Support for the authors includes Massachusetts General Hospital&#39;s Department of Pediatrics, the Executive Committee on Research Interim Support Funding (ISF) award 2022A009041, the National Institute of Allergy and Infectious Diseases grant R21AI146405, and the National Institute of Diabetes and Digestive and Kidney Diseases grant Nutrition Obesity Research Center at Harvard (NORCH) 2P30DK040561-26. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Protocols for Analyzing Shigella Infection in Colonic Epithelial Cells

The authors declare no conflicts of interest.

This protocol describes a set of three standardized assays to study Shigella adherence, invasion, and intracellular replication of intestinal epithelial cells. Although these methods are merely modified versions of classical gentamicin assays used to study the invasion and intracellular replication of various bacterial pathogens within host cells49,50,51, special considerations must be applied when studying Shigella.
Shigella are facultative anaerobes, which grow optimally at 37 &#176;C in rich medium with aeration43. When performing these assays to interrogate the effects of different environmental signals or metabolites on Shigella infections, it is recommended to grow Shigella in rich media overnight, then subculture into defined or supplemented media to mid-log phase prior to infection. Maximal virulence gene expression occurs during the logarithmic phase of growth52,53. Thus, subculturing overnight Shigella cultures and allowing bacterial growth to exponential phase (OD600 ~ 0.7) are necessary steps to properly assess Shigella virulence. Further, virulence gene expression is temperature-dependent. To ensure specificity for expression only during host infection, virulence genes are induced at host physiological temperatures (37 &#176;C) and strictly repressed at lower temperatures (e.g., 30 &#176;C)54. To ensure expression of virulence genes, bacterial subculturing and infections must be carried out at 37 &#176;C, with proper care to ensure the temperature does not drop below 37 &#176;C. A large, 220 kilobase virulence plasmid, which encodes the molecular machinery required for invasion and epithelial cell infection, is an essential virulence determinant for all Shigella spp5. Repeated passaging and prolonged growth at 37 &#176;C promote virulence plasmid instability, resulting in plasmid loss and rendering Shigella cells avirulent55. To ensure virulence plasmid maintenance and expression of crucial virulence genes, it is recommended to restreak bacteria directly from freezer stocks every two weeks onto fresh TSB + Congo red indicator plates. Congo red agar can be used to differentiate between wild-type virulent (red, CR+) colonies and avirulent (white, CR-) colonies that have lost the virulence plasmid45. If virulence plasmid instability is repeatedly observed, overnight cultures of Shigella can be incubated at 30 &#176;C to prevent virulence plasmid loss, followed by subculturing at 37 &#176;C to promote virulence gene expression43. Finally, if analyses with mutants and/or complemented strains are performed, in which antibiotic markers are present in these bacterial strains, it is recommended to use the appropriate antibiotic selection during the bacterial overnight and subculturing steps.
The use of HT-29 epithelial monolayers has many advantages for studying the molecular mechanisms of Shigella pathogenesis in vitro. Since Shigella is a human-adapted pathogen, small animal models do not accurately reflect the characteristic pathogenesis of shigellosis observed in humans36. Thus, the use of standardized protocols for infection of human-derived cell lines enables the quantitative interrogation of individual steps of bacterial pathogenesis to characterize the molecular interplay between this pathogen and its native host. Historically, HeLa cells were predominantly used to study host-pathogen interactions in vitro25,56,57. However, HeLa cells are an immortalized cervical cancer cell line, which are non-native host cells for enteric bacterial pathogens. Thus, in vitro studies of enteric pathogenesis have shifted to the use of colonic adenocarcinoma cell lines (e.g., HT-29, Caco-2, and T84), which more faithfully recapitulate intestinal epithelial morphology, and can be grown on transwells as epithelial monolayers with differentiated apical and basolateral surfaces58,59,60. Although each cell line has its individual strengths and weaknesses, HT-29 cells are used in the assays described here due to phenotypic similarities to intestinal enterocytes and robust expression of cell surface receptors and pro-inflammatory cytokines58,59,61. However, each of these discussed models are cancer-derived cell lines with aberrant metabolic and growth phenotypes that do not accurately represent the natural physiological state of the colonic epithelium in vivo58. Recent advances in human tissue culture techniques have enabled the cultivation of human intestinal stem cells, obtained from tissue biopsies, into organoids or single two-dimensional cell monolayers that recapitulate the various cell types present in the human gastrointestinal (GI) tract62,63,64. Intestinal organoids can be differentiated to include enterocytes, mucus-producing goblet cells, M cells, and other tissue-specific cell types. Recent studies have validated the use of these organoid models for studying enteric pathogens, including various Shigella spp., Salmonella enterica, and Escherichia coli pathovars62,63,64. Although organoids are more accurate models of the human GI epithelium and can even be cultured in varied nutrient conditions to represent the global population65, their relative complexity requires significant training and technical expertise, resulting in higher costs and longer culturing times in comparison with traditional cancer cell lines58.
This protocol describes medium-throughput methods, whereby two 6-well plates are used for a total of 12 monolayers available for testing. However, experiments can easily be scaled to increase the number of monolayers seeded in order to accommodate additional technical replicates, or testing additional Shigella mutants and environmental signals. Tissue culture plates with more wells (e.g.,&#160;12-well or 24-well plates) can also be used with appropriate adjustments to account for wells with smaller diameters. Under the HT-29 growth conditions outlined in step 3.2, HT-29 titers are sufficient for seeding about six 6-well plates following cultivation from one T75 flask, with enough remaining cells to maintain the HT-29 cell culture. Further, the seeding of HT-29 monolayers and preparation of inoculating bacteria steps are identical between bacterial adherence, invasion, and intracellular replication assays. Thus, assays testing the same Shigella strains or subculturing conditions can easily be performed in parallel to simultaneously examine the role of each experimental condition in various steps of Shigella infection.
Recovery titers for adherent (step 4), invaded (step 5), and intracellular bacteria (step 6) are determined by plating serial dilutions of infected HT-29 cells following lysis, which enables quantitative assessment of the efficiency of each stage of Shigella infection. The centrifugation steps in the invasion and intracellular replication assays, based on classical assays49,50,51, promote bacterial contact with the host cells for immediate invasion and enhance invasion rates. Thus, centrifugation &#34;skips&#34; the adherence step, which was later discovered to be an important aspect of Shigella infection17,18,19,66. It is essential to note that the centrifuge step cannot be performed with polarized epithelial cells seeded on transwells. For the adherence assay, however, adherence is not forced by centrifugation, and no gentamicin is added to the infected HT-29 cells prior to lysis, thus allowing the enumeration of adherent bacterial cells. The volume of tissue culture media is reduced to 1 mL (as opposed to 2 mL for the invasion and intracellular replication assays) to enable more efficient contact of the bacteria with the HT-29 cells. Furthermore, depending on the rate of adherence, some invasion and intracellular replication can occur during the 3 h incubation, but the amount is typically negligible (e.g.,&#160;only 0.05% of the recovered bacterial population following host cell lysis). Nevertheless, to properly account for adherent versus intracellular bacteria during the 3 h incubation, it is recommended to perform parallel assays, where, in addition to the stated protocol, a second plate is incubated with 50 &#181;g/mL gentamicin in 2 mL of DMEM per well for 45 min total (15 min incubation, wash, fresh DMEM + gentamycin for 30 min) to thoroughly lyse extracellular bacteria. Following HT-29 cell lysis as noted in the protocol, the intracellular bacteria can be enumerated as noted above, and will represent the number of bacteria that invaded. This value can then be subtracted from the bacteria enumerated from the plate without gentamicin treatment to determine adherent and invaded bacteria appropriately. Parallel analyses can also be performed to give more insight into the intracellular replication of Shigella inside HT-29 cells after invasion, e.g., using multiple time points to perform intracellular growth curves. Finally, along with enumerating intracellular bacteria after an 18 h gentamicin incubation, the culture supernatants can be collected and analyzed for cytokine secretion of infected HT-29 cells. For instance, IL-8 is a chemokine secreted by epithelial cells that largely functions to recruit PMNs to the site of infection. The amount of IL-8 secreted into the culture media of infected HT-29 cells can be analyzed with an IL-8 ELISA assay67.
Bile salts have proved to be an important virulence signal for Shigella during transit through the human GI system, and can be used to supplement bacterial growth media in these protocols to replicate typical GI conditions. Naturally, bile salts are introduced in the duodenum or upper portion of the small intestine to aid in digestion;&#160;and in the terminal ileum or end of the small intestine, 95% of bile salts are removed and recycled back into circulation for ultimate deposit in the gallbladder68. Bile salts usually have a concentration between 0.2%-2.0% (w/v) in the small intestine and are naturally bactericidal. However, Shigella, along with most enteric bacteria, resist bile salts and use the signals to enhance infection47. Several studies have documented how bile salts exposure, at times in conjunction with other small intestinal signals like glucose, affects Shigella survival and virulence regulation prior to infection. Shigella has been shown to resist bile salts, alter gene expression, and form and disperse a biofilm in conditions that reproduce small intestinal transit17,69. The studies have demonstrated that these changes result in a hypervirulent phenotype, in which adherence and invasion are induced upon subsequent colonic infection by Shigella17,18,19,48,66. Thus, the conditions outlined above document how to subculture Shigella in bile salts to mimic small intestinal transit prior to performing the adherence, invasion, or intracellular replication assays. The specified TSB formulation contains added glucose relative to typical Luria broth (LB)17. Thus, if LB is used, it is important to also supplement the media with glucose (0.5%-2.0% [w/v]) to appropriately consider the glucose signals in the small intestine17. Furthermore, as mentioned above, all Shigella subcultures are washed to remove bile salts and mimic the transition to the colon for infection analyses.
Traditionally, and as highlighted in the results above (Figure 1 and Figure 2), infection assays are valuable to determine the role of a particular gene in Shigella infection. The phenotype of various mutants, however, may not be truly appreciated without proper supplementation of the bacterial culture media. As prior research has demonstrated, bile salts significantly alter S. flexneri gene expression, including genes for central metabolism, transcription factors, sugar transporters, drug resistance, and virulence genes either encoded on the chromosome or virulence plasmid17. These genes provide insight into how Shigella uses bile salts as a signal to alter gene expression and prepare for eventual infection of the colon, and subsequent mutational analyses thus need to be performed in the appropriate supplemented bacterial growth media prior to examining effects on virulence in the adherence, invasion, and intracellular replication assays. As seen above in Figure 1 and Figure 2, the &#916;VF mutation did not affect adherence but did affect invasion and intracellular replication. Since the mutation enhanced invasion and intracellular replication, experiments are currently underway to determine how the gene product regulates infection. The mutant analyses serve as an example of how new understandings in Shigella pathogenesis can be studied, especially in conditions that better replicate the human GI tract. Proper complementation analyses are recommended to validate the phenotypes of various mutants.
In combination, these procedures describe quantitative experiments that will provide important insight into the molecular mechanisms of Shigella adherence to, invasion of, and replication inside HT-29 colonic epithelial cells by best mimicking the natural environment of the human GI tract during infection. Future studies can expand on the mutants and experimental conditions to gain a better understanding of how Shigella prepares for and effectively infects human hosts. Despite decades of research, there is still so much more to discover about Shigella infection.

