Malaria Parasite Invasion of the Mosquito Salivary Gland Requires Interaction between the Plasmodium TRAP and the Anopheles Saglin Proteins
Figure 5
Mutational analysis of TRAP-saglin interactions.
(A) Diagram of the TRAP protein and the Thr126 to Ala mutation in the MIDAS domain. (B) Binding of recombinant wild type or mutant TRAP domain-A to saglin. Saglin was captured on wells of a nickel-coated plate and wild type or mutant recombinant TRAP domain-A were added to the wells, incubated and washed. Bound TRAP domain-A was detected by incubation with anti-TRAP domain-A antibody followed by an alkaline phosphatase-conjugated secondary antibody. (1) Wild type TRAP domain-A; (2) Point mutant TRAP domain-A; (3) No TRAP protein added. (C) Direct visualization of the binding of wild type and mutant TRAP domain-A protein to recombinant saglin. Histidine-tagged saglin was immobilized on Ni-agarose beads, washed and allowed to bind to the purified wild type or mutant TRAP domain-A recombinant protein. Beads were washed and incubated with anti-domain-A antibody. Detection was done after incubation with Alexa Flour-568-conjugated anti-rabbit IgG (red). (a) Wild type TRAP domain-A; (b) Point mutant TRAP domain-A; (c) No TRAP protein added. Panels (d), (e) and (f) show light microscopic images of the fields to their left. (D) Binding of recombinant wild type and mutant domain-A to salivary glands. Fixed salivary glands were incubated with wild type or mutant TRAP domain-A protein, washed and incubated with anti-TRAP domain-A antibody. Detection was with anti-rabbit IgG conjugated to Alexa Flour-488 (green). (a) Wild type TRAP domain-A; (b) Point mutant TRAP domain-A; (c) No TRAP protein added. The lower panels (d, e, f) show light microscopic images of the fields on top (a, b, c, respectively).