We thank our fellow Lee Lab colleagues, who provided valuable insight and expertise that greatly assisted this research. This work was supported by the Young Innovator Award from the National Institute of Health, awarded to S.W. Lee (NIH-1DP2OD008468-01). R.A. Flaherty is supported by the Albertus Magnus Fellowship provided by Dr. Robert C. Boguslaski, and a teaching assistantship through the Eck Institute for Global Health at the University of Notre Dame.

1. Bacterial Culture
<ol>
	<li>Generate isogenic mutant strains to be used for the study of the specific secreted factor of interest21. 
	Note: The GASM1T1 5448 strains utilized for these experiments included WT GAS&#39;, a sagA&#916;cat SLS-deficient mutant (abbreviated in text as &#916;sagA), and a sagA complemented strain (abbreviated in text as &#916;sagA+sagA)21.</li>
	<li>Prepare overnight liquid cultures of bacterial strains of interest.&#160; Grow GAS M1T1 5448 strains in&#160; -10 ml of Todd-Hewitt broth at 37 &#176;C for 16-20 hr prior to infecting human cells.</li>
	<li>Centrifuge overnight bacterial cultures (2,400 rcf for 10 min) and resuspend in fresh bacterial medium. 
	Note: Resuspension in the same volume as the overnight cultures were grown in (5-10 ml) generally produces a desirable initial optical density.</li>
	<li>Normalize bacterial cultures to equal starting concentrations by diluting the re-suspended cultures in additional bacterial medium until the same optical density at 600 nm (OD600) is obtained for all strains to be tested (OD600 of approximately 1.0 is recommended for convenience). 
	Note: In these studies, stationary phase bacterial cultures were used to infect host cells immediately following normalization. However, as some strains of bacteria exit stationary phase nonsynchronously it may be more appropriate to begin host infections with log phase cultures if this is found to be the case for the strains of interest.</li>
	<li>Prior to further analysis, perform a colony counting assay to determine the colony forming units (CFU) per milliliter present in the starting cultures so that the multiplicity of infection (MOI), or ratio of bacteria per host cell, can be determined.
	<ol>
		<li>Prepare 1 ml serial dilutions of bacterial cultures that have been normalized to the desired optical density in phosphate buffered saline (PBS) or appropriate bacterial growth medium (Todd-Hewitt broth was used in these studies). Prepare dilutions ranging from 10-1 to 10-6 to produce at least one set of agar plates with countable colonies (30-300 colonies). Perform all dilutions in triplicate.</li>
		<li>Transfer 100 &#181;l of each serial dilution onto an agar plate containing appropriate medium for the bacterial species being analyzed. 
		Note: Todd-Hewitt agar was used in these studies.</li>
		<li>Use a glass or plastic bacterial spreader to evenly distribute the 100 &#181;l aliquot across the entire surface of the agar plate, and continue spreading until the liquid has been absorbed into the agar. Perform under flame using sterile technique.</li>
		<li>Incubate the agar plates at 37 &#176;C overnight or until countable colonies appear.</li>
		<li>Count the colonies that grow on each plate and calculate the CFU/ml in the original culture by the following formula: number of colonies x inverse of the serial dilution / culture volume plated in milliliters. 
		e.g. (200 colonies x 1/10-5 dilution) / 0.1 ml plated = 2.0 x 108 CFU/ml</li>
	</ol>
	</li>
</ol>
2. Host Cell Culture
<ol>
	<li>Obtain an appropriate host cell line for the desired analyses. 
	Note: HaCaT human epithelial keratinocytes29, a kind gift from V. Nizet, were used for these studies.
	<ol>
		<li>Maintain HaCaT cells in 100 mm culture dishes in Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) with 10% heat inactivated fetal bovine serum (FBS). Incubate at 37 &#176;C with 5% CO2.</li>
	</ol>
	</li>
</ol>
3. Host Cell Infection with Permeable Membrane Insert System
<ol>
	<li>Plate cells in appropriate tissue culture dishes and allow them to grow until they reach 90% confluency. 
	Note: The HaCaT cells used in these studies typically reach confluency within 2-3 days after plating.
	<ol>
		<li>For lysate collection (e.g. signaling analysis and adenosine triphosphate (ATP) determination assays), plate HaCaT cells in 6 well dishes at a seeding density of approximately 3 x 105 cells/well.</li>
		<li>For immunofluorescence imaging experiments, plate cells on sterile glass coverslips within 6 well dishes at a seeding density of approximately 3 x 105 cells/well.</li>
		<li>For ethidium homodimer membrane permeabilization and lactate dehydrogenase (LDH) release assays, plate HaCaT cells in 24 well dishes at a seeding density of approximately 5 x 104 cells/well.</li>
	</ol>
	</li>
	<li>Immediately prior to treatment, wash cells with 1x sterile PBS.</li>
	<li>Apply fresh growth medium to cells. For the conditions described here, apply 2 ml medium per well for 6 well plates or 0.5 ml medium per well for 24 well plates. If pharmacological treatments are being tested, mix with fresh medium and apply during this step. 
	Note: Some assays may require alternative media. For example, as phenol red and high serum concentrations interfere with the LDH release assay, phenol red-free DMEM supplemented with 1% bovine serum albumin (BSA) (w/v), 2 mM L-glutamine, and 1 mM sodium pyruvate was utilized for these experiments.</li>
	<li>After fresh medium has been applied, carefully place a sterile 0.4 &#181;m permeable membrane insert in each well. Perform this step in a laminar flow hood, and use sterile forceps to transfer the permeable membrane insert into each well.</li>
	<li>Apply fresh cell culture medium to the upper chamber of each well according to the manufacturer&#39;s instructions. For the conditions described here, apply 1 ml medium per well for 6 well plates or 0.1 ml medium per well for 24 well plates.</li>
	<li>Apply an appropriate volume of normalized bacterial cultures (see section 1) to the upper chamber of the permeable membrane insert system. Use bacterial medium for uninfected control treatments. For the results described here, add 25 &#181;l of the normalized cultures per chamber for 24 well plates and add 100 &#181;l of the cultures per chamber for 6 well plates. 
	Note: In these studies, wild-type or mutant GAS was applied to host cells at a MOI of 10. MOI was calculated based on the final eukaryotic cell numbers (e.g. 2 x 106 cells/well), such that 10 times as many bacteria were added per host cell at the beginning of the infection period (e.g. 2 x 107 CFU per well). Appropriate MOI will vary with different bacterial species and with the desired follow-up analyses. 
	Note: In addition to using bacterial medium for uninfected controls, other useful controls for certain applications of this technique may include heat-killed bacteria or spent bacterial medium for comparison.</li>
	<li>Incubate infected cells at 37 &#176;C with 5% CO2.</li>
</ol>
4. Sample Collection
<ol>
	<li>To collect cell culture medium, host cell lysates, or glass coverslips with intact cells for follow-up analyses, begin by carefully removing the permeable membrane insert with sterile forceps.</li>
	<li>For Assessment of Secreted Host Protiens:
	<ol>
