The Toxoplasma gondii Rhoptry Kinome Is Essential for Chronic Infection

Abstract

Ingestion of the obligate intracellular protozoan parasite Toxoplasma gondii causes an acute infection that leads to chronic infection of the host. To facilitate the acute phase of the infection, T. gondii manipulates the host response by secreting rhoptry organelle proteins (ROPs) into host cells during its invasion. A few key ROP proteins with signatures of kinases or pseudokinases (ROPKs) act as virulence factors that enhance parasite survival against host gamma interferon-stimulated innate immunity. However, the roles of these and other ROPK proteins in establishing chronic infection have not been tested. Here, we deleted 26 ROPK gene loci encoding 31 unique ROPK proteins of type II T. gondii and show that numerous ROPK proteins influence the development of chronic infection. Cyst burdens were increased in the Δrop16 knockout strain or moderately reduced in 11 ROPK knockout strains. In contrast, deletion of ROP5, ROP17, ROP18, ROP35, or ROP38/29/19 (ROP38, ROP29, and ROP19) severely reduced cyst burdens. Δrop5 and Δrop18 knockout strains were less resistant to host immunity-related GTPases (IRGs) and exhibited >100-fold-reduced virulence. ROP18 kinase activity and association with the parasitophorous vacuole membrane were necessary for resistance to host IRGs. The Δrop17 strain exhibited a >12-fold defect in virulence; however, virulence was not affected in the Δrop35 or Δrop38/29/19 strain. Resistance to host IRGs was not affected in the Δrop17, Δrop35, or Δrop38/29/19 strain. Collectively, these findings provide the first definitive evidence that the type II T. gondii ROPK proteome functions as virulence factors and facilitates additional mechanisms of host manipulation that are essential for chronic infection and transmission of T. gondii

Importance: Reactivation of chronic Toxoplasma gondii infection in individuals with weakened immune systems causes severe toxoplasmosis. Existing treatments for toxoplasmosis are complicated by adverse reactions to chemotherapy. Understanding key parasite molecules required for chronic infection provides new insights into potential mechanisms that can interrupt parasite survival or persistence in the host. This study reveals that key secreted rhoptry molecules are used by the parasite to establish chronic infection of the host. Certain rhoptry proteins were found to be critical virulence factors that resist innate immunity, while other rhoptry proteins were found to influence chronic infection without affecting virulence. This study reveals that rhoptry proteins utilize multiple mechanisms of host manipulation to establish chronic infection of the host. Targeted disruption of parasite rhoptry proteins involved in these biological processes opens new avenues to interfere with chronic infection with the goal to either eliminate chronic infection or to prevent recrudescent infections.

FIG 1

Rhoptry kinome molecules are essential for chronic infection. C57BL/6 mice were infected i.p. with 200 tachyzoites of each strain, and brain cyst burdens were measured 21 days postinfection. Data shown are cumulative results from one to three independent experiments for each strain tested. ROPK knockout strains and the Δku80 parent strain are organized in descending order by cyst number for each strain, with cyst numbers shown as means plus standard errors of the means (SEM) (error bars). P values were calculated by a Student’s t test, and a P < 0.05 (shown in purple or red color) was considered significant. Data on the ROPK knockout strains and the Δku80 parent strain follow: Δrop16 (two experiments; n = 6 mice; P = 0.0242), Δku80 parent (three experiments; n = 12 mice), Δrop42/43/44 (one experiment; n = 4 mice; P = 0.7188), Δrop39 (two experiments; n = 7 mice; P = 0.5691), Δrop23 (one experiment; n = 4 mice; P = 0.5232), Δrop45 (one experiment; n = 4 mice; P = 0.4186), Δrop24 (one experiment; n = 4 mice; P = 0.4259), Δrop32 (one experiment; n = 4 mice; P = 0.1651), Δrop11 (two experiments; n = 7 mice; P = 0.0703), Δrop20 (two experiments; n = 6 mice; P = 0.0534), Δrop28 (one experiment; n = 4 mice; P = 0.0928), Δrop26 (two experiments; n = 5 mice; P = 0.0500), Δrop41 (two experiments; n = 5 mice; P = 0.0384), Δrop37 (two experiments; n = 5 mice; P = 0.0154), Δrop4/7 (three experiments; n = 12 mice; P = 0.0198), Δrop36 (two experiments; n = 6 mice; P = 0.0070), Δrop31 (two experiments; n = 6 mice; P = 0.0058), Δrop21 (two experiments; n = 7 mice; P = 0.0036), Δrop40 (one experiment; n = 4 mice; P = 0.0217), Δrop27 (two experiments; n = 6 mice; P = 0.0073), Δrop25 (two experiments; n = 7 mice; P = 0.0013), Δrop22 (two experiments; n = 5 mice; P = 0.0095), Δrop2/8 (one experiment; n = 4 mice; P = 0.0063), Δrop38/29/19 (two experiments; n = 8 mice; P < 0.0001), Δrop35 (two experiments; n = 8 mice; P < 0.0001), Δrop18 (two experiments; n = 8 mice; P < 0.0001), Δrop17 (two experiments; n = 8 mice; P < 0.0001), and Δrop5 (three experiments; n = 9 mice; P < 0.0001).

