Figure 1.

Characterization of the immunogenicities of PcrV subdomains. (A) Schematic illustration of the structure of full-length PcrV and PcrV_NH. The left panel shows that the full-length PcrV was composed of four domains, namely, Nter (Met1-Lys127), H7 (Arg128-Ala158), Cter (Lys159-Pro250), and H12 (Leu251-Ile294). The Nter domain was connected to H12 with a GSGGSG linker to generate ParV_NH. The cartoon image of the 3D structure of PcrV, predicted from I-TASSER Suite, is shown in the right panel. Nter, H7, Cter, and H12 are colored in gray, blue, green, and orange, respectively. (B) The immune response of the subdomain of PcrV to sera from PA-infected patients. The bar represents the relative percentage of OD of each domain to that of full-length PcrV. The immune reactivity of the Nter and H12 domains was significantly stronger than that of the H7 and Cter domains, (*P < 0.05, ns, not significant P > 0.05). (C) The effect of anti-PcrV subdomain antibodies on the secretion of ExoU. PA 103 grown in ExoU-inducing or non-inducing medium were coincubated with anti-Nter, -H7, -Cter, and -H12 antibodies, and the ExoU in in the culture supernatant was detected by Western blot. The content of flagellin in cell pellets was used as control. Treatment of the anti-Nter and anti-H12 domain antibodies led to a marked reduction of the secretion of ExoU when grown in both the two media. (D) The effect of anti-PcrV subdomain antibodies on the T3SS mediated cytotoxicity. Hela cells were infected with PA103 for 3 h and anti-PcrV subdomain antibodies was added into the culture media. The attached cells were stained crystal violet, which was then dissolved in ethanol. The bar represents the optical density at 590 nm of dissolved crystal violet. The data are shown as the means ± SE. *P < 0.05, ns, not significant (P > 0.05).