		<li>Collect the medium from the lower chamber into 1.5 ml tubes. Avoid disturbing the monolayer during collection.</li>
		<li>Centrifuge samples at 14,000 rcf for 10 min at 4 &#176;C to remove cellular debris.</li>
		<li>Remove all but 50 &#181;l of the supernatant to a fresh 1.5 ml tube and store at -20 &#176;C or use immediately.</li>
	</ol>
	</li>
	<li>For Assessment of Host Cell Lystates:
	<ol>
		<li>Gently aspirate the medium above the monolayer of host cells. Avoid disturbing the monolayer, as this may result in sample loss.</li>
		<li>Rinse cells once with 1x PBS.</li>
		<li>Gently aspirate PBS. Immediately apply an appropriate volume of ice-cold lysis buffer to achieve the desired protein concentration, and incubate the samples on ice for 15 min. Apply between 200 &#181;l and 350 &#181;l of lysis buffer per well of each 6 well plate to achieve protein concentrations between 0.5 and 1.5 mg/ml. 
		Note: The lysis buffer used in these studies was composed of deionized water, 1% (v/v) Nonidet P40 Substitute, a phosphatase inhibitor cocktail, and a protease inhibitor cocktail. Specific reagents are listed in the Materials and Equipment section.</li>
		<li>Use a cell scraper to detach the cells from the plate surface of each well and pipette the entire contents of each well into a 1.5 ml tube.</li>
		<li>Centrifuge samples at 14,000 rcf for 20 min at 4 &#730;C.</li>
		<li>To assess soluble lysate components, remove the supernatant to a fresh tube and store at -20 &#730;C or use immediately.</li>
		<li>To assess nuclear or other insoluble lysate components, reserve the pellet and store at -20 &#730;C or use immediately.</li>
	</ol>
	</li>
	<li>For Assessment by Immunofluorescence Imaging:
	<ol>
		<li>Aspirate medium and wash cells in 1x cold PBS.</li>
		<li>Fix cells overnight in 4% paraformaldehyde (PFA) solution in PBS (w/v). 
		Note: In these studies, 1 ml of PFA was added per well of each 6 well plate.</li>
	</ol>
	</li>
</ol>
5. Suggested Applications
<ol>
	<li>Host Signal Transduction Analysis by SDS-Page and Western Blotting
	<ol>
		<li>Collect host cell lysates as described in section 4.3.</li>
		<li>Determine the protein concentration of each sample lysate by bicinchoninic acid (BCA) assay or equivalent using protein standards30.</li>
		<li>Normalize protein concentrations between samples prior to loading and running the samples on a 4-15% polyacrylamide gel or appropriate alternative31.
		<ol>
			<li>Normalize sample concentrations by pipetting the same protein amount from each sample (e.g. 20 &#181;g) into a new 1.5 ml tube and adding variable volumes of lysis buffer (buffer volume = total volume - sample volume added) to each tube to make the total volume (e.g. 50 &#181;l) and final protein concentration equivalent across samples.</li>
		</ol>
		</li>
		<li>Transfer samples to a polyvinylidene fluoride (PVDF) membrane (or equivalent). For the results described here, transfer samples at 25 volts for 2 hr before increasing the voltage to 70 volts for an additional 45 min. Use transfer buffer composed of 200 mM Tris base, 1.5 M glycine and 20% methanol (v/v) in deionized water.</li>
		<li>Block membranes in 5% BSA + 0.1% Tween 20 in Tris buffered saline (TBS). Adjust accordingly based on manufacturer recommendations for the antibody to be used.</li>
		<li>Incubate membranes with primary antibodies overnight at 4 &#176;C. Use at manufacturer recommended dilution.</li>
		<li>Wash membranes for 1.5 hr in TBS+0.1% Tween 20; refresh buffer every 10-15 min.</li>
		<li>Incubate with Horseradish Peroxidase (HRP)-conjugated secondary antibody at a dilution of 1:5,000 for 1.5 hr at room temperature.</li>
		<li>Wash membranes for 1.5 hr in TBS + 0.1% Tween 20; refresh buffer every 10 - 15 min.</li>
		<li>Incubate membranes with chemiluminescence detection reagent prior to developing on film according to the manufacturer&#39;s instructions. Other detection methods may be used as desired.</li>
	</ol>
	</li>
	<li>Host Cell Immunofluorescence Staining and Imaging
	<ol>
		<li>Fix coverslips containing host cells as described in section 4.4.</li>
		<li>Remove PFA solution and wash coverslips containing treated cells two times in PBS, aspirating between washes. 
		Note: PFA is highly toxic and must be disposed of appropriately.</li>
		<li>Block the coverslips for 2 hr at room temperature in PBS with 1% (w/v) normal goat serum, 2% (v/v) Triton X-100, and 0.5% (v/v) Tween 20.</li>
		<li>Wash coverslips with PBS for 30 min, refreshing the buffer every 10 min.</li>
		<li>Incubate the coverslips with primary antibody at a 1:50 ratio (or as recommended by manufacturer) in blocking solution overnight at 4 &#176;C.</li>
		<li>Wash the coverslips for 1.5 hr in PBS, refreshing the buffer every 30 min.</li>
		<li>Incubate the coverslips for 2 hr at room temperature in secondary antibody (Goat anti-rabbit IgG AlexaFluor488 was used for these studies) using a 1:200 ratio of antibody to blocking solution.</li>
		<li>Wash coverslips for 1 hr, refreshing the buffer every 20 min.</li>
		<li>Apply desired stains. 
		Note: These studies utilized DAPI (nuclear stain) and rhodamine-phalloidin (actin stain) at ratios of 1:1,000 in blocking solution.</li>
		<li>Incubate coverslips with nuclear and actin stains for 30 min at room temperature.</li>
		<li>Wash coverslips in PBS for 30 min at room temperature, refreshing the buffer every 10 min.</li>
		<li>Mount the coverslips on glass slides using mounting medium.</li>
		<li>Allow coverslips to set on the slides overnight prior to sealing and imaging. For the results described here, collect images on a fluorescence microscope using a standard 20X objective. Use ImageJ and microscope imaging software to process the captured images.</li>
	</ol>
	</li>
	<li>Ethidium Homodimer Cell Death Assay
	<ol>
		<li>Aspirate medium from each well and wash cells twice with sterile PBS.</li>
		<li>Apply 4 &#181;M ethidium homodimer-1 in PBS.</li>
		<li>Incubate cells at room temperature for 30 min in the dark.</li>
		<li>Determine the level of fluorescence using a plate reader set to 528 nm excitation and 617 nm emission with a cutoff value of 590 nm.</li>
		<li>Add 0.1% (w/v) Saponin to each well following the initial reading and allow the plate to incubate for an additional 20 min at room temperature (with rocking) before reading the plate a second time at the same settings.</li>
		<li>Determine percent membrane permeabilization individually for each well by dividing the initial fluorescence reading (post-treatment) by the second fluorescence reading (post-Saponin).</li>
	</ol>
	</li>
	<li>ATP Determination Assay
	<ol>
		<li>Collect lysates as described in section 4.3 above.</li>
		<li>Normalize protein levels in the lysates via BCA assay or similar30.</li>
		<li>Determine cellular ATP levels using a luminescence-based ATP determination kit or equivalent according to the manufacturer&#39;s instructions.</li>
		<li>Normalize treated values to the corresponding uninfected control samples.</li>
	</ol>
	</li>
	<li>LDH Release Assay
	<ol>
		<li>Following infection, collect supernatants as described in section 4.3.</li>
		<li>Evaluate LDH release using a cytotoxicity detection kit for LDH release according to the manufacturer&#39;s instructions.</li>
	</ol>
	</li>
</ol>

