Fig. 2.

ZG16 alters distal colonic mucus penetration by Gram-positive bacteria in vivo and in vitro. (A) Carnoy-fixed distal colon immunohistochemistry of the mucus layer (anti-MUC2, green) and Gram-positive bacteria (anti-LTA antibody, red) (SI Appendix, Fig. S5). (Scale bars, 50 µm.) (B–F) C57BL/6 WT or Zg16^−/− distal colon tissues were mounted in an imaging chamber; Fluorescent beads were applied apically to visualize the interface between the impenetrable (IM) and penetrable (PM) mucus layers; 10⁸ cfu E. faecalis (Ef) or E. coli (Ec) were stained with BacLight Red then treated with 10 µg rZG16 and applied apically to mucus; 3D z-stacks of the mucus surface were acquired by confocal microscopy. (B) Schematic representation of data acquisition region. (C) Confocal z-stacks of WT mucus surface exposed to untreated Ef/Ec (Left) or Ef/Ec treated with rZG16 (Right); White dashed line indicates IM/PM interface. (D) Distribution of beads and untreated or rZG16-treated Ef (Left) or Ec (Right) along the z-stack z axis at the WT IM/PM interface. Colored dashed lines represent SEM from n = 6 mice; black dashed lines separate IM, interface, and PM regions of the z-stack. (E) Quantification of control and rZG16-treated Ef and Ec from the PM z-stack region indicated by the arrows in D. Error bars represent SEM from n = 6 mice. (F) Confocal z-stacks of Zg16^−/− mucus surface exposed to untreated Ef/Ec (Left) or Ef/Ec treated with rZG16 (Right). White dashed line indicates IM/PM interface. All images are representative of n = 6 mice. Statistical significance calculated with Tukey’s multiple comparison test (ns, not significant; *P < 0.05). (Scale bars, 20 µm.)