Figure 3.

C. difficile R20291 chemotaxes toward and utilizes mucin components. (A) Capillary chemotaxis assay with C. difficile R20291 in chemotaxis buffer (10 M potassium phosphate, 0.1 mM EDTA) with or without 100 mM sugars, 1 mg/mL mucus (pig stomach MUC5AC/6, HT29-MTX derived MUC2, LS174T derived MUC2), or 1 mg/mL O-linked glycans isolated from LS174T MUC2. The amount of C. difficile in the capillaries following incubation was assessed by transferring the capillary contents to 96-well plates where fluorescence was read by a plate reader at excitation 428 nm/emission 528 nm. n = 8 wells per sugar, repeated four independent times. *p < 0.05, one-way ANOVA. (B, C) Live cell imaging analysis of ibidi chemotaxis slide using the ibidi “Motility” FIJI plugin. (C) Velocity and (D) distance traveled was measured from traces of individual C. difficile cells. n = 3 replicates, repeated five independent times. *p < 0.05, Student’s t test. (D) C. difficile R20291 was inoculated in the fully defined minimal medium CDMM at an OD_{600 nm} = 0.1. Samples were analyzed for OD_600nm at 1, 3, 6, 9, 12, and 24 h. CDMM was prepared without sugars or with 10 mM sugars (glucose, fucose, galactose, mannose, N-acetylglucosamine (GlcNAc), N-acetylgalactocosamine (GalNAc), or N-acetylneuraminic acid (NANA)). (E) Growth of C. difficile after 24 h in CDMM lacking sugars (0 mM) or containing 1, 10, or 100 mM of glucose or mannose. (F) Levels of AI-2 were determined by measuring V. harveyi luminescnence. V. harveyi was incubated with cell-free supernatant of C. difficile grown for 24 h in CDMM with 10 mM glucose or 10 mM mannose. Control represents cell-free supernatant AB media alone. (G) C. difficile inoculated at OD_600nm = 0.1 in CDMM containing glucose or mannose (1, 10, 100 mM) and incubated for 72 h in a MUC2 (LS174T)-coated 96-well plate. Crystal violet staining of mucin-based biofilms was assessed at A_600nm. (H, I) Vero cells were grown as 2D monolayers and incubated for 4 h with supernatant from 48 h cultures of C. difficile R20291 grown in CDMM with various carbohydrate sources (glucose, fucose, galactose, mannose, GlcNAc, GalNAc, NANA) or LS174T purified MUC2. Cell rounding was visualized by microscopy on a Nikon TiE with a 20× Plan Apo (NA 0.75) differential interference contrast objective, using a SPECTRA X LED light source and ORCA-Flash 4.0 sCMOS camera. Cell diameter (μm) was calculated from representative images of Vero cells following treatment (scale bar = 100 μm) (n = 3 per experiment, repeated two independent experiments). *p < 0.05, one-way ANOVA.