Diarrheal diseases caused by enteric bacterial pathogens are a significant global health burden. In 2016, diarrheal diseases were responsible for 1.3 million deaths worldwide and were the fourth leading cause of death in children younger than five years of age1,2. The Gram-negative, enteric bacterial pathogen Shigella is the causative agent of shigellosis, a major cause of diarrheal deaths worldwide3. Shigellosis causes significant morbidity and mortality each year in children from lower- and middle-income countries4,5, while infections in high-income countries are linked to daycare center, foodborne, and waterborne outbreaks6,7,8,9. Ineffective vaccine development10 and rising rates of antimicrobial resistance (AMR)11,12 have complicated the management of large-scale Shigella outbreaks. Recent Centers for Disease Control and Prevention data show that nearly 46% of Shigella infections in the United States displayed drug resistance in 202013,14, while the World Health Organization has declared Shigella as an AMR priority pathogen for which new therapies are urgently needed15.
Shigella infections are easily transmitted via the fecal-oral route upon ingestion of contaminated food or water, or through direct human contact. Shigella has evolved to be an efficient, human-adapted pathogen, with an infectious dose of 10-100 bacteria sufficient to cause disease16. During small intestinal transit, Shigella is exposed to environmental signals, such as elevated temperature and bile17. Detection of these signals induces transcriptional changes to express virulence factors that enhance the ability of the bacteria to infect the human colon17,18,19. Shigella does not invade the colonic epithelium from the apical surface, but rather transits across the epithelial layer following uptake into specialized antigen-presenting microfold cells (M cells) within the follicle-associated epithelium20,21,22. Following transcytosis, Shigella cells are phagocytosed by resident macrophages. Shigella rapidly escapes the phagosome and triggers macrophage cell death, resulting in the release of pro-inflammatory cytokines5,23,24. Shigella then invades colonic epithelial cells from the basolateral side, lyses the macropinocytic vacuole, and establishes a replicative niche in the cytoplasm5,25. Pro-inflammatory cytokines, particularly interleukin-8 (IL-8), recruit polymorphonuclear neutrophil leukocytes (PMNs) to the site of infection, which weakens epithelial tight junctions, and enables bacterial infiltration of the epithelial lining to exacerbate basolateral infection5. The PMNs destroy the infected epithelial lining to contain the infection, which results in the characteristic symptoms of bacillary (bloody) dysentery5. Although invasion and intracellular replication mechanisms have been thoroughly characterized, new research is demonstrating important new concepts in Shigella infection, including virulence regulation during gastrointestinal (GI) transit17, adherence19, improved basolateral access through barrier permeability26, and asymptomatic carriage in malnourished children27.
The ability of Shigella spp. to cause diarrheal disease is restricted to humans and non-human primates (NHP)28. Shigella intestinal infection models have been developed for zebrafish29, mice30, guinea pigs31, rabbits21,32,33, and pigs34,35. However, none of these model systems can accurately replicate the disease characteristics observed during human infection36. Although NHP models of shigellosis have been established to study Shigella pathogenesis, these model systems are expensive to implement and require artificially high infectious doses, up to nine orders of magnitude higher than the infectious dose of humans37,38,39,40,41,42. Thus, the remarkable adaptation of Shigella for infection of human hosts necessitates the use of human-derived cell cultures to recreate physiologically relevant models for accurate interrogation of Shigella pathogenesis.
Here, detailed procedures are described to measure the rates of Shigella adherence to, invasion of, and replication within HT-29 colonic epithelial cells. Using these standardized protocols, the molecular mechanisms by which bacterial virulence genes and environmental signals impact each step of Shigella infection can be interrogated to better understand the dynamic host-pathogen interaction relationship.