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E., 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=Infection+with+Rhinovirus+Facilitates+Allergen+Penetration+Across+a+Respiratory+Epithelial+Cell+Layer.">Infection with Rhinovirus Facilitates Allergen Penetration Across a Respiratory Epithelial Cell Layer.</a> Int Arch Allergy Immunol. 166 (4), 291-296 (2015).</li><li>Peng, X., Zhang, Q., Zeng, Y., Li, J., Wang, L., Ai, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evodiamine+inhibits+the+migration+and+invasion+of+nasopharyngeal+carcinoma+cells+in+vitro+via+repressing+MMP-2+expression.">Evodiamine inhibits the migration and invasion of nasopharyngeal carcinoma cells in vitro via repressing MMP-2 expression.</a> Cancer Chemother Pharmacol. 76 (6), 1173-1184 (2015).</li><li>Zheng, Y., Guo, 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=FoxM1+transactivates+PTTG1+and+promotes+colorectal+cancer+cell+migration+and+invasion.">FoxM1 transactivates PTTG1 and promotes colorectal cancer cell migration and invasion.</a> BMC Med Genomics. 8, 49 (2015).</li><li>Losa, D., K&#246;hler, T., 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=Airway+Epithelial+Cell+Integrity+Protects+from+Cytotoxicity+of+Pseudomonas+aeruginosa+Quorum-Sensing+Signals.">Airway Epithelial Cell Integrity Protects from Cytotoxicity of Pseudomonas aeruginosa Quorum-Sensing Signals.</a> Am J Respir Cell Mol Biol. 53 (2), 265-275 (2015).</li><li>Cook, K. W., Letley, D. P., 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=CCL20/CCR6-mediated+migration+of+regulatory+T+cells+to+the+Helicobacter+pylori-infected+human+gastric+mucosa.">CCL20/CCR6-mediated migration of regulatory T cells to the Helicobacter pylori-infected human gastric mucosa.</a> Gut. 63 (10), 1550-1559 (2014).</li><li>Boukamp, P., Petrussevska, R. T., Breitkreutz, D., Hornung, J., Markham, A., Fusenig, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+keratinization+in+a+spontaneously+immortalized+aneuploid+human+keratinocyte+cell+line.">Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.</a> J Cell Biol. 106 (3), 761-771 (1988).</li><li>Krieg, R. C., Dong, Y., Schwamborn, K., Knuechel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+quantification+and+its+tolerance+for+different+interfering+reagents+using+the+BCA-method+with+regard+to+2D+SDS+PAGE.">Protein quantification and its tolerance for different interfering reagents using the BCA-method with regard to 2D SDS PAGE.</a> J Biochem Biophys Methods. 65 (1), 13-19 (2005).</li><li>Brunelle, J. L., Green, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-dimensional+SDS-polyacrylamide+gel+electrophoresis+(1D+SDS-PAGE).">One-dimensional SDS-polyacrylamide gel electrophoresis (1D SDS-PAGE).</a> Methods Enzymol. 541, 151-159 (2014).</li></ol>

The authors have nothing to disclose.

