Figure 5. Vesicle-mediated secretion of BmIPA48 antigen by B. microti.

(A) Distribution of BmIPA48 in the plasma (S), hemolysate (H), and membrane (P) fractions isolated from blood of uninfected or B. microti (PRA99)–infected erythrocytes was determined by Western blotting using polyclonal antibodies against BmIPA48 (48 kD). Preimmune (PI) sera were used as control. (B) Immunoblot analysis using preimmune and anti-BmIPA48 antibodies on either intact plasma (PL) collected from mice infected with B. microti PRA99 strain or on two fractions (supernatant: Us and pellet: Up) of plasma after UC at 120,000g. (C) Immunofluorescence assay using anti-BmIPA48 in PRA99-infected erythrocytes. BmIPA48 was labeled with polyclonal antibodies and could be detected within the parasite and in discrete IVs within the cytoplasm of the infected erythrocyte. Anti–TER-119 monoclonal antibody was used to label the plasma membrane of mouse erythrocytes and DAPI was used to stain the parasite nucleus. Staining of control uninfected red blood cells is shown. Scale bar: 3 μm. (D, E) Representative images of immunoelectron micrographs of B. microti LabS1–infected mouse erythrocytes. Ultrathin sections of high-pressure frozen and Durcupan resin–embedded infected erythrocytes were immunolabeled with anti-BmIPA48 antibodies coupled to 10-nm gold particles. Scale bars: 500 nm (D and E). mRBC, mouse red blood cells; P, parasite; RBCC, red blood cell cytoplasm; RBCM, red blood cell membrane.