<table><tbody><tr><td>0.22 &amp;mu;m PES filter</td><td>Millipore-Sigma</td><td>SCGP00525</td><td>Sterile, polyethersulfone filter for sterilizing up to 50 mL media</td></tr><tr><td>14 mL culture tubes</td><td>Corning</td><td>352059</td><td>17 mm x 100 mm polypropylene test tubes with cap</td></tr><tr><td>50 mL conical tubes</td><td>Corning</td><td>430829</td><td>50 mL clear polypropylene conical bottom centrifuge tubes with leak-proof cap</td></tr><tr><td>6-well tissue culture plates</td><td>Corning</td><td>3516</td><td>Plates are treated for optimal cell attachment</td></tr><tr><td>Bile salts</td><td>Sigma-Aldrich</td><td>B8756</td><td>1:1 ratio of cholate to deoxycholate</td></tr><tr><td>Congo red dye</td><td>Sigma-Aldrich</td><td>C6277</td><td>A benzidine-based anionic diazo dye, &gt;85% purity</td></tr><tr><td>Countess cell counting chamber slide</td><td>Invitrogen</td><td>C10283</td><td>To be used with the Countess Automated Cell Counter</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D8418</td><td>A a highly polar organic reagent</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)</td><td>Gibco</td><td>10569-010</td><td>DMEM is supplemented with high glucose, sodium pyruvate, GlutaMAX, and Phenol Red</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma-Aldrich</td><td>F4135</td><td>Heat-inactivated, sterile</td></tr><tr><td>Gentamicin</td><td>Sigma-Aldrich</td><td>G3632</td><td>Stock concentration is 50 mg/mL</td></tr><tr><td>HT-29 cell line</td><td>ATCC</td><td>HTB-38</td><td>Adenocarcinoma cell line; colorectal in origin</td></tr><tr><td>Paraffin film</td><td>Bemis</td><td>PM999</td><td>Laboratory sealing film</td></tr><tr><td>Petri dishes</td><td>Thermo Fisher Scientific</td><td>FB0875713</td><td>100 mm x 15 mm Petri dishes for solid media</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Thermo Fisher Scientific</td><td>10010049</td><td>1x concentration; pH 7.4</td></tr><tr><td>Select agar</td><td>Invitrogen</td><td>30391023</td><td>A mixture of polysaccharides extracted from red seaweed cell walls to make bacterial plating media</td></tr><tr><td>T75 flasks</td><td>Corning</td><td>430641U</td><td>Tissue culture flasks</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td>A common non-ionic surfactant and emulsifier&amp;nbsp;</td></tr><tr><td>Trypan blue stain</td><td>Invitrogen</td><td>T10282</td><td>A dye to detect dead tissue culture cells; only live cells can exclude the dye</td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>25200-056</td><td>Reagent for cell dissociation for cell line maintenance and passaging</td></tr><tr><td>Tryptic Soy Broth (TSB)</td><td>Sigma-Aldrich</td><td>T8907</td><td>Bacterial growth media</td></tr></tbody></table>

