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Inhibition of the immunomodulatory role of LysC by Ctl24.

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posted on 23.11.2022, 19:00 authored by Ping-Ping Liu, Zhe Wei, Zi-Hua Cheng, Xian-Wei Wang

(A) Inhibition of P. damselae-rLysC binding by rCtl24. P. damselae were pre-treated with rCtl24 or control rGST, and then incubated with rLysC. The amount of rLysC bound to bacterial cells was detected by western blotting using anti-6His antibodies. (B) Inhibition of the deposition of rLysC onto P. damselae by Ctl24. FITC-labeled bacteria (green) were pre-treated with rCtl24, and then incubated with rLysC. rLysC deposition was visualized by anti-6His antibodies and Dylight 549 Goat Anti-Mouse IgG (red). Scale bar = 20 μm. (C) Binding of P. damselae PGN by rCtl24. Gradient amount rCtl24 was added into 96-well plates pre-coated by PGN, and the binding of rCtl24 was detected by an ELISA assay. (D-E) Variation of intestinal immunity after feeding with rLysC-generated products when PGN was either or not pre-treated with rCtl24 before rLysC cleavage. P. damselae PGN was pre-treated with rCTl24 or rGST, and then incubated with rLysC. The digesting products were collected and introduced into intestines. Gene expression (D) and the nucleus level of FoxO and Relish (E) were detected another 6 h later. At least five shrimp were used for a sample. (F) Inhibition of the digestion of PGN by rLysC into muropeptides by rCtl24. P. damselae PGN was pre-treated with rCTl24 or rGST, and then processed with rLysC. The digesting products were analyzed by HPLC. All bar charts show the mean ± SD from three replicates. The images are representative of three repeats. Statistical analysis was performed using the Students’ t test. ***, p < 0.001.

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