Cell-free expression of the outer membrane protein OprF of Pseudomonas aeruginosa for vaccine purposes

(A, B, C) Atomic force microscopy topographic images of an OprF proteoliposomes’ sample absorbed on mica surface, acquired using a tip functionalized with Tris–NTA, without (A, B) and with Triton X-100 (C). The circles in white dotted lines correspond to proteoliposomes. (A′, B′, C′) Corresponding adhesion maps of specific adhesion forces between the Tris–NTA modified tip and OprF His-tag, without (A′, B′) and with Triton X-100 (C′). The white to grey squares represent values of adhesion forces between 80 and 150 pN, as illustrated by the scale bar on the right. The circles in white dotted lines corresponding to proteoliposomes are zoomed on the right. The white arrows indicate specific adhesion points validated by the force distance curves and correspond to 16 transmembrane passages based on the theoretical model Worm-Like Chain.

The result of the trypsin digestion of OprF at different incubation times is visualized by anti-His Western blot (FL, full length). Anti-His antibody was conjugated to HRP and was detected using chemiluminescence.

(A, B) Force distance curves, obtained with a tip functionalized with Tris–NTA in Triton X-100 solution, detecting 64% of specific adhesion events corresponding to eight transmembrane domains (A) and 36% of specific adhesion events corresponding to 16 transmembrane domains (B), based on the theoretical model Worm-Like Chain.

(A) EM image of empty liposomes (incubated using the cell-free cell lysate and reaction mixture = negative control). (B, C, D, E) EM images of OprF proteoliposomes (LC1). The membrane was perforated with a series of holes with an average size of 9.5 ± 4 nm corresponding to OprF pores.

(A) Topographic image of the surface of an OprF proteoliposome absorbed onto a freshly cleaved mica support, acquired using a tip functionalized with Tris–NTA, in presence of Triton X-100. (B) Corresponding adhesion map of specific adhesion forces between the tris–NTA modified tip and OprF His-tag. The white to grey squares represent adhesion forces values between 80 and 150 pN, as illustrated by the scale bar on the right. The circles in white dotted lines highlight pores. (C) Force distance curves detecting specific adhesion events (indicated by arrows).

(A) C57Bl/6 mice were immunized subcutaneously with 25, 50 or 75 μg of OprF in proteoliposome. 1 wk after the fourth immunization, OprF-immunized groups (n = 10 or 11) or control group (n = 11) were subcutaneously challenged with a lethal dosage of Pseudomonas aeruginosa CHA strain (2 × 10⁷ CFU/mouse) and monitored for survival and disease symptoms for up to 80 h. (B) Naïve mice were subcutaneously challenged with a lethal dose of the CHA strain and an intraperitoneal injection with sera containing polyclonal antibodies induced by the OprF proteoliposomes was performed 1 h after the bacterial challenge. Mice were monitored for survival and disease symptoms for up to 80 h.

Antibody titration curves of sera from mice immunized four times with 25, 50, or 75 μg of recombinant OprF proteoliposome. Control mice were immunized with liposomes previously incubated with cell-free lysate. Values are means of three mice per group determined by indirect ELISA using recombinant OprF proteoliposome as coated antigen.

GFP was used as a negative control. The secondary antibody binding polyclonal antibodies was an HRP-conjugated rabbit anti-mouse IgG and it was detected using chemiluminescence.