Abstract
The inner membrane complex (IMC), a series of flattened vesicles at the periphery of apicomplexan parasites, is thought to be important for parasite shape, motility and replication, but few of the IMC proteins that function in these processes have been identified. TgPhIL1, a Toxoplasma gondii protein that was previously identified through photosensitized labeling with 5-[125I] iodonapthaline-1-azide, associates with the IMC and/or underlying cytoskeleton and is concentrated at the apical end of the parasite. Orthologs of TgPhIL1 are found in other apicomplexans, but the function of this conserved protein family is unknown. As a first step towards determining the function of TgPhIL1 and its orthologs, we generated a T. gondii parasite line in which the single copy of TgPhIL1 was disrupted by homologous recombination. The TgPhIL1 knockout parasites have a distinctly different morphology than wild-type parasites, and normal shape is restored in the knockout background after complementation with the wild-type allele. The knockout parasites are outcompeted in culture by parasites expressing functional TgPhIL1, and they generate a reduced parasite load in the spleen and liver of infected mice. These findings demonstrate a role for TgPhIL1 in the morphology, growth and fitness of T. gondii tachyzoites.
Highlights
The Phylum Apicomplexa contains a number of medically important parasites including Cryptosporidium spp., which cause diarrheal illness in children and immunocompromised patients, Plasmodium spp., which are the causative agents of malaria, and Toxoplasma gondii, which causes life-threatening disease in immunocompromised people and the developing fetus
Culture of parasites Wild-type RH strain T. gondii was maintained by serial passage in confluent primary human foreskin fibroblast (HFF) cells in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 1% heat inactivated fetal bovine serum (FBS), 10 units/ml penicillin G, 10 mg/ml streptomycin sulfate, and 10 mM HEPES buffer, as previously described [20]
Ultrastructure of the TgPhIL1 knockout parasites Given their striking difference in shape, we examined the
Summary
The Phylum Apicomplexa contains a number of medically important parasites including Cryptosporidium spp., which cause diarrheal illness in children and immunocompromised patients, Plasmodium spp., which are the causative agents of malaria, and Toxoplasma gondii, which causes life-threatening disease in immunocompromised people and the developing fetus. Mortality and economic loss caused by these pathogens, information is lacking regarding many aspects of their basic biology. T. gondii is a powerful model system for studying conserved aspects of apicomplexan biology, due to the ease with which T. gondii can be cultured and genetically manipulated [1,2]. Apicomplexan parasites contain a number of unusual subcellular structures and organelles [3,4], including the inner membrane complex (IMC), a series of flattened vesicles that are tightly apposed to the cytosolic face of the plasma membrane [5,6]. The plasma membrane and IMC are referred to as the pellicle. Additional cytoskeletal structures associated with the pellicle include the conoid, which is a cone-shaped structure composed of a novel polymeric form of tubulin [9], a pair of polar rings located at either end of the conoid [9,10], and 22 microtubules radiating posteriorly from the lower polar ring [10]
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