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

The IFN-γ-Inducible GTPase, Irga6, Protects Mice against Toxoplasma gondii but Not against Plasmodium berghei and Some Other Intracellular Pathogens

Clearance of infection with intracellular pathogens in mice involves interferon-regulated GTPases of the IRG protein family. Experiments with mice genetically deficient in members of this family such as Irgm1(LRG-47), Irgm3(IGTP), and Irgd(IRG-47) has revealed a critical role in microbial clearance, especially for Toxoplasma gondii. The in vivo role of another member of this family, Irga6 (IIGP, IIGP1) has been studied in less detail. We investigated the susceptibility of two independently generated mouse strains deficient in Irga6 to in vivo infection with T. gondii, Mycobacterium tuberculosis, Leishmania mexicana, L. major, Listeria monocytogenes, Anaplasma phagocytophilum and Plasmodium berghei. Compared with wild-type mice, mice deficient in Irga6 showed increased susceptibility to oral and intraperitoneal infection with T. gondii but not to infection with the other organisms. Surprisingly, infection of Irga6-deficient mice with the related apicomplexan parasite, P. berghei, did not result in increased replication in the liver stage and no Irga6 (or any other IRG protein) was detected at the parasitophorous vacuole membrane in IFN-γ-induced wild-type cells infected with P. berghei in vitro. Susceptibility to infection with T. gondii was associated with increased mortality and reduced time to death, increased numbers of inflammatory foci in the brains and elevated parasite loads in brains of infected Irga6-deficient mice. In vitro, Irga6-deficient macrophages and fibroblasts stimulated with IFN-γ were defective in controlling parasite replication. Taken together, our results implicate Irga6 in the control of infection with T. gondii and further highlight the importance of the IRG system for resistance to this pathogen

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

Antimicrobial effects of murine mesenchymal stromal cells directed against Toxoplasma gondii and Neospora caninum: role of immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs)

Mesenchymal stromal cells (MSCs) have a multilineage differentiation potential and provide immunosuppressive and antimicrobial functions. Murine as well as human MSCs restrict the proliferation of T cells. However, species-specific differences in the underlying molecular mechanisms have been described. Here, we analyzed the antiparasitic effector mechanisms active in murine MSCs. Murine MSCs, in contrast to human MSCs, could not restrict the growth of a highly virulent strain of Toxoplasma gondii (BK) after stimulation with IFN-γ. However, the growth of a type II strain of T. gondii (ME49) was strongly inhibited by IFN-γ-activated murine MSCs. Immunity-related GTPases (IRGs) as well as guanylate-binding proteins (GBPs) contributed to this antiparasitic effect. Further analysis showed that IFN-γ-activated mMSCs also inhibit the growth of Neospora caninum, a parasite belonging to the apicomplexan group as well. Detailed studies with murine IFN-γ-activated MSC indicated an involvement in IRGs like Irga6, Irgb6 and Irgd in the inhibition of N. caninum. Additional data showed that, furthermore, GBPs like mGBP1 and mGBP2 could have played a role in the anti-N. caninum effect of murine MSCs. These data underline that MSCs, in addition to their regenerative and immunosuppressive activity, function as antiparasitic effector cells as well. However, IRGs are not present in the human genome, indicating a species-specific difference in anti-T. gondii and anti-N. caninum effect between human and murine MSCs

Repertoires and differential expression of known and predicted host-interaction genes and AP2 transcription factors.

<p>It was only possible to reliably identify orthologues for 22 SRS genes due to the way they have expanded, often being in large tandem arrays subject to gene conversion. While the AP2 transcription factors are not directly involved in host-parasite interaction they may be important in regulating expression of invasion genes. Each report card details the comparative repertoires of a particular group of genes in these species, the names of the genes specific to each organism and those which are differentially expressed between organisms. Further details of these relationships, including reference numbers, are included in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002567#ppat.1002567.s013" target="_blank">Table S4</a>. Arrows show the fold change in expression (RPKM; Reads Per Kilobase per Million mapped reads) between the two species on a log₂ scale. The ticks are 2, 6 and 8 on this scale. Green arrows highlight increased expression in <i>N. caninum</i> tachyzoites. Red arrows highlight genes with increased expression in <i>T. gondii</i> tachyzoites vs. <i>N. caninum</i> tachyzoites. A fold change is infinite where the gene is not expressed at all in one organism.</p

Protein-coding gene content and metabolic activity are largely conserved between the two species.

