Phosphorylation of Mouse Immunity-Related GTPase (IRG) Resistance Proteins Is an Evasion Strategy for Virulent Toxoplasma gondii

GTPases of the mouse IRG protein family, mediators of resistance against Toxoplasma gondii in the mouse, are inactivated by a polymorphic kinase of the parasite, resulting in enhanced parasite virulence

Comparative Genomics of the Apicomplexan Parasites Toxoplasma gondii and Neospora caninum: Coccidia Differing in Host Range and Transmission Strategy

Toxoplasma gondii is a zoonotic protozoan parasite which infects nearly one third of the human population and is found in an extraordinary range of vertebrate hosts. Its epidemiology depends heavily on horizontal transmission, especially between rodents and its definitive host, the cat. Neospora caninum is a recently discovered close relative of Toxoplasma, whose definitive host is the dog. Both species are tissue-dwelling Coccidia and members of the phylum Apicomplexa; they share many common features, but Neospora neither infects humans nor shares the same wide host range as Toxoplasma, rather it shows a striking preference for highly efficient vertical transmission in cattle. These species therefore provide a remarkable opportunity to investigate mechanisms of host restriction, transmission strategies, virulence and zoonotic potential. We sequenced the genome of N. caninum and transcriptomes of the invasive stage of both species, undertaking an extensive comparative genomics and transcriptomics analysis. We estimate that these organisms diverged from their common ancestor around 28 million years ago and find that both genomes and gene expression are remarkably conserved. However, in N. caninum we identified an unexpected expansion of surface antigen gene families and the divergence of secreted virulence factors, including rhoptry kinases. Specifically we show that the rhoptry kinase ROP18 is pseudogenised in N. caninum and that, as a possible consequence, Neospora is unable to phosphorylate host immunity-related GTPases, as Toxoplasma does. This defense strategy is thought to be key to virulence in Toxoplasma. We conclude that the ecological niches occupied by these species are influenced by a relatively small number of gene products which operate at the host-parasite interface and that the dominance of vertical transmission in N. caninum may be associated with the evolution of reduced virulence in this species

The activation interface of Irga6 largely contributes to interaction with ROP5.

<p>(A) In vitro pull-down of ROP5 with bacterially expressed and purified wt or mutant GST-Irga6 (at 25 µg protein/100 µl 1∶1 bead suspension) from RH-YFP strain <i>T. gondii</i>-lysates. GST was used as a negative control. Values reflect the mean ROP5 signal intensity and standard deviations of 2–3 pull-down experiments based on quantitated Western blots using 3E2 antibody (anti-ROP5). The signal given by GST was subtracted and the value obtained for each mutant normalized against wt Irga6. The values were correlated with a color in a spectrum ranging from green (100% of wt intensity) to red (0% of wt intensity). (B) Monomeric structural model of Irga6 based on Irga6-M173A in the GDP-bound form (PDB 1TQ6, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001358#pbio.1001358-Ghosh1" target="_blank">[29]</a>). The G-domain with the residues analyzed in (A) is depicted as spheres colored according to the values given in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001358#pbio-1001358-g008" target="_blank">Figure 8A</a>. Residues not assayed for inhibition of ROP5 binding are shown in blue. The rest of the protein is shown in ribbon structure (N-terminal helical domain in light blue, C-terminal helical domain in cyan). (C) Irga6-M173A-GDP in the same orientations as <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001358#pbio-1001358-g008" target="_blank">Figure 8B</a>, with the residues contributing to the activation interface <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001358#pbio.1001358-Uthaiah1" target="_blank">[30]</a> shown in red. (D) ROP5B_I inhibits oligomerization of Irga6 in vitro. GTP-dependent oligomerization of 20 µM wt Irga6 was monitored by dynamic light scattering (DLS) in the presence or absence of equal or 4-fold molar excess amounts of ROP5B_I pseudokinase domain protein. Values shown give the mean hydrodynamic radius of molecular complexes in solution. Monomeric Irga6 in the presence of GDP has a hydrodynamic radius of 3.4 nm. Oligomerization of wt Irga6 is almost completely inhibited by addition of a 4-fold molar excess of ROP5B_I.</p

A <em>Toxoplasma gondii</em> Pseudokinase Inhibits Host IRG Resistance Proteins

<div><p>The ability of mice to resist infection with the protozoan parasite, <em>Toxoplasma gondii</em>, depends in large part on the function of members of a complex family of atypical large GTPases, the interferon-gamma-inducible immunity-related GTPases (IRG proteins). Nevertheless, some strains of <em>T. gondii</em> are highly virulent for mice because, as recently shown, they secrete a polymorphic protein kinase, ROP18, from the rhoptries into the host cell cytosol at the moment of cell invasion. Depending on the allele, ROP18 can act as a virulence factor for <em>T. gondii</em> by phosphorylating and thereby inactivating mouse IRG proteins. In this article we show that IRG proteins interact not only with ROP18, but also strongly with the products of another polymorphic locus, <em>ROP5</em>, already implicated as a major virulence factor from genetic crosses, but whose function has previously been a complete mystery. ROP5 proteins are members of the same protein family as ROP18 kinases but are pseudokinases by sequence, structure, and function. We show by a combination of genetic and biochemical approaches that ROP5 proteins act as essential co-factors for ROP18 and present evidence that they work by enforcing an inactive GDP-dependent conformation on the IRG target protein. By doing so they prevent GTP-dependent activation and simultaneously expose the target threonines on the switch I loop for phosphorylation by ROP18, resulting in permanent inactivation of the protein. This represents a novel mechanism in which a pseudokinase facilitates the phosphorylation of a target by a partner kinase by preparing the substrate for phosphorylation, rather than by upregulation of the activity of the kinase itself.</p> </div

Peptides of ROP5 isoforms identified by MS after GST-Irga6 pull-down.

<p>The first three sections (ROP5A, ROP5B, and ROP5C) show peptides that specifically discriminate between the three ROP5 isoforms. Residues in bold type are unique for the specific isoform indicated in the table. Sections 4 (ROP5A+ROP5C) and 5 (ROP5B+ROP5C) show peptides that fit to both indicated isoforms only. The peptides displayed in Section 6 (ROP5A+ROP5B+ROP5C) show peptides that match to all isoforms except that marked with *, which is found in all ROP5 isoforms but ROP5C1. Altogether 51 peptides were found for ROP5C comprising a sequence coverage of 86.2%, 47 peptides for ROP5B comprising a sequence coverage of 70.3%, and 34 peptides for ROP5A comprising a sequence coverage of 55.9%. In total 65 individual peptides for RH-ROP5 were found. Many of those were limited digests of the ones presented in the table and are not shown. The next best hit for any <i>T. gondii</i> protein yielded 17 peptides. All together 44 additional <i>T. gondii</i> peptides were recovered from this band.</p