Herein is described a method using a permeable membrane insert-based infection system to examine the effects of the bacterial toxin Streptolysin S on human epithelial keratinocytes in an in vitro system. This protocol can be adapted for the study of other secreted bacterial virulence factors as well as alternative host cell types. This recently developed system provides several advantages over experimental methods that utilize purified toxins or filtered bacterial supernatants1-3,8,18-20. The permeable membrane insert-based system provides a constantly maintained dose of the relevant bacterial toxin to host cells as it is produced, which allows maximal toxin activity to be maintained and also increases consistency between experiments. Additionally, this system more closely mimics physiological conditions by allowing the secreted factor to accumulate over time as the infection progresses and by eliminating the need to arbitrarily select specific toxin concentrations to apply to host cells. Furthermore, this system would also provide means for investigators to assess whether a bacterial factor of interest is delivered to host cells in a contact-dependent manner. Contact-dependence is often tested through the generation of isogenic bacterial mutants deficient in adherence or through the use of reagents that prevent adherence. The system described here would provide a simple alternative to complement or replace these traditional approaches.
In addition to the tested applications described in section 5 of the protocol, other applications to which this infection system could be easily adapted include the collection of samples for cytokine arrays and ELISA assays. In both of these cases, a similar protocol to that described for LDH release assays may be followed. Though not shown here, this system has been used to effectively collect host cell samples to be analyzed by flow cytometry. In this application, the permeable membrane insert is removed following the infection period, the cells are washed to remove cell culture medium, and trypsin is applied to allow collection of the cells for analysis. The physical separation of bacteria from host cells is particularly useful for this application because it helps to prevent the formation of large aggregates comprised of adhered bacteria and host cells that could otherwise interfere with accurate cell counting by the flow cytometer.
Though very consistent host responses have been observed when comparing the results of the permeable membrane insert-based infection studies with traditional direct infection studies, some notable differences in the kinetics of these host responses have been observed21. For example, changes in host signaling and membrane-based cytotoxicity take 30-50% longer to occur in the permeable membrane insert-based infection model than corresponding direct infection models. Because the permeable membrane insert-based system requires medium to be applied to the upper and lower compartments of each well, the system developed for the studies described here required a 30% increase in total medium volume per well compared to corresponding direct infection models used previously21. This difference in total medium volume, coupled with the increased distance between bacteria and host cells when direct contact is prohibited, likely increases the time it takes for SLS to diffuse through the medium to reach host cells and elicit the observed effects. Additionally, the permeable membrane insert-based system removes many GAS virulence factors that are likely to contribute to host cell damage; the absence of these additional factors in this system is also likely to contribute to the delay in host cell death and initiation of host signaling events compared to direct infection21. These factors should be considered when designing experiments to assess other secreted bacterial components.
To identify the most meaningful conditions for testing the effects of secreted bacterial factors on host cells, testing a range of time points for each application of the permeable membrane insert-based infection system is recommended. It is important to consider under what conditions the specific virulence factor of interest is optimally produced (e.g. log phase, stationary phase, in response to certain environmental signals, etc.) in order to successfully observe its effects. In experiments focused on evaluating changes in host signaling proteins, it was necessary to select time points that allowed adequate time for SLS to reach host cells and to be produced in sufficient amounts to induce signaling changes 21. At the same time, assessment of changes in host signaling needed to be conducted prior to SLS-induced membrane permeabilization, as this effect complicates the collection of host cell lysates for analysis. Cell death induced by SLS could be optimally observed in both direct and permeable membrane insert-based infection models several hours after the initiation of the reported signaling events21. Furthermore, in selecting time points of interest, it is also important to ensure that the bacteria being studied are unable to penetrate the porous insert membrane during the period of study. The production of certain bacterial components over an extended period may disrupt the integrity of the membrane and allow the passage of bacteria to the lower compartment. To determine whether this is a potential problem in the system of interest, the bacterial strains to be studied can be applied to the upper chamber of the permeable membrane insert-based system at a range of time points. At each time point, the insert can be carefully removed and the medium from the bottom chamber can be collected and utilized in a standard colony counting assay (see section 1.5). If no colonies are formed from the cell culture medium in the lower compartment, the insert membrane may be assumed to be effectively preventing the passage of bacteria at the time point and bacterial load tested.
Another experimental design component that is likely to require optimization for the effective analysis of secreted bacterial factors is the multiplicity of infection (MOI). The MOI refers to the ratio of bacterial cells per host cell, and is therefore influenced by host cell confluency at the time of infection and by the number of bacterial colony forming units (CFU) applied to the cells. In these studies, keratinocyte cells were grown to 90% confluency, which allowed these cells to form a cohesive monolayer with intact tight junctions. Thoughtful consideration of the physiological organization of the host cells to be studied is necessary to select an appropriate confluency for infection experiments. Once an appropriate number of host cells per well is determined, a suitable bacterial CFU can be calculated based off the desired MOI. In the studies described here, GAS was applied to host cells at a MOI of 10. Appropriate MOI will vary with different bacteria and with the desired follow-up analyses. A low beginning MOI is typically more physiologically relevant and allows for the bacterial factor of interest to accumulate slowly enough to capture subtle changes in host cell signaling before dramatic changes in host cell viability are evident. Higher MOIs are used in many studies assessing cytotoxicity, but this effect can also be achieved by beginning with a low MOI and allowing the infection to progress for a longer period of time. It is important to determine an accurate CFU to optical density corollary for all bacterial strains to be tested, as isogenic mutants may have an altered growth rate compared to wild-type bacteria and may therefore require that a higher or lower CFU be added per well to ensure an appropriate comparison of the host response between strains. In cases where the mammalian host cells being studied are capable of killing bacteria or inhibiting their growth or if nonsynchronous growth between bacterial strains is suspected, it may also be useful to perform studies to assess the final bacterial load at the end of the infection period. This could be accomplished by collecting the contents of the permeable insert and performing a colony counting assay similar to that described in section 1.5 of the procedure.
Selecting appropriately sized wells for the desired assay is also important for obtaining optimal results. Permeable membrane inserts are available in a variety of sizes, but the most consistent results for the studies shown here were observed using inserts designed for 24 well and 6 well tissue culture plates. These insert sizes are relatively easy to handle with sterile forceps, and ease of manipulation is critical in preventing unwanted transfer of bacteria from the upper compartment to the lower compartment of the well. For experiments involving the collection of host cell lysates, 6 well dishes provide an appropriate cell number per condition for most analyses and are large enough to accommodate cell scrapers for sample collection. For cytotoxicity assays in which the host cells remain adherent and are labeled with a fluorescent or colorometric dye that can be measured on a plate reader (e.g. ethidium homodimer assay, trypan blue exclusion assay), use of a 24 well plate with the corresponding inserts is recommended. For experiments in which cell culture medium will be collected and analyzed (e.g. LDH release, cytokine studies) either the 24 well or 6 well plates may be used, though the smaller wells typically provide adequate sample volumes for these analyses and minimize use of the required reagents. Overall, the method is highly versatile and may be adapted as needed to prepare samples for numerous follow-up applications.

