Targeted Disruption of TgPhIL1 in Toxoplasma gondii Results in Altered Parasite Morphology and Fitness

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.

Global Analysis of Palmitoylated Proteins in Toxoplasma gondii

“…Consistent with this, our chemical proteomic approach identified PhIL1, which has been previously implicated in parasite morphology (Barkhuff et al, 2011), as being palmitoylated. We metabolically labeled parasites expressing a GFP-tagged PhIL1 (Gilk et al, 2006) with 17-ODYA.…”

“…We biochemically confirm the palmitoylation of several candidates from our proteomic analysis, including Myosin Light Chain 1 (MLC1) and Myosin A (MyoA), components of the glideosome, the parasite's motility complex (Herm-Gotz et al, 2002; Meissner et al, 2002). We confirm that the morphology-associated photosensitized INA-labeled protein (PhIL1) (Barkhuff et al, 2011; Gilk et al, 2006) is palmitoylated. Unexpectedly, we found that Apical Membrane Antigen 1 (AMA1), a protein that functions during invasion (Hehl et al, 2000; Lamarque et al, 2014; Mital et al, 2005), is palmitoylated.…”

The inner membrane complex through development ofToxoplasma gondiiandPlasmodium

“…At the present time, no alveolin proteins have been disrupted in T. gondii and so any potential functional redundancy within this family is currently unknown. However, the IMC-associated protein TgPhIL1 has been deleted, resulting in viable parasites which were shorter and wider than the wild type, although with no observable ultrastructural IMC defect (Barkhuff et al, 2011). These mutants replicated normally, but had subtly impaired motility and a fitness defect in mixed infection (Gilk et al, 2006;Barkhuff et al, 2011;Leung et al, 2014).…”

Cytoskeleton Assembly in Toxoplasma gondii Cell Division

“…The meshwork component IMC15 is also enriched in the apical cap (Anderson-White et al, 2011). Another protein, which is not essential, localizing predominantly to the apical cap is Photosensitized INA-labeled protein 1 (PhIL1) (Barkhuff et al , 2011; Gilk et al , 2006). Finally, a component of the glideosome, gliding-associated protein 70 (GAP70), localizes specifically to the apical cap (Fig.…”