TgPhIL1, which was originally discovered in photolabeling experiments designed to identify novel proteins of the T. gondii
, is conserved among members of the Phylum Apicomplexa but exhibits very little sequence similarity to non-apicomplexan proteins currently in the sequence databases. Formulating a hypothesis on the function of a protein that contains no identifiable protein domains or homologs of known function is challenging. Based on its localization to the IMC and properties suggestive of both a cytoskeletal and membrane-associated protein, TgPhIL1 could serve to organize or physically connect elements of the parasite cytoskeleton with the membranes of the IMC 
As a first step in elucidating the function of TgPhIL1, we generated a TgPhIL1
knockout parasite line. The knockout parasites have a conspicuously altered morphology, being shorter and wider than the wild-type parasites from which they were derived. Given that the shape of T
is thought to be important for motility 
, it was surprising that the RHΔTgPhIL1
parasites were capable of normal motility in vitro
, although we cannot rule out some more subtle effect on in vitro
motility or a defect that would only be apparent in an infected organism, where the parasites are required to travel longer distances and through various biological barriers 
. Because PhIL1 localizes to a ring-like structure at the posterior end of the conoid, we also hypothesized that the protein might play some role in conoid extension; however, conoid extension was unaffected by disruption of TgPhIL1
Although indistinguishable from wild-type parasites in a number of respects, the knockout parasites showed significantly slower growth in culture. The growth defect observed in culture translated into a significant decrease in parasite fitness in a mouse model of infection. The number of RHΔTgPhIL1 parasites detected in the liver, spleen and peritoneal fluid of infected mice was consistently lower with RHΔTgPhIL1 parasites than with wild-type parasites. No difference was observed in the survival of mice infected with wild-type and RHΔTgPhIL1 parasites, with all mice dying around day 8 (data not shown). However, these experiments were done with a single dose of 1×104 parasites; a more extensive analysis, using a range of parasite inocula, would be required to establish whether the decreased fitness of the knockout parasites affects host survival.
An alternative approach to identifying the biological function of TgPhIL1 would be to identify proteins with which it interacts. However, TgPhIL1's highly insoluble nature 
makes coimmunoprecipitation studies to identify interacting proteins technically challenging. In P. falciparum,
a genome-wide yeast-two-hybrid screen has been performed, with the results available online (www.plasmodb.org
). In this screen, the P. falciparum
homologue of TgPhIL1 (PFA0440w) was shown to interact with another P. falciparum
protein (PFF0325c), and a homolog of this P. falciparum
PhIL1-binding protein exists in T. gondii
(38.m01070). While PFF0325c shows no homology to non-apicomplexan proteins, it was found to interact in the yeast-two-hybrid screen with five proteins in addition to PfPhIL1 (PFA0110w, PFA0285c, PF10_0378, PF08_0137, PFD0320c). Two of these are DnaJ-domain containing proteins, one is predicted to be exported, and several have homologs in T. gondii.
As additional functional annotation of genes from other organisms becomes available, new insights into the function of the potential PhIL1-binding proteins and of PhIL1 itself may become apparent.
If one of the primary functions of TgPhIL1 is to provide structural stability to the parasite, as suggested by the data reported here, a reduced ability of the knockout parasites to tolerate osmotic or mechanical stresses encountered during in vivo
infection might be an underlying cause of the parasite's reduced fitness. Intriguingly, disruption of the IMC-associated proteins IMC1a and IMC1b in Plasmodium berghei
also alters the shape and reduces the mechanical stability of sporozoites and ookinetes, respectively 
. The rate of parasite proliferation early during infection may affect parasite loads later, as the parasite has a limited time window in which to proliferate before the immune system becomes fully activated. The development of drugs that target components of the IMC and cytoskeleton may therefore be a potentially useful approach to disease management.
In the context of a population of parasites, even relatively subtle growth defects such as the one reported here are likely to present a significant selective disadvantage over time, since parasites exhibiting such a defect will be outcompeted during infection by parasites whose growth is not impaired. Several other T. gondii
proteins have recently been identified that are non-essential, but make a clear contribution to parasite growth and fitness (e.g., 
and GEW, unpublished data). With the recent development of highly efficient methods for gene replacement in T. gondii 
, many more such genes are likely to be identified. In these cases, it may be that we simply do not know the relevant assay, host, or environmental conditions to reveal a clear phenotype when the gene is disrupted. The overall fitness of a given parasite strain will be determined by both its essential genes and the additive effect of genes which may not be essential but provide some degree of fitness benefit to the parasite in the context of particular host organisms or environmental conditions. A broader experimental context is likely to be required for elucidating the biological function of genes such as TgPhIL1, which are beneficial to the parasite but not strictly essential.