Understanding the function of bacterial virulence factors in the context of host cell infection is a major focus of bacterial pathogenesis research. Many bacterial pathogens actively secrete toxins and other soluble factors 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 infection1-10. Though methods have been developed to successfully purify and produce many of these important virulence factors for study, some bacterial products have unique structures or extensive post-translational modifications that make them recalcitrant candidates for purification methods and thus cannot be studied in isolation using in vitro systems. For example, Group A Streptococcus, the bacterial pathogen responsible for a myriad of infections ranging from pharyngitis to necrotizing fasciitis and toxic shock syndrome, produces a secreted bacterial toxin known as Streptolysin S (SLS)11-17. This ribosomally produced peptide is encoded by the Streptolysin S associated gene (sag) cluster, and the mature product is estimated to be 2.7 kDa in size14-17. The protoxin, encoded by sagA, is modified post-translationally by several enzymes (SagB, SagC, and SagD) to produce the mature, functional form15-17. The complexity of these post-translational modifications coupled with the toxin&#39;s unusual amino acid sequence have rendered the toxin incompatible with all purification and structure elucidation efforts that have been attempted to date15,17. These challenges have complicated efforts to determine the specific role of this toxin in host pathogenesis.
In cases where the preparation of purified toxins or other secreted factors is either complex or not possible, mechanisms to elucidate the function of these products have traditionally been studied through the preparation of filtered bacterial supernatants, which are then applied to host cells18-20. There are several challenges associated with this technique. First, many of these secreted factors, including SLS, do not maintain maximal or consistent activity when stored and applied to host cells at a later time. Additionally, when supernatants are collected at a single time point and then applied to host cells, it is difficult to determine physiological relevance and to draw direct conclusions about the natural infection process, where secreted factors are allowed to accumulate during the course of infection to a physiologically relevant concentration. This second challenge applies not only to the use of bacterial supernatants, but also to the use of purified toxins in host cell studies1-3,8. To address these issues, a permeable membrane insert-based infection system has been developed to assess the effects of SLS on host cells in a manner that maintains optimal toxin activity and also eliminates the variable of direct contact between bacteria and host cells. In this system, human epithelial cells are grown in a monolayer in the bottom chamber of a two chamber well, and bacteria are introduced into the upper chamber of the same well. A porous membrane (0.4 &#181;m pores) separates the upper and lower chambers, allowing secreted factors to be exchanged between the two chambers but preventing the passage of bacteria. This system has allowed for effective evaluation of host responses that are solely due to sustained secreted bacterial components while eliminating any responses that may occur through direct contact during Group A Streptococcal infection. Though other secreted bacterial factors besides SLS can also pass through the porous membrane, the use of an isogenic mutant panel including wild-type (WT), an SLS-knockout (&#916;sagA) and a sagA complemented strain (&#916;sagA+sagA) allows for precise evaluation of host responses that are strictly SLS-dependent21.
Although similar permeable membrane insert systems have been employed for the study of secreted factors involved in viral infections, cancer biology, and immune cell migration22-26, few studies involving bacterial interactions with host cells have utilized this approach6,27,28. Even studies that have employed such systems to explore interactions between bacteria and host cells have primarily focused on migration of inflammatory cells or bacteria through an epithelial or endothelial monolayer plated on the permeable membrane insert. The permeable membrane insert-based infection system described herein is a simple and effective method that relies on the separation of bacteria and host cells via a porous membrane to assess the effects of the secreted toxin, SLS, on host membrane integrity, cellular viability, cellular signal transduction, and secreted host cell factors. This technique can be adapted to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the role of a specific bacterial factor over the course of an infection.