<p>(A) Most protein-coding genes in <i>N. caninum</i> have a one-to-one orthologous relationship (yellow) with a gene of <i>T. gondii</i>. A larger proportion of the <i>T. gondii</i> genome consists of genes with no <i>N. caninum</i> homologue than vice versa (organism-specific genes in red). The increase in shared multi-gene families (blue) in <i>N. caninum</i> reflects the expansion of SRS genes in this organism. The increase in organism-specific multigene families (red) in <i>T. gondii</i> reflects, for instance, the TSF gene family identified by us in this work. (B) Of the one-to-one orthologues shared by <i>T. gondii</i> and <i>N. caninum</i> we identified those which have orthologues in three or more non-apicomplexan eukaryotes (yellow), are not present in three or more apicomplexans but in all apicomplexan groups sequenced to date (grey), are in at least one other apicomplexan group (blue) or are specific to <i>T. gondii</i> and <i>N. caninum</i> (red). (C) Pooled day three and four RNAseq experiments were used to determine orthologous genes differentially expressed between <i>T. gondii</i> and <i>N. caninum</i>. Differentially expressed genes were examined for enrichment with enzymes from different KEGG pathways as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002567#s4" target="_blank">methods</a>. No pathways were identified with a p-value less than 0.01, although putative differences were identified at a p-value cutoff of 0.05. The width of bars in the chart relates to the number of genes in the pathway which were differentially expressed, e.g. <i>Porphyrin metabolism</i> had three enzymes differentially expressed, while <i>Nitrogen metabolism</i> had two and <i>Tyrosine metabolism</i> one. Only pathways with a p-value (adjusted for multiple hypothesis testing) below 0.1 are shown. (D) Gene Ontology terms over represented amongst genes upregulated in <i>N. caninum</i> and <i>T. gondii</i>. All terms shown are significantly upregulated (P<0.05). The terms ‘membrane’, ‘regulation of transcription, DNA-dependent’ and ‘ATP binding’ are found more often than expected in genes upregulated in <i>T. gondii</i>. SRS surface antigens, rhoptry kinases and AP2 transcription factors respectively are associated with these terms, suggesting that SRSs, ROPs and AP2s are amongst the most highly upregulated groups of genes in <i>T. gondii</i> relative to <i>N. caninum</i>. The term ‘protein amino acid phosphorylation’ is overrepresented amongst genes upregulated in <i>N. caninum</i> relative to <i>T. gondii</i>. Many of the genes in this group are rhoptry kinases suggesting that while some are upregulated in <i>T. gondii</i>, others are upregulated in <i>N. caninum</i>. These findings are explored in more detail in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002567#ppat-1002567-g003" target="_blank">Figure 3</a>.</p

Chromosomal alignment of <i>N. caninum</i> Nc-Liv and <i>T. gondii</i> Me49 highlighting surface antigen gene families.

<p>(A) Aligned chromosomes of <i>N. caninum</i> (above) and <i>T. gondii</i> (below) showing conservation of synteny and distribution of SRS and SUSA surface antigen gene families. Tandemly repeated genes are shown clustered together. Uncoloured genes had less than 20% unique sequence and expression levels could not be accurately determined. 49 additional NcSRSs were found in UnAssigned Contigs (UACs), while three further TgSRSs were not assigned to chromosomes. (B) Shows putative rearrangements between <i>N. caninum</i> and <i>T. gondii</i> chromosomes. Large (>30 kb) insertions in one genome relative to the other are numbered on the chromosomes of <i>N. caninum</i> (orange) and <i>T. gondii</i> (blue). Red ribbons show regions of protein sequence similarity between these regions. The plot shows that most insertions have a pairwise relationship, e.g. region 13 from <i>T. gondii</i> chromosome VIIa is putatively orthologous to region 24 in <i>N. caninum</i> chromosome IX. Thus these regions are shared and not specific to one organism. The arrow symbol refers to sequence similarity with parts of the comparator genome not currently assigned to chromosomes (UACs). A capital ‘T’ identifies a region with no similarity in the comparator genome. These regions include genes belonging to novel families (TSF and KRUF).</p