Immunomodulatory role of LysC in intestinal immunity.
(A) Schematic illustration of the generation of muropeptides. (B) HPLC characterization of LysC-generated muropeptides. Muropeptide solution (20 μl) was characterized by the reversed-phase column. UV detection was performed at 206 nm. (C) Induction of AMPs by oral treatment with muropeptides. Muropeptides (10 μg) were introduced into intestines. qRT-PCR was performed to detect gene expression 6 h later. (D) Induction of AMPs by feeding with rLysC. rLysC (5 μg) was introduced into intestines, AMPs expression was detected 12 h later. (E) Influence of the abundance of intestinal total bacteria by knockdown of three Alfs. The bacterial abundance was determined by detecting 16S rDNA using qPCR 4 d after RNAi. (F) Influence of the abundance of intestinal total bacteria by AlfB1 oral treatment. AlfB1 (2 μg) was administered into intestines, the bacterial abundance was detected 1 d later. (G) Bactericidal activity of AlfB1, revealed by PI staining. P. damselae were treated with 5 μM of AlfB1 for 2 h at 28°C, and labeled with Hoechst (staining all cells) and PI (staining dead cells). The cells were observed under a confocal microscopy or analyzed using flow cytometry. Scale bar = 10 μm. Flow cytometry collected 10,000 events. Statistical analysis was performed by calculating Q2/(Q1+Q2) × 100%. (H) Induction of the nucleus level of FoxO and Relish by muropeptides, revealed by western blotting. Muropeptides (5 μg) were introduced into intestines. The protein level in nucleus was detected with indicated antibodies 6 h later. Histone 3 and β-actin were detected as internal references for nuclear proteins and cytoplasmic proteins, respectively. (I) Induction of the nucleus level of FoxO and Relish by muropeptides, revealed by immunocytochemical assay. Muropeptides (2 μg) were injected into shrimp hemocoel. Hemocytes were collected 6 h later for immunocytochemical analysis, and observed under confocal microscopy. Scale bar = 10 μm. (J) Expression of the immune effectors after FoxO or Relish knockdown and muropeptides challenge. Muropeptides (5 μg) were introduced into intestines at 24 h after dsRNA application. Gene expression was detected another 6 h later. All bar charts show the mean ± SD from three replicates. The images are representative of three repeats. One dot represents one shrimp in the scatter graphs. Statistical analysis was performed using the Students’ t test. *, 0.01 < p < 0.05; ***, p < 0.001.