<table><tbody><tr><td>Todd-Hewitt broth</td><td>Acumedia</td><td>7161A</td><td>Use appropriate broth for bacterial species of interest.</td></tr><tr><td>BioSpectrometer</td><td>Eppendorf</td><td>basic model</td><td>Any UV-Vis spectrophotometer is suitable.&amp;nbsp;</td></tr><tr><td>Petri Dishes</td><td>Fisher Scientific</td><td>FB0875713</td><td>Other brands are suitable.</td></tr><tr><td>Agar</td><td>Sigma</td><td>A1296-1kg</td><td>Other brands are suitable.</td></tr><tr><td>HaCaT human epithelial keratinocytes</td><td></td><td></td><td>Gift from lab of Victor Nizet.</td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>Life Technologies</td><td>11995-073</td><td>Use appropriate media for cell line of interest.</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Biowest</td><td>S162H</td><td>Use appropriate media additives for cell line of interest.</td></tr><tr><td>10 cm tissue culture dishes</td><td>Nunc</td><td>150350</td><td>Other brands are suitable.</td></tr><tr><td>6 well tissue culture dishes</td><td>CytoOne</td><td>cc7682-7506</td><td>Other brands are suitable but need to be compatible with the Corning insert.</td></tr><tr><td>24 well tissue culture dishes</td><td>CytoOne</td><td>cc7682-7524</td><td>Other brands are suitable but need to be compatible with the Corning insert.</td></tr><tr><td>Glass coverslips</td><td>Fisherbrand</td><td>12-541-B</td><td>These must be autoclaved prior to use for cell plating.</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>Phenol Red Free DMEM</td><td>Life Technologies</td><td>31053028</td><td>Phenol Red Free media is only required for the LDH release assay.</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Fisher Scientific</td><td>BP1600-100</td><td></td></tr><tr><td>L-glutamine</td><td>Gibco</td><td>25030149</td><td>Only required to supplement cell culture media for LDH release assay.</td></tr><tr><td>Sodium pyruvate</td><td>Gibco</td><td>BP356100</td><td>Only required to supplement cell culture media for LDH release assay.</td></tr><tr><td>Collagen-coated 0.4 &amp;mu;m Transwell inserts (6 well)</td><td>Corning</td><td>3540</td><td></td></tr><tr><td>Collagen-coated 0.4 &amp;mu;m Transwell inserts (24 well)</td><td>Corning</td><td>3595</td><td></td></tr><tr><td>Forceps</td><td>Fisher</td><td>3120018</td><td>These must be sterilized with ethanol prior to handeling Transwell inserts.</td></tr><tr><td>Cell scrapers</td><td>Fisherbrand</td><td>08-100-241</td><td>These are only required for the collection of cell lysates.</td></tr><tr><td>Paraformaldehyde</td><td>Fisher Scientific</td><td>04042-500</td><td>TOXIC.&amp;nbsp; This is only required for immunofluorescence imaging.</td></tr><tr><td>Bicinchoninic acid (BCA) assay kit</td><td>Pierce</td><td>23227</td><td>Other protein quantification assays may be used.</td></tr><tr><td>Tris-glycine 4 - 15% polyacrylamide gels</td><td>BioRad</td><td>4561083</td><td>Buffer system and percent polyacrylamide should be selected based on proteins of interest.</td></tr><tr><td>Electrophoresis cassette supplies</td><td>BioRad</td><td>1658063</td><td>Only required for Western Blotting.</td></tr><tr><td>Electrophoresis power supply</td><td>BioRad</td><td>1645050</td><td>Only required for Western Blotting.</td></tr><tr><td>Western blot transfer cassette</td><td>BioRad</td><td></td><td>Only required for Western Blotting.</td></tr><tr><td>Polyvinylidene (PVDF) Membrane</td><td>EMD Millipore</td><td>IPVH00010</td><td>Only required for Western Blotting.</td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P1379-500ml</td><td></td></tr><tr><td>Rabbit IgG-HRP secondary antibody</td><td>Santa Cruz Biotechnology</td><td>sc2030</td><td>The secondary antibody should be determined based on the selected method of detection.&amp;nbsp;</td></tr><tr><td>Mouse IgG-HRP secondary antibody</td><td>Santa Cruz Biotechnology</td><td>sc2031</td><td>The secondary antibody should be determined based on the selected method of detection.&amp;nbsp;</td></tr><tr><td>Phospho-p38 (T180 + T182) MAPK antibody</td><td>Cell Signaling</td><td>4511</td><td>Select appropriate primary antibodies based on proteins of interest.</td></tr><tr><td>Total p38 MAPK antibody</td><td>Cell Signaling</td><td>8690</td><td>Select appropriate primary antibodies based on proteins of interest.</td></tr><tr><td>Goat anti-rabbit IgG Alexafluor 488</td><td>Molecular Probes</td><td>A-11034</td><td>Other secondary antibodies may be used for immunofluoresence imaging detection.</td></tr><tr><td>LumiGLO Detection Reagent</td><td>KPL</td><td>54-61-00</td><td>Only required for Western Blotting with detection on film.</td></tr><tr><td>Detection film</td><td>Biodot</td><td>BDB57</td><td>Only required for Western Blotting with detection on film.</td></tr><tr><td>DeltaVision Nikon 90i fluoresence microscope</td><td>Nikon</td><td></td><td>Other fluorescence microscopes are suitable for these analyses.</td></tr><tr><td>Normal goat serum</td><td>Thermo Scientific</td><td>31873</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T9284-500mL</td><td></td></tr><tr><td>DAPI nuclear stain</td><td>Cell Signaling</td><td>4083</td><td>Select stains based on specific immunofluorescence applications of interest.</td></tr><tr><td>Rhodamine-phalloidin actin stain</td><td>Molecular Probes</td><td>R415</td><td>Select stains based on specific immunofluorescence applications of interest.</td></tr><tr><td>Fluoromount-G</td><td>Southern Biotech</td><td>0100-01</td><td>Only required for immunofluorescence imaging.</td></tr><tr><td>Ethidium homodimer 1</td><td>Molecular Probes</td><td>E1169</td><td>Only required for membrane permeabilization cytotoxicity assay.</td></tr><tr><td>Spectramax M5 Microplate Reader</td><td>Molecular Devices</td><td></td><td>Other microplate readers capable of detecting UV-vis, fluorescence and luminescence may be suitable.</td></tr><tr><td>Saponin</td><td>Sigma</td><td>47036-50G-F</td><td>Only required for membrane permeabilization cytotoxicity assay.</td></tr><tr><td>Molecular Probes ATP Determination Kit</td><td>Life Technologies</td><td>A22066</td><td>Other kits are likely to be suitable.</td></tr><tr><td>Cytotoxicity Detection Kit for LDH release</td><td>Roche</td><td>11644793001</td><td>Other kits are likely to be suitable.</td></tr><tr><td>Nonidet P40 Substitute</td><td>Sigma</td><td>74385-1L</td><td>Other suppliers are suitable.</td></tr><tr><td>HALT Phosphatase Inhibitor Cocktail</td><td>Thermo Scientific</td><td>78420B</td><td>Other cocktails are likely to be suitable.</td></tr><tr><td>SIGMAFAST Protease Inhibitor Cocktail Tablets, EDTA-free</td><td>Sigma</td><td>S8830-2TAB</td><td>Other cocktails are likely to be suitable.</td></tr></tbody></table>

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.