epithelial cell infection analyses with shigella

The human-adapted enteric bacterial pathogen Shigella causes millions of infections each year, creates long-term growth effects among pediatric patients, and is a leading cause of diarrheal deaths worldwide. Infection induces watery or bloody diarrhea as a result of the pathogen transiting the gastrointestinal tract and infecting the epithelial cells lining the colon. With staggering increases in antibiotic resistance and the current lack of approved vaccines, standardized research protocols are critical to studying this formidable pathogen. Here, methodologies are presented to examine the molecular pathogenesis of Shigella using in vitro analyses of bacterial adherence, invasion, and intracellular replication in colonic epithelial cells. Prior to infection analyses, the virulence phenotype of Shigella colonies was verified by the uptake of the Congo red dye on agar plates. Supplemented laboratory media can also be considered during bacterial culturing to mimic in vivo conditions. Bacterial cells are then used in a standardized protocol to infect colonic epithelial cells in tissue culture plates at an established multiplicity of infection with adaptations to analyze each stage of infection. For adherence assays, Shigella cells are incubated with reduced media levels to promote bacterial contact with epithelial cells. For both invasion and intracellular replication assays, gentamicin is applied for various time intervals to eliminate extracellular bacteria and enable assessment of invasion and/or the quantification of intracellular replication rates. All infection protocols enumerate adherent, invaded, and/or intracellular bacteria by serially diluting infected epithelial cell lysates and plating bacterial colony forming units relative to infecting titers on Congo red agar plates. Together, these protocols enable independent characterization and comparisons for each stage of Shigella infection of epithelial cells to study this pathogen successfully.

