Daughter Cell Assembly in the Protozoan ParasiteToxoplasma gondii

Abstract

The phylum Apicomplexa includes thousands of species of obligate intracellular parasites, many of which are significant human and/or animal pathogens. Parasites in this phylum replicate by assembling daughters within the mother, using a cytoskeletal and membranous scaffolding termed the inner membrane complex. Most apicomplexan parasites, including Plasmodium sp. (which cause malaria), package many daughters within a single mother during mitosis, whereas Toxoplasma gondii typically packages only two. The comparatively simple pattern of T. gondii cell division, combined with its molecular genetic and cell biological accessibility, makes this an ideal system to study parasite cell division. A recombinant fusion between the fluorescent protein reporter YFP and the inner membrane complex protein IMC1 has been exploited to examine daughter scaffold formation in T. gondii. Time-lapse video microscopy permits the entire cell cycle of these parasites to be visualized in vivo. In addition to replication via endodyogeny (packaging two parasites at a time), T. gondii is also capable of forming multiple daughters, suggesting fundamental similarities between cell division in T. gondii and other apicomplexan parasites. INTRODUCTIONToxoplasma gondii is a ubiquitous protozoan parasite, chronically infecting 10 -90% of human populations worldwide. Sexual differentiation occurs only in the cat, but asexual T. gondii parasites can invade, proliferate, and encyst in virtually any nucleated cell (Frenkel, 1973;Bonhomme et al., 1992;Smith, 1995;Dubey, 1998;Dubey et al., 1998). Primary infection during pregnancy poses a risk of abortion or severe birth defects. Reactivation of dormant parasite tissue cysts (bradyzoites) gives rise to rapidly replicating tachyzoites, which may be fatal in immunocompromised individuals. Pathogenesis in both congenital infection and immunosuppressed patients is directly attributable to parasite proliferation (Frenkel, 1973;Dubey, 1998). Understanding the replication process of this parasite is therefore essential for the development of improved treatment but little is known about cell cycle control in these parasites.Like other members of the phylum Apicomplexa, T. gondii is an obligate intracellular parasite. Haploid tachyzoites invade into host cells, establishing a parasitophorous vacuole whose membrane is derived from the host plasma membrane (Joiner et al., 1994;Suss-Toby et al., 1996;Mordue et al., 1999; Figure 1A). Two parasites are typically produced in each mitotic cell cycle (ϳ7-10 h), and replication proceeds synchronously, resulting in geometric expansion of clonal progeny until the host cell is lysed, ϳ48 h postinfection.In contrast to replication by binary fission (as observed in most animal, plant, and bacterial cells), parasite replication proceeds via assembly of daughters within the mother (Figure 1B). Because asexual replication of T. gondii tachyzoites typically produces two parasites per mitotic cell cycle, this process is often termed "endodyogeny" (Sheffield and Melton, 1968). In c...

Differential Localization of Alternatively Spliced Hypoxanthine-Xanthine-Guanine Phosphoribosyltransferase Isoforms in Toxoplasma gondii

“…Molecular Methods-HXGPRT-I or HXGPRT-II expression vectors and plasmids pdhfrCAT and ptubIMC1-YFP have been described previously (5,15,18). For fluorescent protein fusions, the open reading frames of HXGPRT-I and HXGPRT-II were PCR-amplified using sense primer HXBglF, 5Ј-GAagatctATGGCGTCCAAACCCATTG-3Ј, and antisense primer HXAvrR, 5Ј-GGCcctaggCTTCTCGAACTTTTTGCGA-G-3Ј (restriction sites indicated in lowercase).…”

Toxoplasma gondii Rab6 Mediates a Retrograde Pathway for Sorting of Constitutively Secreted Proteins to the Golgi Complex

“…in both adult and forming daughter parasites, thus serving as a hallmark of daughter cell formation by microscopy (34). In nearly all untransfected tachyzoites, parasites budded by endodyogeny, with two forming daughters distinguishable (Fig.…”

Inhibition of Toxoplasma gondii growth by pyrrolidine dithiocarbamate is cell cycle specific and leads to population synchronization

“…Fluorescent patterns of inner membrane complex (IMC1, to assess daughter formation) [23], proliferating cellular nuclear antigen (PCNA1, to assess nuclear morphology) [3], centrin (to assess centrosome duplication) [24], and genomic DNA (stained with dapi) were evaluated by IFA in tachyzoites grown in HFF monolayers (9cm 2 slideflask, #170920, Nalge Nunc International, Roskilde, Denmark) and synchronized by PDTC treatment. Microscopic slideflasks were used throughout these studies to better match the culture format employed in parallel experiments to determine DNA content by FACS analysis.…”