The permeable membrane insert-based infection system protocol developed for the study of secreted bacterial factors is detailed in Figure 1. This system relies on the separation of bacteria and host cells via a porous membrane to assess the effects of secreted bacterial factors, in this case Streptolysin S (SLS), on host responses such as host membrane integrity, cellular viability, cellular signal transduction, and secreted host cell factors. Figure 2 provides representative Western Blot data demonstrating that this system may be used to assess changes in the activation of contact-independent host signaling proteins. Specifically, the representative data show significantly enhanced p38 MAPK activation in the presence of SLS-producing GAS strains. This system can also be applied to visualize the effects of secreted bacterial factors on host protein localization by immunofluorescence microscopy (Figure 3). The data show SLS-dependent activation of the key inflammatory mediator Nuclear Factor kappa B (NF&#954;B), which translocates from the cytoplasm to the nucleus upon activation21. The results in Figures 2 and 3 indicate that both SLS-dependent inflammatory signaling responses do not require direct contact between the bacteria and host cells. As previous experiments have already demonstrated SLS-dependent p38 and NF&#954;B activation in direct infection models21, similar levels of p38 or NF&#954;B activation among the three GAS strains in the permeable membrane insert-based system would have indicated that the response required direct contact between the bacteria and host cells. Figure 4 demonstrates that this infection system may be applied to assess toxin-dependent changes in host cytotoxicity via ethidium homodimer and LDH release assays. This is evidenced by the significant increase in both membrane permeabilization and in the release of LDH from host cells exposed to GAS strains containing SLS compared to uninfected cells or cells exposed to the SLS-deficient strain. These changes in cytotoxicity are not immediate, as significant effects are not evident until 12 hr post infection in the case of membrane permeabilization and 16 hr in the case of LDH release. The representative data illustrate the importance of selecting appropriate time points and infection conditions for the evaluation of these host responses. In addition to allowing for the assessment of host signaling changes and cytotoxicity, the permeable membrane insert-based infection system is applicable to the study of metabolic changes such as accurate determination of host ATP levels by preventing bacterial contamination of host cell lysates (Figure 5). These data show a significant loss of keratinocyte ATP in response to GAS infection by 16 hours post infection, with enhanced ATP loss in the presence of SLS. These results are consistent with the observed toxin-dependent increases in host stress-response signaling and cytotoxicity.
<img alt="Figure 1" src="/files/ftp_upload/54406/54406fig1.jpg" /> 
Figure 1: Diagram of Permeable Membrane Insert-Based Infection System Protocol to Assess the Effects of Secreted Bacterial Factors on Host Cells. Human keratinocytes are plated in the bottom compartment and grown to 90% confluence. A collagen coated membrane with 0.4 &#181;m pores separates the upper and lower chambers, and bacteria are added to the upper chamber of the permeable membrane insert system for the desired infection period. Cell culture supernatants and host cells may be collected following the infection period and utilized for a variety of analyses. <a href="https://www.jove.com/files/ftp_upload/54406/54406fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54406/54406fig2.jpg" /> 
Figure 2: The Permeable Membrane Insert-Based Infection System can be Utilized for Host Cell Lysate Analysis by SDS-PAGE and Western Blotting. The representative data demonstrate that SLS enhances activation of the p38 MAPK pathway in infected keratinocytes. (A) HaCaTs were infected with GAS for 7 hr via the permeable membrane insert infection system (at MOI = 10) and lysates were assessed for activation of p38. (B) Densitometry from three independent Western Blots was performed to quantify the relative activation of p38 in response to GAS&#39;infection. Averages from three biological replicates are shown, with error bars representing standard deviation. Relative activation of p38 is represented as phosphorylated/total protein level. Statistical significance was determined compared to uninfected cells. The overall p-value was determined by ANOVA (p = 0.0063). Dunnett&#39;s tests were performed post hoc to compare each condition to the corresponding uninfected control mean. *, p = 0.01-0.05; **, p = 0.001-0.01; ***, p = 0.0001-0.001; ****, p &#60;0.0001. This figure has been modified from Flaherty et al. 201521. <a href="https://www.jove.com/files/ftp_upload/54406/54406fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54406/54406fig3.jpg" /> 
Figure 3: The Permeable Membrane Insert-Based Infection System Allows for the Visualization of Host Signaling Changes by Immunofluorescence Microscopy. The representative data show that Streptolysin S enhances pro-inflammatory signaling through activation of NF&#954;B. HaCaT human keratinocytes were infected with GAS at an MOI of 10 for 8 hr using the permeable membrane insert-based infection system. (A and B) Nuclear localization of NF&#954;B was assessed by immunofluorescence imaging. The percentage of nuclear localized cells was calculated by counting the number of cells in which NF&#954;B had translocated from the cytoplasm to the nucleus for a given field and dividing that number by the total number of cells for the same field. Scale bars indicate 100 &#181;m. (A) The average of three biological replicates are represented for each condition with error bars representing standard deviation. The overall p-value was determined by ANOVA; p &#60;0.0001. Dunnett&#39;s tests were performed to compare each condition to the corresponding uninfected control condition. *, p = 0.01-0.05; **, p = 0.001-0.01; ***, p = 0.0001-0.001; ****, p &#60;0.0001. This figure has been modified from Flaherty et al. 201521. <a href="https://www.jove.com/files/ftp_upload/54406/54406fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54406/54406fig4.jpg" /> 
Figure 4: The Permeable Membrane Insert-Based Infection System Allows for the Determination of Bacterial-Mediated Membrane Permeabilization and Cytotoxicity in the Absence of Direct Contact between Bacteria and Host Cells. The representative data demonstrate that keratinocyte viability decreases in the presence of active SLS toxin. GAS-induced cell death was assessed in HaCaT cells in the presence of WT, SLS-deficient or sagA complemented GAS. Keratinocytes were exposed to GAS using the permeable membrane insert-based infection system for 8-16 hr at an MOI of 10. (A) Viability was assessed by ethidium homodimer assay or (B) LDH release assay. In both panels, 3 replicates are averaged and error bars represent standard deviation. Significance for each time point was determined by ANOVA (A) 8 hr, p = 0.0241; 12 hr, p &#60;0.0001 (B) 8 hr, p = 0.0287; 12 hr, p = 0.1977; 16 hr, p = 0.0031. Dunnett&#39;s tests were performed to compare means from each condition to the wild-type infection for the corresponding time point. *, p = 0.01-0.05; **, p = 0.001-0.01; ***, p = 0.0001-0.001; ****, p &#60;0.0001. This figure has been modified from Flaherty et al. 201521. <a href="https://www.jove.com/files/ftp_upload/54406/54406fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54406/54406fig5.jpg" /> 
Figure 5: The Permeable Membrane Insert-Based Infection System Allows for Accurate Determination of Host ATP Levels by Preventing Bacterial Contamination of Host Cell Lysates. The representative data demonstrate SLS-dependent loss of ATP during Group A Streptococcal infection. HaCaT cells were infected with GAS for 8-16 hr using the permeable membrane insert-based infection system. Technical replicates (n = 3) from one representative biological replicate (2 x 106 cells per sample) were averaged for each condition, with error bars representing standard deviation. The overall p-values were determined by ANOVA (12 hr, p &#60;0.0001; 16 hr, p &#60;0.0001). Dunnett&#39;s tests were performed to compare each condition with wild-type infection for the corresponding time point. *, p = 0.01-0.05; **, p = 0.001-0.01; ***, p = 0.0001-0.001; ****, p &#60;0.0001. This figure has been modified from Flaherty et al. 201521. <a href="https://www.jove.com/files/ftp_upload/54406/54406fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells at JoVE.com

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

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 ...

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JoVE Journal

Immunology and Infection

11.0K Views.  University of Notre Dame.  Hier wordt een methode waarbij een permeabel membraan-insert gebaseerde infectiesysteem de effecten van streptolysine S, een uitgescheiden toxine geproduceerd door Groep A Streptococcus bestuderen op keratinocyten beschreven. Dit systeem kan eenvoudig worden toegepast op de studie van andere uitgescheiden bacteriële eiwitten op verschillende types gastheercel tijdens infectie. 

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

Invasion of Human Cells by a Bacterial Pathogen

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

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

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

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Electroporation of Functional Bacterial Effectors into Mammalian Cells

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

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

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

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 ...

A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The BCSFB functions in separating the cerebrospinal fluid (CSF) from the blood and restricting the molecular exchange to a minimum extent. An in vitro model of the BCSFB is based on cells derived from a human choroid plexus papilloma (HIBCPP). HIBCPP cells display typical barrier functions including formation of tight junctions (TJs), development of a transepithelial electrical resistance (TEER), as well as minor permeabilities for macromolecules. There are several pathogens that can enter the central nervous system (CNS) via the BCSFB and subsequently cause severe disease like meningitis. One of these pathogens is Neisseria meningitidis (N. meningitidis), a human-specific bacterium. Employing the HIBCPP cells in an inverted cell culture filter insert system enables to study interactions of pathogens with cells of the BCSFB from the basolateral cell side, which is relevant in vivo. In this article, we describe seeding and culturing of HIBCPP cells on cell culture inserts. Further, infection of the cells with N. meningitidis along with analysis of invaded and adhered bacteria via double immunofluorescence is demonstrated. As the cells of the CP are also involved in other diseases, including neurodegenerative disorders like Alzheimer`s disease and Multiple Sclerosis, as well as during the brain metastasis of tumor cells, the model system can also be applied in other fields of research. It provides the potential to decipher molecular mechanisms and to identify novel therapeutic targets.

The epithelial cells of the choroid plexus (CP) form the blood-cerebrospinal fluid barrier (BCSFB). An in vitro model of the BCSFB employs human choroid plexus papilloma (HIBCPP) cells. This article describes culturing and basolateral infection of HIBCPP cells using a cell culture filter insert system.

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The ...

Medicine

Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach

The intestinal epithelial barrier separates the host from the microbiota that is normally tolerated or ignored. The breach of this barrier results in the entrance of bacteria or bacteria-derived products into the host, accessing the host circulation and inner organs leading to the uncontrolled inflammation as observed in patients with inflammatory bowel disease (IBD), that are characterized by an increased intestinal epithelial permeability.
To mimic the entrance of bacterial-derived compounds into the host, an endotoxemia model has been adopted in which lipopolysaccharide (LPS), a component of the outer cell wall of Gram-negative bacteria, were injected into mice. In this study, a sublethal dose of LPS was intraperitoneally injected and the mice were subsequently monitored for 8 h using a disease score. Furthermore, the expression levels of the inflammatory cytokines Il6, Il1b, and Tnfa were analyzed in the spleen, liver and colon by qPCR at different time points post LPS injection. This model could be useful for the studies involving investigation of immune responses after the invasion of microorganisms or bacterial-derived products caused by a barrier breach of body surfaces.

Here a protocol to mimic the entrance of bacterial-derived compounds after intestinal barrier breach is presented. A low sublethal dose of lipopolysaccharide was injected systemically into mice, which were monitored for 24 h post-injection. The expression of pro-inflammatory cytokines was determined at several time points in spleen, liver, and colon.

The intestinal epithelial barrier separates the host from the microbiota that is normally tolerated or ignored. The breach of this barrier results in the ...

Analyzing the Permeability of the Blood-Brain Barrier by Microbial Traversal through Microvascular Endothelial Cells

The human blood-brain barrier (BBB) is characterized by a very low permeability for biomolecules in order to protect and regulate the metabolism of the brain. The BBB is mainly formed out of endothelial cells embedded in collagen IV and fibronectin-rich basement membranes. Several pathologies result from dysfunction of the BBB followed by microbial traversal, causing diseases such as meningitis. In order to test the effect of multiple parameters, including different drugs and anesthetics, on the permeability of the BBB we established a novel human cell culture model mimicking the BBB with human brain microvascular endothelial cells. The endothelial cells are grown on collagen IV and fibronectin-coated filter units until confluence and can then be treated with different compounds of interest. In order to demonstrate a microbial traversal, the upper chamber with the apical surface of the endothelial cells is inoculated with bacteria. After an incubation period, samples of the lower chamber are plated on agar plates and the obtained colonies are counted, whereby the number of colonies correlate with the permeability of the BBB. Endogenous cellular factors can be analyzed in this experimental set-up in order to elucidate basic cellular mechanisms of the endothelial cells contributing to the BBB. In addition, this platform allows performing a screen for compounds that might affect the permeability of the endothelial cells. Finally, bacterial traversal can be studied and linked to different pathologies, such as meningitis. It might be possible to extend the model and analyze the pathways of the bacteria through the BBB. In this article, we provide a detailed protocol of the described method to investigate the permeability of the BBB.

The human blood-brain barrier selectively prevents penetration of hydrophilic molecules and pathogens into the brain. Several pathologies, including meningitis and postoperative delirium, are associated with an increased permeability of the blood-brain barrier. Here, we describe an endothelial cell culture model to test the barrier permeability by microbial traversal.

The human blood-brain barrier (BBB) is characterized by a very low permeability for biomolecules in order to protect and regulate the metabolism of the ...

Introduction of Bacterial Effector Proteins into Mammalian Cells by Electroporation

Source: Sontag, R. L. et al., <a href="https://app.jove.com/v/52296">Electroporation of Functional Bacterial Effectors into Mammalian Cells.</a> J. Vis. Exp. (2015)
In this video, we demonstrate the introduction of bacterial effector proteins into mammalian cells by electroporation. The intracellular localization of the bacterial effector proteins is detected by confocal microscopy following staining with specific fluorophore-conjugated antibodies.

Source: Sontag, R. L. et al., Electroporation of Functional Bacterial Effectors into Mammalian Cells. J. Vis. Exp. (2015)
In this video, we demonstrate ...

JoVE Encyclopedia of Experiments

Immunology

Immunopathology

In Vitro Models

A Permeable Membrane Insert-Based Infection System to Study the Impact of Bacterial Toxins

Source: Flaherty, R. A., et al. <a href="https://app.jove.com/v/54406">Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells.</a> J. Vis. Exp. (2016).
This video demonstrates a permeable membrane insert-based infection system to assess the impact of secreted bacterial toxins. By eliminating direct contact between the bacterial pathogen and the host cells using a permeable membrane, this technique allows for studying the effect of toxins secreted by pathogenic bacteria on mammalian host cells.

1. Bacterial Culture

	Generate isogenic mutant strains to be used for the study of the specific secreted factor of interest.
	Note: The GAS (Group A ...

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

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