Using a human colonoid-derived monolayer to study bacteriophage translocation

Figures & data

Table 1. Phage characteristics.

Phage	Morpho- logy	Genome material	Phage size (width and length)	Bacterial host	Phage titre (PFU/ml)	Endotoxin level (Unit//ml media)	GenBank Accession no./Sequence
Ec70	Myovirus	163 Kbp DNA	87.505 nm 239.654 nm	MDR E. coli JIE 3454	10^11-10^14	1.8–3.9 x 10^−4	SRA accession: PRJNA764821; SAMN40258554
Kp127	Siphovirus	113.7 Kbp DNA	82.966 nm 374.799 nm	MDR K. pneumonia ATCC 13,883	10^10-10^13	2.3–5.5 x 10^−4	MN434096

Figure 1. Development of colon-derived epithelial monolayer for phage translocation study.

a. Colon crypts (left) were isolated from colon biopsies and grown into 3D colonoids (centre) which were then dissociated into single cells and seeded on transwells to form a colon-derived epithelial monolayer (right). b. Expression of colon epithelial cell markers in expanding and differentiated layers. Markers: LRG5, stem cells; MUC2, Goblet cells; LYZ, Paneth cells; CHGA, endocrine cells; SI, enterocytes. Wilcoxon test, n=6, * p < .05, ** p < .01. c. Optimising transwell-coating substances to allow free diffusion of translocated phages. Black bar scale,200 nm; red bar scale, 50 nm.

Figure 2. Characterisation of colon epithelial monolayer models.

a. Immunofluorescent staining for MUC2, LYZ and CHGA markers for goblet cells, Paneth cells and endocrine cells, respectively, on differentiated (Diff+) and undifferentiated (Diff-) colonoid-derived monolayers and Caco-2 monolayer cultured in 21 days (21 Day) and 5 days (5 Day). b. Haematoxylin and eosin (H&E), Alcian blue and Periodic acid–Schiff (PAS) staining on vertical section of the monolayer models. c. Immunofluorescent staining for Zonula Occludin(ZO-1) on the monolayer models. Scale bar = 20 µm.

Figure 3. Permeability of colon epithelial models and phage translocation across these models.

a. Trans-epithelial electrical resistance (TEER) was measured before phage treatment showing increased resistance in differentiated/mature monolayers. b. Following the phage translocation assay, FITC-dextran was used to measure the degree of permeability of cell monolayers by measuring FITC fluorescence intensity in the basolateral chamber media. c, d. Ec70 phage titre collected from the basolateral chamber at 2 h (c) and 24 h (d) after administration of EC70 (10⁷ PFU) to the transwell. Unpaired t-test, * p< .5, ** p< .01, ***p< .005. Diff-, undifferentiated, Diff+, differentiated.

Figure 4. Monolayer integrity and phage translocation following mucus stimulation and depletion.

Histological staining and immunofluorescent labelling of ZO-1 in Caco-2 monolayers (a, b) and colonoid monolayers (c, d) to assess monolayer composition, mucus production and tight junction expression. Trans-epithelial electrical resistance (TEER) was measured in all treatments, with only EDTA causing a significant reduction in both models (e, g). Phages Ec70 and Kp127 were added to Caco-2 (f) and colonoid monolayers (h), and phage translocation was quantified following stimulation of mucus production (semi-wet), mucus depletion (N-Ac, N-acetylcysteine treatment) and EDTA treatment. Mann-Whitney U test, * p< .5, ** p< .01.

Figure 5. Monolayer integrity and phage translocation following stimulation of intestinal permeability with alcohol, lipid and cytokines.

Histological staining and immunofluorescent labelling of ZO-1 in Caco-2 monolayers (a, b) and colonoid monolayers (c, d) following stimulation for 24 hours with aetiological drivers of intestinal permeability including 0.2% ethanol (EtOH), 0.3mM 2 oleic: 1 palmitic acid (OA:PA) and inflammatory cytokines (10 µg/ml each TNF, IL1β, IL6). TEER was measured in all treatments, indicating no significant change (e, g). Phages Ec70 and Kp127 were added to Caco-2 (f) and colonoid monolayers (h), with phage translocation measured after 2 and 24 hours. Mann-Whitney U test, * p< .5, ** p< .01.