Journal: mSystems

Article Title: Adaptation of Enterococcus faecalis to intestinal mucus revealed by a human colonic organoid model

doi: 10.1128/msystems.01304-25

Figure Lengend Snippet: Colon epithelial monolayers with histological signatures for studying Enterococcus faecalis mucosal colonization. ( A ) Schematic depicting the experimental setup. Human colon organoids grown in Matrigel were split into single cells and seeded on top of collagen-coated Transwell membranes. Once the epithelial monolayer formed, the cells were differentiated at the air-liquid interface. Transwells were used on day 7 after seeding. ( B ) Immunostaining images of differentiated organoid monolayers with MUC2 protein (yellow), nuclei stained with DAPI (blue), and mucus labeled using Jacalin-biotin and streptavidin-Cy5 (magenta). ( C ) Colony-forming units (CFU/mL) analysis of Ef growth in mucus 6 h after inoculation. Both time points represent the colony-forming units of bacteria present in the Transwell in four independent biological replicates. The horizontal black lines mark the mean. ( D ) Maximum intensity projection of Jacalin-labeled mucus (magenta) and Ef WT expressing pDasherGFP (green). ( E ) Ef WT expressing pDasherGFP (green) in the same z-plane as the Jacalin-labeled mucus (magenta) and does not colocalize with the colonoid epithelium labeled with CellMask-DeepRed (red). ( F ) Ef colony volume calculated from confocal images represented in panel C . A minimum of 148 clusters was quantified from each image of four independent biological replicates.

Article Snippet: Streptavidin-Cy5 , Vector Laboratories , SA-1500-1.

Techniques: Immunostaining, Staining, Labeling, Bacteria, Expressing

Journal: mSystems

Article Title: Adaptation of Enterococcus faecalis to intestinal mucus revealed by a human colonic organoid model

doi: 10.1128/msystems.01304-25

Figure Lengend Snippet: Glycosyltransferase BgsB in E. faecalis is essential for growth in colonic mucus. ( A ) Table of log2FC values calculated from the Tn-seq experiment for the glycosyltransferase bgsB and a biofilm-forming factor bph . ( B ) Representative images of Ef colonies in colonic mucus at 9 h of growth from three biological replicates. Mucus was labeled using Jacalin-biotin and streptavidin-Cy5. Wild-type Ef fluorescently expressed pDasher-GFP, and bgsB and bph mutants expressed tdTomato. ( C ) Colony-forming unit (CFU/mL) quantification of the deletion mutants grown in mucus compared with the wild-type Ef after 6 h of growth. Each dot is a biological replicate (at least three independent biological replicates for each mutant), and the horizontal black lines mark the mean. ( D ) Competition between WT (green) and mutant samples (orange) with over three biological replicates, where the WT vs bgsB::Tn is marked with a filled circle and WT vs Δ bph with an unfilled triangle. Volume ratio is calculated as a fraction of the mutant signal occupying the total (mutant + WT) volume in the image. Volume expansion is calculated as a ratio to the initial volume in the respective channel. Mean and standard deviation are shown. ( E ) The same mixed samples were grown in mucus in a flow chip shown in , under the flow of 5 µL/min of minimal medium ( n = 3). Arrows indicate the same place at the start and the end of the time-lapse. Statistics in panel C were calculated using one-way ANOVA with post hoc Dunnett’s multiple comparison test (*** P < 0.001).

Article Snippet: Streptavidin-Cy5 , Vector Laboratories , SA-1500-1.

Techniques: Labeling, Mutagenesis, Standard Deviation, Comparison