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

Behnke MS, Khan A, Lauron EJ, Jimah JR, Wang Q, Tolia NH, Sibley LD - PLoS Genet. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

ROP5 is a virulence factor in South American strains TgCtBr5 and TgCtBr18.(A) Population genetic network analysis using genome-wide SNPs from 16 Toxoplasma gondii reference strains plus strain TgCtBr18. Numbers represent the haplogroup of the strain and colors represent the Clade: Clade A (pink), Clade B (gray), Clade C (blue), Clade D (green), Clade E (yellow), Clade F (purple). (B) Western blot of the TgCtBr5Δrop5 clones probing with rabbit α-ROP5 (IRDye 680: red) and mAb DG52 against SAG1 (IRDye 800: green–control), Stds: protein standards. (C) CD-1 mice were i.p. injected with indicated number of parasites, five mice per clone. (D) Western blot of the TgCtBr18Δrop5 clones probing with rabbit α-ROP5 (IRDye 680: red) and mAb DG52 against SAG1 (IRDye 800: green–control), Stds: protein standards. (E) CD-1 mice were i.p. injected with indicated number of parasites, five mice per clone. The percentage of surviving mice was adjusted for those that were not infected, based on serological testing.
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pgen.1005434.g003: ROP5 is a virulence factor in South American strains TgCtBr5 and TgCtBr18.(A) Population genetic network analysis using genome-wide SNPs from 16 Toxoplasma gondii reference strains plus strain TgCtBr18. Numbers represent the haplogroup of the strain and colors represent the Clade: Clade A (pink), Clade B (gray), Clade C (blue), Clade D (green), Clade E (yellow), Clade F (purple). (B) Western blot of the TgCtBr5Δrop5 clones probing with rabbit α-ROP5 (IRDye 680: red) and mAb DG52 against SAG1 (IRDye 800: green–control), Stds: protein standards. (C) CD-1 mice were i.p. injected with indicated number of parasites, five mice per clone. (D) Western blot of the TgCtBr18Δrop5 clones probing with rabbit α-ROP5 (IRDye 680: red) and mAb DG52 against SAG1 (IRDye 800: green–control), Stds: protein standards. (E) CD-1 mice were i.p. injected with indicated number of parasites, five mice per clone. The percentage of surviving mice was adjusted for those that were not infected, based on serological testing.

Mentions: Genetic crosses have now implicated ROP5 as a major virulence factor using type 1 GT1 [9] (Clade A), type 2 ME49 (Clade D), type 3 VEG (Clade C) [7,10] and type 10 VAND (Clade F) parasites as parental strains. These strain types represent a broad, yet incomplete, spectrum of the global T. gondii genetic diversity (Fig 3A) [21,22]. Given this, we were interested to test the contribution of ROP5 to virulence in strains from Clade B, a genetically distinct group not represented by the currently available genetic crosses. Using the double-CRISPR strategy applied to generate the VANDΔrop5 strain, we created Δrop5 parasites for virulent South American type 8 TgCtBr5 and type 4 TgCtBr18 strains. Knockouts were confirmed by PCR screening of clones for integration of the selection cassette (S3A and S3B Fig), loss of the ROP5 coding region (S1B and S1C Fig), and loss of ROP5 protein expression by Western blotting (Fig 3B and 3D). Mice succumbed to infection with wild type strains of TgCtBr5 or TgCtBr18, yet when mice were infected with either TgCtBr5Δrop5 or TgCtBr18Δrop5 they survived the course of the experiment (Fig 3C and 3E). These data demonstrate that ROP5 is also a major contributor to virulence in Clade B strains of T. gondii.


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

Behnke MS, Khan A, Lauron EJ, Jimah JR, Wang Q, Tolia NH, Sibley LD - PLoS Genet. (2015)

ROP5 is a virulence factor in South American strains TgCtBr5 and TgCtBr18.(A) Population genetic network analysis using genome-wide SNPs from 16 Toxoplasma gondii reference strains plus strain TgCtBr18. Numbers represent the haplogroup of the strain and colors represent the Clade: Clade A (pink), Clade B (gray), Clade C (blue), Clade D (green), Clade E (yellow), Clade F (purple). (B) Western blot of the TgCtBr5Δrop5 clones probing with rabbit α-ROP5 (IRDye 680: red) and mAb DG52 against SAG1 (IRDye 800: green–control), Stds: protein standards. (C) CD-1 mice were i.p. injected with indicated number of parasites, five mice per clone. (D) Western blot of the TgCtBr18Δrop5 clones probing with rabbit α-ROP5 (IRDye 680: red) and mAb DG52 against SAG1 (IRDye 800: green–control), Stds: protein standards. (E) CD-1 mice were i.p. injected with indicated number of parasites, five mice per clone. The percentage of surviving mice was adjusted for those that were not infected, based on serological testing.
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getmorefigures.php?uid=PMC4546408&req=5

pgen.1005434.g003: ROP5 is a virulence factor in South American strains TgCtBr5 and TgCtBr18.(A) Population genetic network analysis using genome-wide SNPs from 16 Toxoplasma gondii reference strains plus strain TgCtBr18. Numbers represent the haplogroup of the strain and colors represent the Clade: Clade A (pink), Clade B (gray), Clade C (blue), Clade D (green), Clade E (yellow), Clade F (purple). (B) Western blot of the TgCtBr5Δrop5 clones probing with rabbit α-ROP5 (IRDye 680: red) and mAb DG52 against SAG1 (IRDye 800: green–control), Stds: protein standards. (C) CD-1 mice were i.p. injected with indicated number of parasites, five mice per clone. (D) Western blot of the TgCtBr18Δrop5 clones probing with rabbit α-ROP5 (IRDye 680: red) and mAb DG52 against SAG1 (IRDye 800: green–control), Stds: protein standards. (E) CD-1 mice were i.p. injected with indicated number of parasites, five mice per clone. The percentage of surviving mice was adjusted for those that were not infected, based on serological testing.
Mentions: Genetic crosses have now implicated ROP5 as a major virulence factor using type 1 GT1 [9] (Clade A), type 2 ME49 (Clade D), type 3 VEG (Clade C) [7,10] and type 10 VAND (Clade F) parasites as parental strains. These strain types represent a broad, yet incomplete, spectrum of the global T. gondii genetic diversity (Fig 3A) [21,22]. Given this, we were interested to test the contribution of ROP5 to virulence in strains from Clade B, a genetically distinct group not represented by the currently available genetic crosses. Using the double-CRISPR strategy applied to generate the VANDΔrop5 strain, we created Δrop5 parasites for virulent South American type 8 TgCtBr5 and type 4 TgCtBr18 strains. Knockouts were confirmed by PCR screening of clones for integration of the selection cassette (S3A and S3B Fig), loss of the ROP5 coding region (S1B and S1C Fig), and loss of ROP5 protein expression by Western blotting (Fig 3B and 3D). Mice succumbed to infection with wild type strains of TgCtBr5 or TgCtBr18, yet when mice were infected with either TgCtBr5Δrop5 or TgCtBr18Δrop5 they survived the course of the experiment (Fig 3C and 3E). These data demonstrate that ROP5 is also a major contributor to virulence in Clade B strains of T. gondii.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus