An organellar segregation defect in the apicomplexan parasite Toxoplasma gondii

The apicomplexan plastid, or ‘apicoplast’ is a non-photosynthetic plastid organelle found in parasites of the phylum apicomplexa, including Plasmodium (the causative agents of malaria) and Toxoplasma gondii (a prominent congenital pathogen and a common opportunistic infection associated with AIDS). This remarkable organelle was acquired by secondary endosymbiosis of a eukaryotic alga, and retention of the algal chloroplast as a distinctive structure enclosed by four membranes. Drugs that specifically target the apicoplast (macrolides, lincosamides, rifamipicins, fluoroquinolones, etc) inhibit growth only after parasites escape from the cell in which they were treated and establish a new infection in another host cell. This ‘delayed death’ phenotype links apicoplast function with parasite survival, validating this organelle as a target for parasiticidal drug design. But what does the apicoplast do? Purification of the apicoplast by various subcellular fractionation methods has proved difficult, due to the heterogeneity and fragility of this organelle. In the course of studies designed to target heterologous reporters to the apicoplast surface for organelle affinity purification, however, we inadvertently constructed a fusion between a nuclear-encoded apicoplast protein, the green fluorescent protein reporter, and a rhoptry protein ROP1. The protein product of this fusion construct targets specifically to the apicoplast membranes, grossly disturbs the organellar morphology and blocks apicoplast segregation during parasite mitosis. These parasites grow normally within the first host cell, yielding parasitophrous vacuoles containing as many as 128 parasites, only one of which contains a (giant) apicoplast. In subsequent infections, parasites retaining the ‘super’-apicoplast remain infectious, while plastid-deficient parasites die, providing a cell biological validation of the ‘delayed death’ phenotype. The apicoplast segregation mutants can be sorted by fluorescence-activated cell sorting to yield pure populations of plastid-deficient and ‘super’-apicoplast-containing parasites, permitting a variety of cell biological, biochemical, pharmacological and genetic studies. These studies provide proof that the processing of nuclear-encoded apicoplast proteins occurs within the organelle itself, confirm the feasibility of using apicoplast segregation mutants to screen for drugs targeting the apicoplast, and provide valuable insight into the apicoplast functions