Rhoptry Proteins ROP5 and ROP18 Are Major Murine Virulence Factors in Genetically Divergent South American Strains of Toxoplasma gondii

Abstract

Toxoplasma gondii has evolved a number of strategies to evade immune responses in its many hosts. Previous genetic mapping of crosses between clonal type 1, 2, and 3 strains of T. gondii, which are prevalent in Europe and North America, identified two rhoptry proteins, ROP5 and ROP18, that function together to block innate immune mechanisms activated by interferon gamma (IFNg) in murine hosts. However, the contribution of these and other virulence factors in more genetically divergent South American strains is unknown. Here we utilized a cross between the intermediately virulent North American type 2 ME49 strain and the highly virulent South American type 10 VAND strain to map the genetic basis for differences in virulence in the mouse. Quantitative trait locus (QTL) analysis of this new cross identified one peak that spanned the ROP5 locus on chromosome XII. CRISPR-Cas9 mediated deletion of all copies of ROP5 in the VAND strain rendered it avirulent and complementation confirmed that ROP5 is the major virulence factor accounting for differences between type 2 and type 10 strains. To extend these observations to other virulent South American strains representing distinct genetic populations, we knocked out ROP5 in type 8 TgCtBr5 and type 4 TgCtBr18 strains, resulting in complete loss of virulence in both backgrounds. Consistent with this, polymorphisms that show strong signatures of positive selection in ROP5 were shown to correspond to regions known to interface with host immunity factors. Because ROP5 and ROP18 function together to resist innate immune mechanisms, and a significant interaction between them was identified in a two-locus scan, we also assessed the role of ROP18 in the virulence of South American strains. Deletion of ROP18 in South American type 4, 8, and 10 strains resulted in complete attenuation in contrast to a partial loss of virulence seen for ROP18 knockouts in previously described type 1 parasites. These data show that ROP5 and ROP18 are conserved virulence factors in genetically diverse strains from North and South America, suggesting they evolved to resist innate immune defenses in ancestral T. gondii strains, and they have subsequently diversified under positive selection.

Genetic Mapping of Pathogenesis Determinants inToxoplasma gondii

“…The binding of ROP5 to IRGs alters the conformation of SW1, preventing oligomerization (97) and favoring phosphorylation by ROP18 (36) (Figure 4). The interfaces of ROP5 and IRGs that interact show evidence of strong selective pressure, suggesting they have coevolved as virulence and defense effectors in a gene-for-gene manner (7,36,97). Consistent with this model, the heightened resistance of a wild-derived house mouse (Mus musculus) has been associated with expression of a novel IRG haplotype that appears to block the ability of ROP5 to recognize Irga6 (70).…”

“…Finally, recent studies extended this analysis by crossing the South American type 10 VAND strain with the type 2 ME49 strain (60), leading to the identification of ROP5 as the major determinant of virulence differences (7). Other data also support a wider role for these same few genetic determinants in mediating the acute virulence of South American strains.…”

“…This break can then be repaired by nonhomologous end joining to create frame-shift or stop mutants, or by homologous recombination to create allelic replacements or knockouts when targeting constructs are employed. CRISPR has been successfully applied to all these genetic manipulation strategies in T. gondii, and in strains that were previously refractory to genetic manipulation (7,8,71,107,112). The power of this very efficient approach promises to open a myriad of possibilities for the study of this parasite.…”

“…For example, gain-offunction studies have shown that ROP18 alleles in South American strains, which resemble the type 1 allele, are capable of conferring acute virulence when expressed in the type 3 background (62). Additionally, using CRISPR/Cas9 to disrupt the ROP18 or ROP5 loci, Behnke et al, (7) showed that these factors are also critical for the acute virulence of South American strains TgCtBr5 and TgCtBr18, which belong to clade B (see sidebar on Development of CRISPR in T. gondii ). Collectively, ROP5 and ROP18 alleles correlate with the acute virulence phenotype in all four crosses and have been functionally implicated in representative strains for five of the six known clades of T. gondii (Figure 1).…”

Serial Dissection of Parasite Gene Families

“…A double-guide-RNA approach was used previously to precisely target complete single-gene deletions in T. gondii using a CRISPR/Cas9 system (22). The new scheme devised by Long and coworkers additionally incorporates several features to enable more-reliable development of mutant parasite strains that possess multiple targeted gene deletions.…”