Binding of a Monoclonal Antibody to Sporozoites of Sarcocystis singaporensis Enhances Escape from the Parasitophorous Vacuole, Which Is Necessary for Intracellular Development

Early intracellular development in vitro of the cyst-forming protozoon Sarcocystis singaporensis and the influence of a monoclonal antibody on invasion, intracellular localization, and development of sporozoites were studied. As revealed by immunofluorescence using parasite-specific antibodies which 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 compartment within the first 8 h postinfection to enter the host cell cytosol. By semiquantitative analysis of acetyl-histone H4 expression of sporozoites, a marker linked to early gene expression of eukaryotic cells, we show (supported by ultrastructural analysis) that escape from the PV appears to be necessary for early intracellular development. More than 90% of sporozoites located in the cytosol expressed high levels of acetylated histone H4 in the nucleus, whereas only a quarter of the intravacuolar sporozoites exhibited a similar signal. As revealed by ultrastructural analysis, young schizonts all resided in the cytosol. Specific binding of a monoclonal antibody (11D5/H3) to sporozoites before invasion significantly enhanced their escape from the PV, whereas cell invasion itself remained unaffected. The antibody actually increased proliferation of the parasites in vitro, providing a further link between residence in the cytosol and successful intracellular development. Monoclonal antibody 11D5/H3 precipitated a major 58-kDa antigen from oocyst-sporocyst extracts and reacted with the cytoplasm and the surface of sporozoites in immunofluorescence assays. Collectively, the observed antibody-parasite interaction suggests the existence of a signaling event that influences intracellular development of Sarcocystis.

本研究针对形成包囊的原生动物新加坡肉孢子虫（Sarcocystis singaporensis）的体外早期胞内发育过程，以及单克隆抗体（monoclonal antibody）对子孢子（sporozoites）入侵、胞内定位与发育的影响展开了系统探究。通过使用靶向寄生泡膜（parasitophorous vacuole membrane, PVM）的寄生虫特异性抗体开展免疫荧光标记，并结合超微结构分析，结果显示：子孢子通过形成寄生泡（parasitophorous vacuole, PV）入侵大鼠肺细胞。感染后前8小时内，约半数子孢子会脱离该囊泡结构，进入宿主细胞的胞质溶胶中。通过对真核细胞早期基因表达标志物乙酰化组蛋白H4（acetyl-histone H4）进行半定量分析，结合超微结构验证结果表明：子孢子从寄生泡中逃逸，是其早期胞内发育的必要条件。定位在胞质溶胶中的子孢子，有超过90%在细胞核内呈现高水平的乙酰化组蛋白H4表达；而仅四分之一的泡内子孢子可检测到类似的信号。超微结构分析进一步显示，幼年裂殖体均定位于胞质溶胶中。入侵前将单克隆抗体11D5/H3与子孢子特异性结合，可显著提升其从寄生泡中的逃逸效率，但对细胞入侵过程本身无显著影响。该抗体还可显著促进寄生虫的体外增殖，进一步印证了胞质溶胶定位与成功胞内发育之间的关联性。单克隆抗体11D5/H3可从卵囊-孢子囊提取物中沉淀得到一个分子量为58 kDa的主要抗原，且在免疫荧光实验中可与子孢子的胞质及表面发生特异性结合。综上，本次观察到的抗体-寄生虫相互作用提示，新加坡肉孢子虫的胞内发育过程可能受到某种信号事件的调控。