A Novel Strategy to Study the Invasive Capability of Adherent-Invasive Escherichia coli by Using Human Primary Organoid-Derived Epithelial Monolayers

Abstract

Over the last decades, Adherent-Invasive Escherichia coli (AIEC) has been linked to the pathogenesis of Crohn's Disease. AIEC's characteristics, as well as its interaction with the gut immune system and its role in intestinal epithelial barrier dysfunction, have been extensively studied. Nevertheless, the currently available techniques to investigate the cross-talk between this pathogen and intestinal epithelial cells (IECs) are based on the infection of immortalized cell lines. Despite their many advantages, cell lines cannot reproduce the conditions in tissues, nor do they reflect interindividual variability or gut location-specific traits. In that sense, the use of human primary cultures, either healthy or diseased, offers a system that can overcome all of these limitations. Here, we developed a new infection model by using freshly isolated human IECs. For the first time, we generated and infected monolayer cultures derived from human colonic organoids to study the mechanisms and effects of AIEC adherence and invasion on primary human epithelial cells. To establish the optimal conditions for AIEC invasion studies in human primary organoid-derived epithelial monolayers, we designed an infection-kinetics study to assess the infection dynamics at different time points, as well as with two multiplicities of infection (MOI). Overall, this method provides a model for the study of host response to AIEC infections, as well as for the understanding of the molecular mechanisms involved in adhesion, invasion and intracellular replication. Therefore, it represents a promising tool for elucidating the cross-talk between AIEC and the intestinal epithelium in healthy and diseased tissues.

Keywords: adherent-invasive E. coli (AIEC); bacterial infection; inflammatory bowel disease (IBD); intestinal epithelial cells (IECs); organoid-derived epithelial monolayers (ODM).

Figure 1

Illustrated experimental workflow of the most critical steps of the d-ODMs-E. coli infection protocol. (A) Mechanical dissociation of EpOCs with the help of a 5 ml syringe with a 21G needle until achievement of single-cells. (B) Single-cell counting and seeding (2x10⁵ cells/well on pre-coated 48-well plates with diluted Matrigel (1:20). Cells were incubated until ODM formation for further differentiation. (C) Characterization of ODMs and d-ODMs by qPCR and immunostaining (only during the protocol set-up). (D) ON growth of E. coli LF82 and K12 strains were grown in liquid LB. (E) Infection of d-ODMs with E. coli strains performed by gently releasing the drop. Infection times were from 4-7 hours. (F) Gentamicin (100 µg/ml) addition for 1 hour to eliminate adherent bacteria. (G) Cell treatment with 1% Triton X-100 to facilitate intracellular bacteria release. The bacterial suspension was then serially diluted and seeded. (H) ON incubation of bacterial dilutions in LB agar plates. (I) After colony counting, the Invasion Index for each strain was determined. This figure was created using BioRender.com.

Figure 2

Organoid-Derived Monolayers (ODMs) characterization. (A) ODMs (left panel) 24 hours after seeding showed a confluence of around 70-80% and d-ODMs (right panel) 48 hours after differentiation, showed 100% confluence. (B) Gene expression analysis of ODMs and d-ODMs (n = 5 for each culture type). AXIN2, MYC, MKI67, TFF3, MUC2 and TJP3 genes were analyzed by qPCR to determine their expression levels in ODM vs. d-ODMs. A paired t-test was performed to examine statistically different expression patterns between the two groups (ODMs/d-ODMs). A P value of <0.05 was considered statistically significant. AXIN2: ** indicates P = 0.0012. MYC: *indicates P = 0.0135. MKI67: *indicates P = 0.0335. TJP3: *indicates P = 0.0365. (C) Protein expression analysis by immunofluorescence. KI67 and MUC2 were analyzed to confirm the proliferation and differentiation status of both ODMs and d-ODMs. E-Cadherin and EpCAM were used as epithelial cell-wall markers. DAPI was used to counterstain the cell nuclei. Scale bars: 25 µm. Images are representative of n = 3 independent experiments performed with samples from two different donors. (D) Box-plot distribution of the fluorescent signal of KI67 and MUC2 proteins in ODMs and d-ODMs, expressed as Mean Intensity. Fluorescence was quantified in 5 different fields per sample. A paired t-test was performed to examine statistically different expression patterns between the two groups (ODMs/d-ODMs). A P value of <0.05 was considered statistically significant. KI67: **indicates P = 0.0013.

Figure 3

Graphic representation of E. coli LF82 and K12 invasion indexes on d-ODMs. INV-I% of both E. coli strains (n = 5 for each represented point in the graph) at MOI 20 (A) and 100 (B) relative to the increasing infection time points. The dashed line represents the established threshold (0,1) over which E. coli strains were considered to be invasive. The error bars correspond to the SEM. (C) Mean, SEM and adjusted p-values obtained by a 2-way ANOVA test to examine statistical significance between LF82 and K12 INV-I% for each infection timepoint. This analysis was followed by a Tukey test correction for multiple testing. A P value of <0.05 was considered statistically significant, and it is highlighted in bold.

Figure 4

E. coli LF82 and K12 invasion of d-ODMs as determined by the gentamicin protection assay. Phalloidin staining was performed to visualize the non-invasive control strain K12 (A) and the invasive LF82 (B) in d-ODMs after 5 hours of infection and 1 hour of gentamicin treatment at MOI 100. Phalloidin marked the eukaryotic actin filaments while DAPI bound to the DNA of both epithelial and bacterial cells. White arrows show bacterial localization inside the IECs. Scale bars: 25 µm. Images are representative of n = 3 independent experiments performed with samples from two different donors.