Binding of a Monoclonal Antibody to Sporozoites ofSarcocystis singaporensisEnhances Escape from the Parasitophorous Vacuole, Which Is Necessary for Intracellular Development

Abstract

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.Cyst-forming coccidia of the genus Sarcocystis are among the most prevalent parasites of livestock and are responsible for considerable economic losses (10, 15). Furthermore, recent outbreaks of Sarcocystis-induced disease among humans in tropical countries (3) underscore the increasing importance of these parasites for public health (31).A peculiar aspect of Sarcocystis infection has been known for a long time but has received little attention: the possible escape of sporozoites from the parasitophorous vacuole (PV) after invasion of host cells. Early observations in vivo and ultrastructural studies of infected cell cultures of bovine pulmonary artery endothelial cells and bovine monocytes revealed that Sarcocystis cruzi sporozoites as well as the resulting schizonts were located free in the host cell cytoplasm, i.e., not surrounded by a PV (11, 34). We have extended this observation to Sarcocystis singaporensis, a species that infects rats as intermediate hosts and specifically develops inside endothelial cells and pneumonocytes, a characteristic that renders it a suitable model for Sarcocystis infections in the laboratory (21). To date, however, nothing is known about a possible escape from ...

Fussing About Fission: Defining Variety Among Mainstream and Exotic Apicomplexan Cell Division Modes

“…Both the tissue cyst inhabiting bradyzoite stages of T. gondii and S. neurona (pre-bradyzoite metrocytes as well) undergo endodyogeny; only the acute stage of S. neurona divides by endopolygeny (Dubey et al, 2001). In addition, although the acute stage of S. neurona dissolves the vacuolar compartment and replicates in the host cell cytoplasm, bradyzoite multiplication and cyst formation occur within a vacuole (Jakel et al, 2001). Cyst expansion has to occur gradually so as not to compromise the cyst wall, which obviously is more compatible with endodyogeny than endopolygeny.…”

The IRG proteins: A function in search of a mechanism

“…However, while disruption of the PVM may expose the enclosed organism to the cytosol, it is unknown whether the cytosol is an inimical compartment for T. gondii survival. Another closely related Apicomplexan, Sarcocystis, lives and replicates in the cytosol (Jakel et al, 2001), while Plasmodium sporozoites penetrate the cytosol of several hepatocytes before forming a stable parasitophorous vacuole (Mota et al, 2001). We looked for but failed to find a consistent association of LC3-positive, putatively autophagic, membranes around disrupting vacuoles, although undoubtedly LC3 was activated in many infected cells (Martens et al, 2005).…”

Fussing about fission: defining variety among mainstream and exotic apicomplexan cell division modes

“…In 769 contrast, during S. neurona endopolygeny the micronemes disappear halfway through the 770 division process, which depletes their invasion capacity (Vaishnava et al, 2005); hence, this 771 model does not hold true for the polyploid internal budding modes. Alternatively, it can be 772 argued that the maintenance of a cortical cytoskeleton in the mother makes the parasites more 773 resistant to mechanical stress, but it is not immediately obvious how this sets the tissue cyst- formation occur within a vacuole (Jakel et al, 2001). Cyst expansion has to occur gradually so as 786 not to compromise the cyst wall, which obviously is more compatible with endodyogeny than endopolygeny.…”

“…Both the tissue cyst 782 inhabiting bradyzoite stages of T. gondii and S. neurona (pre-bradyzoite metrocytes as well) 783 undergo endodyogeny; only the acute stage of S. neurona divides by endopolygeny (Dubey et 784 al., 2001). In addition, although the acute stage of S. neurona dissolves the vacuolar 785 compartment and replicates in the host cell cytoplasm, bradyzoite multiplication and cyst 786 formation occur within a vacuole (Jakel et al, 2001). Cyst expansion has to occur gradually so as 787 not to compromise the cyst wall, which obviously is more compatible with endodyogeny than endopolygeny.…”