Adherence, invasion, and intracellular replication assays were performed comparing S. flexneri 2457T wild type (WT) to S. flexneri &#916;VF (&#916;VF), a mutant hypothesized to negatively regulate Shigella virulence. Since Shigella uses bile salts as a signal to regulate virulence17,18,47, experiments were performed after bacterial subculture in TSB media as well as TSB supplemented with 0.4% (w/v) bile salts18. Bile salts exposure during the subculture step acts as a pre-treatment to replicate small intestinal transit prior to colonic infection17,18,47. Figure 1 analyzes the&#160;effect of the &#916;VF mutation on the ability of S. flexneri to adhere to HT-29 colonic epithelial cells. Percent adherence is plotted on the y-axis and represents the ratio of recovered bacteria following HT-29 lysis standardized to the number of input bacteria. As expected, both S. flexneri WT and &#916;VF strains had a significant increase in adherence when subcultured with bile salts supplementation in comparison to TSB without bile salts supplementation18. However, there was no difference in adherence to HT-29 cells between WT and &#916;VF mutant strains within each subculture condition. These data indicate that the &#916;VF mutation has no effect on the ability of S. flexneri to adhere to HT-29 epithelial cells with or without the bile salts pre-treatment.
In Figure 2, the effect of the &#916;VF mutation on the ability of S. flexneri to invade (Figure 2A) and replicate (Figure 2B) inside of HT-29 colonic epithelial cells with or without bile salts pre-treatment was analyzed. Percent recovery is plotted on the y-axis and represents the ratio of recovered bacterial cells following HT-29 cell lysis standardized to the number of input bacteria. In Figure 2A, there was an expected significant increase in the ability of WT S. flexneri 2457T to invade HT-29 cells after pre-exposure to bile salts48, while the S. flexneri &#916;VF mutant displayed a smaller increase in invasion following bile salts pre-exposure compared to the WT strain.&#160;The &#916;VF mutant had increased invasion rates of HT-29 cells compared to the WT subcultured in TSB, but had similar invasion rates as WT when subcultured in TSB supplemented with bile salts (Figure 2A). These results suggest that the &#916;VF mutation enhances the ability of S. flexneri to invade HT-29 cells, which lessens the effect of the bile salts pre-exposure even though the invasion ability of the &#916;VF mutant did increase further following bile salts subculture.
Overall, 10-fold more bacteria were recovered following overnight incubation (Figure 2B) compared to the 90 min incubation (Figure 2A), which demonstrates the differences in monitoring intracellular growth versus invasion, respectively. When infected HT-29 cells were incubated for 18 h to allow for intracellular replication of the bacteria (Figure 2B), the impact of the bile salts pre-treatment decreased for both the WT and &#916;VF strains. However, the reduced effect of bile salts pre-treatment during intracellular replication was more dramatic for the &#916;VF mutant. Since the increase in intracellular replication of both strains when pre-exposed to bile salts was smaller than the increase in invasion rates in the same conditions, we hypothesize that bile salts have a greater impact on the early steps in S. flexneri pathogenesis. The &#916;VF mutant displayed an increase in percent recovery from overnight replication inside HT-29 cells compared to WT (Figure 2B) following both subculture conditions. However, the percent recoveries of the &#916;VF mutant were similar regardless of bile salts pre-exposure. These data trends suggest that the &#916;VF mutant replicates more efficiently inside HT-29 cells compared to WT, and that bile salts pre-exposure does not impact the ability of the &#916;VF mutant to replicate intracellularly, as observed for WT (Figure 2B). Since the difference between the mutant and WT strains in the bile salts pre-exposure condition was not observed during the 90 min invasion assay, we hypothesize that the product encoded by the deleted VF gene may also regulate S. flexneri replication inside HT-29 cells. Combined, both analyses demonstrate that the &#916;VF mutant is more virulent relative to WT, which suggests that the VF gene product is a negative regulator of S. flexneri virulence.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66426/66426fig01.jpg" /> 
Figure 1: Bile salts pre-exposure induced adherence of S. flexneri to HT-29 cells. S. flexneri 2457T WT and &#916;VF mutant cells were subcultured in either TSB or TSB supplemented with 0.4%&#160;(w/v) bile salts (TSB+BS) media. The bacteria were then applied to HT-29 cells at a multiplicity of infection (MOI) of 100 and incubated for 3 h to examine adherence. After incubation, infected HT-29 cells were washed and lysed, and serial dilutions of recovered bacteria were plated to enumerate colony-forming units per mL (CFU/mL). The number of adherent bacteria is plotted relative to the input bacteria titers to establish the percent adherence. Data are representative of one biological replicate with three technical replicates (individual dots). Error bars indicate the standard error of the mean (SEM). Statistical significance was determined by a Student&#39;s t-test (*p &#60; 0.05; ***p &#60; 0.001). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66426/66426fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66426/66426fig02.jpg" /> 
Figure 2: Bile salts pre-exposure increased WT S. flexneri invasion and intracellular replication. S. flexneri 2457T WT and &#916;VF mutant cells were subcultured in either TSB or TSB supplemented&#160;with bile salts (TSB+BS) media. The bacteria were then applied to HT-29 cells at an MOI of 100, centrifuged onto the cells, and incubated at 37 &#176;C with 5% CO2 for 45 min. Cells were washed&#160;with PBS, and extracellular bacteria were lysed by the addition of gentamicin to the DMEM to exclusively recover intracellular bacteria. After 90 min (A, bacterial invasion) or 18 h (B,&#160;intracellular replication) incubations, infected HT-29 cells were washed and lysed, and serial dilutions of recovered bacteria were plated to enumerate colony-forming units per mL (CFU/mL). The number of intracellular bacteria is plotted relative to the input bacterial titers to establish the percent recovery. Data are representative of one biological replicate, each with three technical replicates (individual dots). Error bars indicate SEM. Statistical significance was determined by a Student&#39;s t-test (*p &#60; 0.05). Please note the differences in the y-axis scales&#160;between panels (A)&#160;and (B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66426/66426fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Epithelial Cell Infection Analyses with Shigella at JoVE.com

