or GST- control beads outcompete V. parahaemolyticus for attachment to host cell surface receptors (Figure 4).
<img alt="Figure 4" src="/files/ftp_upload/53400/53400fig4.jpg" /> 
Figure 4. Characterization of bacterial attachment during competition experiments. Hela cells were infected with V. parahaemolyticus POR2 (V.p. +), in the absence (-) or presence of competing entities (comp.) as follows: MAM7 beads (MAM7), MAM1 beads (MAM1) or GST control beads (GST). Bacterial adhesion was measured after 1 hr, as described in section 3. Results are means &#177; s.e.m from triplicate experiments. Means (f.l.t.r. in CFU/ml) are 2.30106, 1.53105, 2.05106, 2.12106. <a href="https://www.jove.com/files/ftp_upload/53400/53400fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Adhesins coupled to fluorophore labeled beads result in the generation of materials that mimic bacterial adhesion to host cells and are powerful tools for cellular imaging (Figure 5). Using MAM7-coupled fluorescent blue beads, we characterized the process of MAM7-mediated attachment to epithelial cells. Attachment of MAM7 to host cells resulted in actin rearrangements and formation of stress fibers, which were co-visualized using rhodamine-phalloidin to stain for F-actin (Figure 5B).
<img alt="Figure 5" src="/files/ftp_upload/53400/53400fig5.jpg" /> 
Figure 5. Preparation and use of fluorophore labeled biomimetic beads for imaging purposes.(A) Suspension of fluorescent blue biomimetic beads (right tube, 10x stock) and buffer control (left tube). (B) Attachment of MAM7-coupled beads to Hela cells results in actin rearrangements and stress fiber formation. Attachment of fluorescent blue beads and resulting actin stress fibers (red, stained with rhodamine-phalloidin) were imaged by microscopy. Bar, 10 &#956;m. Images in panel B were adapted from Lim et al.1 and reproduced under the Creative Commons Attribution license. <a href="https://www.jove.com/files/ftp_upload/53400/53400fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Biomimetic Materials to Characterize Bacteria-host Interactions at JoVE.com

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

biomimetic-materials-to-characterize-bacteria-host-interactions

Research

JoVE Journal

Immunology and Infection

9.3K Views.  University of Birmingham. Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors. 

Video: Biomimetic Materials to Characterize Bacteria-host Interactions

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations&#39; survivability.

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

Biochemistry

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical imaging. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry represents a label-free, in situ imaging modality capable of generating spatial maps for a wide variety of metabolites. While thinly sectioned tissue samples are now routinely analyzed via this technology, imaging mass spectrometry analyses of non-traditional substrates, such as bacterial colonies commonly grown on agar in microbiology research, remain challenging due to the high water content and uneven topography of these samples. This paper demonstrates a sample preparation workflow to allow for imaging mass spectrometry analyses of these sample types. This process is exemplified using bacterial co-culture macrocolonies of two gastrointestinal pathogens: Clostridioides difficile and Enterococcus faecalis. Studying microbial interactions in this well-defined agar environment is also shown to complement tissue studies aimed at understanding microbial metabolic cooperation between these two pathogenic organisms in mouse models of infection. Imaging mass spectrometry analyses of the amino acid metabolites arginine and ornithine are presented as representative data. This method is broadly applicable to other analytes, microbial pathogens or diseases, and tissue types where a spatial measure of cellular or tissue biochemistry is desired.

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

A novel sample preparation method is demonstrated for the analysis of agar-based, bacterial macrocolonies via matrix-assisted laser desorption/ionization imaging mass spectrometry.

Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical ...

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many ...

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time.
Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method ...

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota and the vertebrate gut. The spatial landscape of the gut microbiota is tightly controlled by physical features - intestinal mucus, crypts, and folds - and is affected by host-controlled properties such as pH, oxygen availability, and immune factors. These properties limit the ability of commensal microbes and pathogens alike to colonize the gut stably. At the micron-scale, microbial organization determines the close-range interactions between different microbes as well as the interactions between microbes and their host. These interactions then affect large-scale organ function and host health.
This protocol enables the visualization of the gut microbiota spatial organization from distances between cells to organ-wide scales. The method is based on fixing gut tissues while preserving intestinal structure and mucus properties. The fixed samples are then embedded, sectioned, and stained to highlight specific bacterial species through fluorescence in situ hybridization (FISH). Host features, such as mucus and host cell components, are labeled with fluorescently labeled lectins. Finally, the stained sections are imaged using a confocal microscope utilizing tile-scan imaging at high magnification to bridge the micron to centimeter length scales. This type of imaging can be applied to intestinal sections from animal models and biopsies from human tissues to determine the biogeography of the microbiota in the gut in health and disease.

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

This streamlined protocol details a workflow to detect and image bacteria in complex tissue samples, from fixing the tissue to staining microbes with fluorescent in situ hybridization.

