Figure 4
Secretion and display of antigens by attenuated Salmonella Typhimurium. (A-B) Secretion and display of antigens fused to the Hbp passenger. S. Typhimurium SL3261 (unlabeled) and derivatives expressing Hbp-Ag85B[C+N]-ESAT6-Rv2660c or HbpD-Ag85B[C+N]-ESAT6-Rv2660c were analyzed by SDS-PAGE and Coomassie staining (A) or immunoblotting using the indicated antibodies (B). The equivalent of 0.03 OD660 units cells (c) and corresponding culture medium (m) samples was analyzed. (C-D) Exposure of antigens at the S. Typhimurium cell surface. (C) SL3261 cells (lane 1-2) and derivatives expressing HbpD-Ag85B[C+N]-ESAT6-Rv2660c from A were treated with Proteinase K (+ pk) or mock-treated (- pk). (D) Samples described under C were analyzed by immunoblotting. Cell integrity during the procedure was demonstrated by showing the inaccessibility of the periplasmic chaperone SurA towards Proteinase K using anti-SurA (cf. lanes 1, 3, 5 and 2, 4, 6, resp.). Cleaved Hbp passenger (>), non-cleaved Hbp species (*), the cleaved β-domain (β) and Proteinase K (pk) are indicated. An unrelated protein that cross-reacts with the Hbp β-domain antiserum is indicated (x). A proteolytic fragment of HbpD-Ag85B[C+N]-ESAT6-Rv2660c is indicated (f). Molecular weight markers (kDa) are shown at the left side of the panels.