Figure 1. BmGPI12 is secreted into the erythrocyte cytoplasm and subsequently into the extracellular environment of the B. microti–infected erythrocyte.

(A) Immunoblotting analysis using preimmune (PI) and anti-BmGPI12 immune rabbit sera on fractions of uninfected erythrocytes (UI) or erythrocytes infected with either PRA99 or LabS1 strain of B. microti. In uninfected erythrocytes, the P fraction consists primarily of erythrocyte membranes. In B. microti–infected erythrocytes, the P fraction includes both erythrocyte membranes and protein extracts from isolated parasites. The erythrocyte membrane protein TER-119 (52 kD) was detected only in the P fractions from uninfected and B. microti–infected red blood cells using anti–TER-119 monoclonal antibody. (B) Immunofluorescence assay on mouse erythrocytes infected with the PRA99 strain of B. microti. BmGPI12 was labeled with anti-BmGPI12 polyclonal antibodies and could be observed within the parasite cytoplasm, the PPM as well as in the erythrocyte cytoplasm and within IV and TOVs (indicated by arrowheads). Anti–TER-119 monoclonal antibody was used to label the plasma membrane of the infected mouse erythrocytes. The DAPI staining was applied to verify the presence of parasites within the erythrocytes by labeling parasitic nuclear DNA. Staining of control uninfected red blood cells using the same antibodies is shown (lower panel). Scale bar: 3 μm. H, hemolysate; mRBC, mouse red blood cells; P, membrane fractions; S, mouse plasma.