Figure 1.
MDX strongly enhances E. coli biofilm formation.
(A) Growth curves of LF82 grown in medium supplemented with the indicated polysaccharide or sugar. (B) Specific biofilm formation of LF82. Average ±SD shown. **p<0.01, ***p<0.001, n.d. = none detected (C) Micrographs of LF82 biofilms from B stained with Congo red to detect exopolysaccharide formation (pink) with bacteria counterstained with carbol fusion (blue).
Table 1.
Bacterial strains.
Figure 2.
MDX promotes biofilm formation of multiple E. coli strains in a process dependent on MDX metabolism.
(A) Specific biofilm formation of a panel of E. coli strains. (B) Micrographs of crystal violet stained biofilms from A. (C) Specific biofilm formation of LF82 in medium supplemented with MDX of different chain lengths. (D) Micrographs of crystal violet stained biofilms from C. Average ±SD shown. *p<0.05, **p<0.01, n.d. = none detected.
Figure 3.
MDX increases biofilm formation via type 1 pili.
(A) Scanning electron micrographs of LF82 biofilms. Arrowheads indicate bacteria-plastic (white) or inter-bacterial (black) adhesins. (B) Assessment of the LF82 fim operon by PCR to determine type 1 pili expression. (C) Specific biofilm formation of LF82 in the presence or absence of 2% mannose. (D) Micrographs of crystal violet stained biofilms from C. (E) Specific biofilm formation of LF82 and isogenic mutant strains. (F) Micrographs of crystal violet stained biofilms from E. Average ±SD shown. *p<0.05, **p<0.01.
Figure 4.
MDX selectively enhances LF82 adhesion to intestinal epithelial cell lines.
(A) Adhesion of LF82 to Caco2 monolayers. (B) Immunofluorescent confocal micrographs of LF82 adhered to Caco2 monolayers. Green = LF82, blue = nuclei (C) Adhesion of LF82 to HT29 monolayers. (D) Immunofluorescent confocal micrographs of LF82 adhered to HT29 monolayers. Green = LF82, blue = nuclei (E) Intracellular LF82 recovered from HT29 monolayers. (F) Immunofluorescent confocal micrographs demonstrating the localization of LF82 (red) on the surface of HT29 cells (green). Nuclei = blue. Average ±SD shown. *p<0.05, **p<0.01 relative to glucose.
Figure 5.
MDX selectively enhances LF82 adhesion to Raw264.7 macrophages.
(A) Total amount of Raw264.7 cell-associated LF82. (B) Intracellular LF82 recovered from Raw264.7 cells. (C) Immunofluorescent confocal micrographs of LF82 (red) infected Raw264.7 cells (green). Nuclei = blue. Average ±SD shown. **p<0.01, ***p<0.001 relative to glucose.
Figure 6.
MDX enhances epithelial cell adhesion in a type 1 pili-dependent manner.
(A) Adhesion of LF82 to HT29 monolayers pre-incubated with 2% mannose. (B) Adhesion of LF82 isogenic mutants to HT29 monolayers. Average ±SD shown. *p<0.05, **p<0.01 relative to glucose.
Figure 7.
MDX-enhanced LF82 adhesion to epithelial cells occurs via a mechanism independent of CEACAM6.
(A) Immunoblots of CEACAM6 expression. (B) Adhesion of LF82 to Caco2 cell lines stably expressing shRNAs. Average ±SD shown. **p<0.01, ***p<0.001 relative to glucose. (C) Immunoblots of CEACAM6 expression. (D) Immunoflurescent confocal micrographs of LF82 adhered to Caco2 cells used in B. Green = LF82, blue = nuclei.
Figure 8.
Bacteria with the malX gene are more prevalent in the mucosa of ileal CD.
(A) Prevalence of the malX gene normalized to total Eubacterial DNA (Eub) amplified from mucosal samples by qPCR. (B) Prevalence of E. coli 16S DNA (E. coli) as measured in A. Mean indicated with bar. *p<0.0175.