Epithelial Cell Infection Analyses with Shigella

Summary

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

Abstract

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.

Introduction

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 age^1,2. 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 countries^4,5, while infections in high-income countries are linked to daycare center, foodborne, and waterborne outbreaks^6,7,8,9. 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 2020^13,14, 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 colon^17,18,19. 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 epithelium^20,21,22. 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 cytokines^5,23,24. Shigella then invades colonic epithelial cells from the basolateral side, lyses the macropinocytic vacuole, and establishes a replicative niche in the cytoplasm^5,25. 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, rabbits^21,32,33, and pigs^34,35. 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 humans^{37,38,39,40,41,42}. 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.

Protocol

1. Preparation of reagents and materials NOTE: All volumes are consistent with an assay using two 6-well plates. 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. 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…

Representative Results

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…

Discussion

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 cells^49,50,51, special considerations must be applied when studying Shigella…

Materials

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