Inhibition of the antimicrobial activity of AlfB1 by Ctl24.
(A) Inhibition of bactericidal activity of AlfB1 by Ctl24. rCtl24-pretreated P. damselae were treated with AlfB1 (5 μM), labeled with Hoechst (staining all cells) and PI (staining dead cells), and observed using confocal microscopy or analyzed by flow cytometry. Scale bar = 10 μm. Flow cytometry collected 10,000 events. Statistical analysis was performed by calculating Q2/(Q1+Q2) × 100%. Data are representative of three repeats. (B) Inhibition of the antimicrobial activity of AlfB1 in vivo by rCtl24. Shrimp were treated orally with indicated proteins (2 μg). The abundance of intestine total bacteria was determined by detecting 16S rDNA using qPCR 3 d later. One dot represents three shrimp intestines. Data are representative of two repeats. ***, p < 0.001. (C) Inhibition of the deposition of AlfB1 onto P. damselae by Ctl24. FITC-labeled bacteria (green) were pre-treated with rCtl24 in the presence or absence of LPS, and then incubated with AlfB1-6His. AlfB1 deposition was visualized by anti-6His antibodies and Dylight 549 Goat Anti-Mouse IgG (red). Scale bar = 20 μm. Data are representative of three repeats.