A major role for proteolytic activity and proteinase-activated receptor-2 in the pathogenesis of infectious colitis

Abstract

Citrobacter rodentium is a bacterial pathogen that causes a murine infectious colitis equivalent to enterohemorrhagic Escherichia coli infection in humans. Colonic luminal fluid from C. rodentium-infected mice, but not from sham-infected mice, contains active serine proteinases that can activate proteinase-activated receptor-2 (PAR2). We have identified granzyme A and murine trypsins to be present in C. rodentium-infected luminal fluid, as determined by mass spectrometry and Western blot analysis. Inflammatory indices (colonic mucosa macroscopic damage score, increased intestinal wall thickness, granulocyte infiltration, and bacterial translocation from the colonic lumen to peritoneal organs) were all increased in C. rodentium-infected mice, compared with sham-infected mice. Soybean trypsin inhibitor-treated wild-type mice and untreated PAR2-deficient (PAR2-/-) mice (compared with their wild-type littermates) both had substantially reduced levels of C. rodentium-induced inflammation. These data point to an important role for both pathogen-induced host serine proteinases and PAR2 in the setting of infectious colitis.

Fig. 1.

C. rodentium-infected luminal fluid exhibits trypsin-like proteinase activity. The rates of cleavage of the substrate Boc-Gln-Ala-Arg-AMC (75 μM) by luminal fluids collected at different time points (3, 7, and 10 days) from sham- and C. rodentium-infected, wild-type (PAR₂^+/+) mice, treated or not with STI, compared to the rate of cleavage by trypsin (4 nM). ^*, Significantly different from sham; Ψ, significantly different from C. rodentium-infected PAR₂^+/+, P < 0.05, n = 8 in each group.

Fig. 2.

Proteinase(s) released upon C. rodentium infection activate PAR₂. (A) Activation of calcium signaling in PAR₂-transfected KNRK cells by sham-infected (n = 12) and C. rodentium-infected (n = 26) luminal fluid, C. rodentium-infected luminal fluid plus STI (n = 3), and sham- and C. rodentium-infected colonic homogenates (n = 4) at 10 days after infection. (B) Desensitization of the C. rodentium-infected luminal fluid calcium signal by prior desensitization of PAR₂. The left side of the tracing shows a continuous recording of calcium-mediated fluorescence (E₅₃₀) of a suspension of rPAR₂-transfected KNRK cells exposed to receptor-desensitizing concentrations of the PAR₂-activating peptide SLIGRL-NH₂ before treatment with C. rodentium-infected luminal fluid (n = 4). The calcium signal caused by a sample of C. rodentium-infected luminal fluid in a cell suspension that had not been previously exposed to SLIGRL-NH₂ is shown on the right side of the tracing (n = 4). (C) There exists a direct correlation between the trypsin-like activity and the calcium signaling in PAR₂-transfected KNRK cells by C. rodentium-infected luminal fluid.

Fig. 3.

Detection of trypsin-like activity and the presence of trypsin and granzyme A in the luminal fluid of C. rodentium-infected mice. (A and B) Fluorescent in-gel assays using the substrate Boc-Gln-Ala-Arg-AMC copolymerized in the SDS/PAGE gel. (C) Labeling using the activity-based probe, Biotin-Pro-Lys-diphenylphosphonate. (D) Western blot analysis of trypsin expression. (E) Western blot analysis of granzyme A expression. All pictures are representative of three to four samples.

Fig. 4.

Indices of inflammation and bacterial translocation. Shown are macroscopic damage score (A), wall thickness (B), myeloperoxydase activity (C), and bacterial translocation (number of colony-forming units when blood is plated) (D) at different time points after sham or C. rodentium (C. rod.) infection in PAR₂-deficient (PAR₂^-/-) or wild-type (PAR₂^+/+) mice treated or not with orally administered STI. Only the 10-day time point is shown for bacterial translocation. ^*, Significantly different from sham; Ψ, significantly different from infected PAR₂^+/+, P < 0.05, n = 8 in each group.