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Immunology and Infection

Epithelial Cell Infection Analyses with Shigella

Published: February 9th, 2024

DOI:

10.3791/66426

1Mucosal Immunology and Biology Research Center, Division of Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital, 2Department of Pediatrics, Harvard Medical School
* These authors contributed equally

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, 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.

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,....

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1. Preparation of reagents and materials

NOTE: All volumes are consistent with an assay using two 6-well plates.

  1. 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.
  2. 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.......

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Adherence, invasion, and intracellular replication assays were performed comparing S. flexneri 2457T wild type (WT) to S. flexneri ΔVF (ΔVF), 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.......

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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.......

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Support for the authors includes Massachusetts General Hospital'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.

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NameCompanyCatalog NumberComments
0.22 μm PES filterMillipore-SigmaSCGP00525Sterile, polyethersulfone filter for sterilizing up to 50 mL media
14 mL culture tubesCorning35205917 mm x 100 mm polypropylene test tubes with cap
50 mL conical tubesCorning43082950 mL clear polypropylene conical bottom centrifuge tubes with leak-proof cap
6-well tissue culture platesCorning3516Plates are treated for optimal cell attachment
Bile saltsSigma-AldrichB87561:1 ratio of cholate to deoxycholate
Congo red dyeSigma-AldrichC6277A benzidine-based anionic diazo dye, >85% purity
Countess cell counting chamber slideInvitrogenC10283To be used with the Countess Automated Cell Counter
Dimethyl sulfoxide (DMSO)Sigma-AldrichD8418A a highly polar organic reagent
Dulbecco’s Modified Eagle Medium (DMEM)Gibco10569-010DMEM is supplemented with high glucose, sodium pyruvate, GlutaMAX, and Phenol Red
Fetal Bovine Serum (FBS)Sigma-AldrichF4135Heat-inactivated, sterile
GentamicinSigma-AldrichG3632Stock concentration is 50 mg/mL
HT-29 cell lineATCCHTB-38Adenocarcinoma cell line; colorectal in origin
Paraffin filmBemisPM999Laboratory sealing film
Petri dishesThermo Fisher ScientificFB0875713100 mm x 15 mm Petri dishes for solid media
Phosphate-buffered saline (PBS)Thermo Fisher Scientific100100491x concentration; pH 7.4
Select agarInvitrogen30391023A mixture of polysaccharides extracted from red seaweed cell walls to make bacterial plating media
T75 flasksCorning430641UTissue culture flasks
Triton X-100Sigma-AldrichT8787A common non-ionic surfactant and emulsifier 
Trypan blue stainInvitrogenT10282A dye to detect dead tissue culture cells; only live cells can exclude the dye
Trypsin-EDTAGibco25200-056Reagent for cell dissociation for cell line maintenance and passaging
Tryptic Soy Broth (TSB)Sigma-AldrichT8907Bacterial growth media

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Tags

Immunology and Infection
Shigella
adherence
invasion
intracellular replication
pathogenesis
virulence factors
epithelial cells
bile salts

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