Figure 1.
CRAMP-deficient mice have a thinner layer of mucus in the colon.
(A and B) Mouse colon tissues from a representative colonic segment taken from 129/SvJ (A, wild-type) and Camp−/− (B) mice, fixed by water-free Methanol-Carnoy's fixative, stained with PAS and visualized by light microscopy. Stars indicate thickness of the inner mucus layer. Scale bar indicates 25 µm. (C) The thickness of the inner stratified layer of colonic mucus in µm. The individual values and means are displayed for each group of mice (N = 6 and 5, respectively). The inner mucus layer in Camp−/− mice was thinner by 32.7% (p<0.01) (D) The total amount of colonic mucus expressed as the amount of Alcian blue bound to mg of colonic tissue. The individual values and medians are shown (N = 4 in each group). The amount of mucus in Camp−/− mice was lower by 43.8% compared with the wild-type animals (p<0.05).
Figure 2.
CRAMP-deficient mice are more easily colonized with E. coli O157:H7.
(A) Bacterial colonization one day after inoculation with E. coli O157:H7 expressed as CFU/g feces. Colonization of cathelicidin-deficient animals was significantly higher, p<0.001. Data show individual values and means from three independent experiments, N = 11 in each group. (B) Dynamics of bacterial colonization up to 12 days after inoculation with E. coli O157:H7 expressed as CFU/g feces. From the 3rd day of the experiment, the degree of colonization of CRAMP-deficient animals decreased and reached the levels observed in wild-type mice. Data show means and SD from three independent experiments, N = 11 mice in each group.
Figure 3.
E. coli O157:H7 penetrate the mucus of CRAMP-deficient mice and form attaching and effacing lesions.
(A–D) (A and C) PAS-stained and (B and D) corresponding immunofluorescence sections from mouse colon of wild-type (A and B) and CRAMP-deficient mice (C and D) inoculated with E. coli O157:H7. (B) In CRAMP-producing wild-type mice, E. coli O157:H7 (green) was localized in the intestinal content and did not penetrate the inner stratified mucus layer (arrow). (C) In CRAMP-deficient mice the inner mucus layer was thinner and less homogenous and (D) E. coli O157:H7 could be seen within the inner mucus layer and or even in close proximity to the intestinal epithelial cells (arrow). Green indicates O157LPS (antibody), red F-actin (phalloidin) and blue cell nuclei (DAPI). The scale bar indicates 50 µm (E and F) Immunofluorescence staining of colonic segments from a CRAMP-deficient mouse infected with E. coli O157:H7 (E) and from a CRAMP-deficient non-infected mouse (F). In CRAMP-deficient mice, attaching and effacing lesions (arrow) were observed with bacteria adhered to intestinal epithelial cells (green) and actin polymerization (red). Green indicates O157LPS (antibody), red F-actin (phalloidin) and blue cell nuclei (DAPI). The scale bar indicates 25 µm.
Figure 4.
CRAMP-deficient mice develop symptomatic disease after inoculation with E. coli O157:H7.
(A) Relative weight of wild-type and CRAMP-deficient (Camp−/−) animals after inoculation with E. coli O157:H7. Weight is expressed in relation to weight at the beginning of the experiment (before fasting) which was set to be 100%. Data show means and SD from three independent experiments, with a total of N = 11 mice in each group. (B) Morbidity of wild-type and Camp−/− mice depicted as a survival curve. Data represent a summary of three independent experiments and 11 mice in each group. Of the knock-out animals (9/11, 82%) gradually developed clinical signs of disease while all wild-type animals remained asymptomatic (p<0.001). (C) Hemoglobin (g/l) and (D) platelet counts (/µl) 14 days after E. coli O157:H7 inoculation or when evident signs of disease were observed. Means and individual values are presented (N = 11 in each group, three independent experiments, p<0.05 and p<0.0001, respectively). (E) Plasma creatinine (mg/dl) from the same mice as in (C) and (D) (p>0.05). (F) Renal cortex from a representative Camp−/− mouse. Desquamation of tubular epithelial cells with typical apoptotic features such as chromatin condensation, cell shrinkage and membrane blebbing (arrows). (G) Renal cortex from a representative infected wild-type mouse showing normal histology. (H) Normal renal cortex from a representative Camp−/− mouse treated with sterile sucrose/NaHCO3 solution instead of bacteria. Sections are stained with hematoxylin and eosin. The scale bar indicates 50 µm.