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.

1. Preparation of reagents and materials
NOTE: All volumes are consistent with an assay using two 6-well plates.
<ol>
	<li>TSB medium: Add 0.5 L of deionized (DI) water to 15 g of Tryptic Soy Broth (TSB, see Table of Materials) medium and autoclave. Store at room temperature.</li>
	<li>Bile salts medium (TSB + BS): To prepare TSB containing 0.4% (w/v) bile salts, resuspend 0.06 g of bile salts (BS, see Table of Materials) in 15 mL of autocla...

Protocols for Analyzing Shigella Infection in Colonic Epithelial Cells

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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#956;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, &#62;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&#8217;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&#160;</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.

Epithelial Cell Infection Analyses with Shigella

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 w...

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.

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