Binding of a monoclonal antibody to sporozoites of Sarcocystis singaporensis enhances escape from the parasitophorous vacuole, which is necessary forintracellular development

Early intracellular development in vitro of the cyst-forming protozoon Sarc ocystis singaporensis and the influence of a monoclonal antibody on invasio n, intracellular localization, and development of sporozoites were studied. As revealed by immunofluorescence using parasite- specific antibodies whic h labeled the parasitophorous vacuole membrane (PVM) and by ultrastructural analysis, sporozoites invaded pneumonocytes of the rat via formation-of a parasitophorous vacuole (PV). About half of the sporozoites left this compa rtment within the first 8 h postinfection to enter the host cell cytosol. B y semiquantitative analysis of acetyl-histone H4 expression of sporozoites, a marker linked to early gene expression of eukaryotic cells, we show (sup ported by ultrastructural analysis) that escape from the PV appears to be n ecessary for early intracellular development. More than 90% of sporozoites located in the cytosol expressed high levels of acetylated histone H4 in th e nucleus, whereas only a quarter of the intravacuolar sporozoites exhibite d a similar signal. As revealed by ultrastructural analysis, young schizont s all resided in the cytosol. Specific binding of a monoclonal antibody (11 D5/H3) to sporozoites before invasion significantly enhanced their escape f rom the PV, whereas cell invasion itself remained unaffected. The antibody actually increased proliferation of the parasites in vitro, providing a fur ther link between residence in the cytosol and successful intracellular dev elopment. Monoclonal antibody 11D5/H3 precipitated a major 58-kDa antigen f rom oocyst-sporocyst extracts and reacted with the cytoplasm and the surfac e of sporozoites in immunofluorescence assays. Collectively, the observed a ntibody-parasite interaction suggests the existence of a signaling event th at influences intracellular development of Sarcocystis.