Contributed Papers: Oral Presentations Cell Biology

Plastid Replication in Apicomplexan Parasites

1Shipra Vaishnava, 2Marc-Jan Gubbels, 3Raj Gaji,
4John Murray, 3Daniel Howe and 1,2Boris Striepen
1Department of Cellular Biology and
2Center for Tropical and Emerging Global Diseases,
University of Georgia, Athens, GA 30602, USA;
3Gluck Equine Research Center, Department of
Veterinary Science, University of Kentucky, Lexington 40546, USA;
4Department of Cell and Development Biology, University
of Pennsylvania, PA 19104, USA

Apicomplexan parasites harbor a secondary plastid which is essential for development and pathogenesis. Several plastid localized metabolic and housekeeping enzymes have emerged as promising drug targets in Toxoplasma and Plasmodium. We are studying the replication and segregation of this important organelle and its genome. Our genomic analysis reveals that Apicomplexa have lost all elements of the conserved chloroplast/cyanobacterial division machinery including FtsZ, and several Min and Arc genes. We hypothesize that in contrast to plants and algae the plastid in Apicomplexa, is segregated using a genuinely eukaryotic mechanism, association with the centrosomes of the mitotic spindle. We have tested this hypothesis in T. gondii and S. neurona, which we show to have highly organized linear plastids. We show tight association of the plastid and spindle poles throughout the cell cycle. Using pharmacological experiments ablating the parasite’s microtubules we show that spindles are essential for plastid organization. Using several molecular markers we further show that fission of the single plastid occurs late and concurs with daughter cell formation. Using transgenic S. neurona and in-vivo microscopy and laser bleaching experiments we have further tested our model of organellar fission in living cells. FISH analysis employing probes specific for the 35 kb plastid genome suggest that the organellar genome might be positioned and segregated by centrosomes as well. In contrast to the fission machinery the genome replication apparatus of apicomplexans seems of prokaryotic origin. To develop robust molecular markers for the genome’s position we have cloned the putative T. gondii plastid histone-like HU, PolA and helicase genes. We show that the T. gondii HU protein complements the respective mutant in E. coli. Antibodies raised against the recombinant protein show that HU is indeed localized to the plastid and associates with the plastid genome. Overexpression of a HU-YFP fusion protein in T. gondii has a pronounced dominant negative effect on organellar genome replication yielding plastids devoid of a genome.

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