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota ...

Automated, High-Throughput Detection of Bacterial Adherence to Host Cells

Identification of emerging bacterial pathogens is critical for human health and security. Bacterial adherence to host cells is an essential step in bacterial infections and constitutes a hallmark of potential threat. Therefore, examining the adherence of bacteria to host cells can be used as a component of bacterial threat assessment. A standard method for enumerating bacterial adherence to host cells is to co-incubate bacteria with host cells, harvest the adherent bacteria, plate the harvested cells on solid media, and then count the resultant colony forming units (CFU). Alternatively, bacterial adherence to host cells can be evaluated using immunofluorescence microscopy-based approaches. However, conventional strategies for implementing these approaches are time-consuming and inefficient. Here, a recently developed automated fluorescence microscopy-based imaging method is described. When combined with high-throughput image processing and statistical analysis, the method enables rapid quantification of bacteria that adhere to host cells. Two bacterial species, Gram-negative Pseudomonas aeruginosa and Gram-positive Listeria monocytogenes and corresponding negative controls, were tested to demonstrate the protocol. The results show that this approach rapidly and accurately enumerates adherent bacteria and significantly reduces experimental workloads and timelines.

Detection of host-bacterial pathogen interactions based on phenotypic adherence using high-throughput fluorescence labeling imaging along with automated statistical analysis methods enables rapid evaluation of potential bacterial interactions with host cells.

Identification of emerging bacterial pathogens is critical for human health and security. Bacterial adherence to host cells is an essential step in ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

Biology

Organotypic Tissue Model Systems for Investigating Host-Pathogen Interactions In Vitro

Recapitulating the host-pathogen interface at the epithelial or mucosal barrier in vitro remains a challenging prospect for infection biologists. While in-house grown 2D epithelial monolayers lack true representation of the in vivo situation, commercially available tissue models are often overlooked due to their cost and practicality. However, with careful planning, such models provide reproducible platforms for a vast array of different applications. Here, we report the use of epithelial models that can be utilized for a wide variety of experimental purposes to investigate host-pathogen interactions in various ecological niches, such as the oral cavity, skin, and vaginal mucosa. From simple planktonic cells to complex biofilm co-culture, epithelial models are used to assess microbial adherence and invasion, and to evaluate the host response at a transcriptional and/or protein level, with scope for more detailed profiling using different omics approaches. Furthermore, these biological systems can be used as more accurate test beds for evaluating conventional and novel antimicrobial activity in a complex host-pathogen microenvironment in vitro. The protocols described herein document how models are handled upon arrival and prepared in the laboratory for co-culture stimulation with biofilm communities. The methods detail how experimental outputs are achieved from the model systems, including the processing of tissue, the co-culture setup, and data generation. These experiments include host gene expression through single- and multiplex qPCR analyses and inflammatory protein detection using ELISAs. In conclusion, epithelial models provide useful in vitro systems for preclinical investigatory studies into simple or complex host-pathogen interactions.

The present protocol outlines the application of organotypic tissue models for investigating host-pathogen interactions in vitro. Specifically, the model system described uses a multi-layered oral epithelium exposed to microorganisms associated with biofilms (dental plaque) within the context of oral health versus disease.

Recapitulating the host-pathogen interface at the epithelial or mucosal barrier in vitro remains a challenging prospect for infection biologists. While ...

Measuring the Migration and Biofilm Formation of Various Bacteria

As microbes that thrive in the host body primarily have adaptive abilities that facilitate their survival, methods for classifying and identifying their nature would be beneficial in facilitating their characterization. Currently, most studies focus only on one specific characterization method; however, the isolation and identification of microorganisms from the host is a continuous process and usually requires several combinatorial characterization methods. Herein, we describe methods of identifying the microbial biofilm-forming ability, the microbial respiration state, and their chemotaxis behavior. The methods are used to identify five microbes, three of which were isolated from the bone tissue of Sprague-Dawley (SD) rats (Corynebacterium stationis, Staphylococcus cohnii subsp. urealyticus, and Enterococcus faecalis) and two from the American Type Culture Collection (ATCC)-Staphylococcus aureus ATCC 25923&#160;and Enterococcus faecalis V583. The microbes isolated from the SD rat bone tissue include the gram-positive microbes. These microbes have adapted to thrive under stressful and nutrient-limiting environments within the bone matrix. This article aims to provide the readers with the specific know-how of determining the nature and behavior of the isolated microbes within a laboratory setting.

Here, we present a practical method for the isolation and identification of microorganisms within the host. In this way, the physicochemical properties of microorganisms and possible ways of living in the host are clearly described.

As microbes that thrive in the host body primarily have adaptive abilities that facilitate their survival, methods for classifying and identifying their ...

Biomimetic Materials to Characterize Bacteria-host Interactions

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