Author Spotlight: Advancements in Understanding and Combatting Shigella Infections

The present protocol describes infection assays to interrogate Shigella adherence, invasion, and intracellular replication using in vitro epithelial cell lines.

Epithelial Cell Infection Analyses with Shigella

The human-adapted enteric bacterial pathogen Shigella causes millions of infections each year, creates long-term growth effects among pediatric patients, ...

epithelial-cell-infection-analyses-with-shigella

Nanyang Technological University

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JoVE Journal

Immunology and Infection

712 Views.  Massachusetts General Hospital. The present protocol describes infection assays to interrogate Shigella adherence, invasion, and intracellular replication using in vitro epithelial cell lines.

Video: Author Spotlight: Advancements in Understanding and Combatting Shigella Infections

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

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 ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

An Imaging Technique to Study the Calcium Response in Bacteria-Infected Cells

Source: Smail, Y., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57728">Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells.</a> J. Vis. Exp. (2018).
This video demonstrates a technique for detecting local and global calcium ion response upon Shigella infection of human epithelial cells. Shigella invades host cells and releases invasion effectors, impacting intracellular calcium levels. The amplitude and frequency of the local and global cytosolic calcium are studied using a fluorescent indicator.

Epithelial Cell Infection Analyses with Shigella

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Application of a Mouse Ligated Peyer’s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Imaging Ca²⁺ Responses During Shigella Infection of Epithelial Cells

A High-throughput Shigella-specific Bactericidal Assay

Technika obrazowania do badania odpowiedzi wapnia w komórkach zakażonych bakteriami