FIG 2

ROPK gene deletions with the most severe defects in cyst burdens differentiate normally in vitro. (A) Infected host cells were treated with bradyzoite inducing conditions (pH 8.1 and CO₂ depletion in ambient air) for 3 days. The cyst wall was stained with Dolichos biflorus agglutinin (DBA) (shown in red). Bradyzoites were visualized by expression of GFP (shown in green), which is under control of the bradyzoite stage-specific LDH2 promoter. Host cell and parasite nuclei were stained with DAPI (shown in blue). Samples were imaged by confocal microscopy, and vacuoles were located using differential interference contrast (DIC) microscopy. Representative results are shown for each strain. (B) C57BL/6 mice were infected i.p. with 200 tachyzoites of each strain (low-dose infection) or with 2 × 10⁵ or 2 × 10⁶ tachyzoites of each strain (high-dose infection), and brain cyst burdens were measured. The values shown represent means plus SEM (error bars). Data shown are cumulative results from one to three independent experiments for each strain tested. Data on the ROPK knockout strains and the Δku80 parent strain follow: Δku80 parent low dose (three experiments; n = 12 mice), Δrop5 low dose (three experiments; n = 9 mice), Δrop17 low dose (two experiments; n = 8 mice), Δrop18 low dose (two experiments; n = 8 mice), Δrop5 high dose (three experiments; n = 6 mice), Δrop17 high dose (one experiment; n = 4 mice), and Δrop18 high dose (two experiments; n = 4 mice).

FIG 3

Complementation of Δrop17 and Δrop18 with the corresponding wild-type gene alleles rescues cyst burdens. C57BL/6 mice were infected i.p. with 200 tachyzoites of each strain, and brain cyst burdens were measured 21 days postinfection. Data shown are cumulative results from one to three independent experiments for each strain tested. Data on the Δrop17 and Δrop18 knockout strains and the Δku80 parent strain follow: Δku80 parent (three experiments; n = 12 mice), Δrop5 (three experiments; n = 9 mice), Δrop5::ROP5A (two experiments; n = 8 mice), Δrop5::ROP5C (two experiments; n = 8 mice), Δrop17 (two experiments; n = 8 mice), Δrop17::ROP17^FLHA (one experiment; n = 4 mice), Δrop18 (two experiments; n = 8 mice), Δrop18::ROP18 (two experiments; n = 8 mice), Δrop18::ROP18^KD (two experiments; n = 8 mice), Δrop18::ROP18^RAH2(ATF) (two experiments; n = 8 mice). The values shown represent means ± SEM. The P values were calculated with a Student’s t test. P values that were significantly different are indicated by a bar and four asterisks (P < 0.0001). P values that were not significantly different (ns) are also indicated.

FIG 4

Deletion of ROP17 reduces plaque size and induces early egress. (A) Monolayers of human foreskin fibroblast (HFF) host cells were infected with ~200 tachyzoites of the indicated strains. PFU were visualized 13 days after infection by staining with Coomassie brilliant blue and photographed. The results shown are representative of three independent experiments. (B) Monolayers of HFF cells were infected with the indicated strains, and noninvaded parasites were removed from cultures at 30 or 60 min postinfection and PFU were determined. The infection rate was calculated by comparison to cultures where parasites were not removed (total PFU), and the percent infection rate was determined. Results are from cumulative data obtained in three independent experiments and are shown as means ± SEM (error bars). The values were not significantly different (ns) at 30 min or at 60 min postinfection, calculated with a Student’s t test. (C) Monolayers of HFF host cells were infected at an MOI of ~0.01, and noninvaded parasites were removed 1 h postinfection. At 21, 45, 68, or 72 h after infection, cultures were scored microscopically to measure the percentage of secondary infection sites. Results are from cumulative data obtained in three independent experiments and are shown as means ± SEM. The P values were calculated with a Student’s t test and are indicated as follows: **, P < 0.01; ***, P < 0.001; ns, not significant.

FIG 5

Type II ROP5 and ROP18 resist Irgb6 coating and killing of the PV. (A) MEFs were stimulated with IFN-γ, and parasite survival (measured as PFU) was determined in comparison to nonstimulated MEFs. Results are from at least four independent experiments and are shown as means plus SEM. Significant P values were calculated with a Student’s t test. The values for all strains shown in red were not significantly different from the value for the Δku80 parent. The values for all strains shown in green were significantly different (P < 0.0001) from the value for the Δku80 parent strain. (B) Quantification of Irgb6 coating of PVs 45 min after infection of IFN-γ-stimulated bone marrow-derived macrophages. Representative images are shown of PVs stained with anti-Irgb6 (shown in green) and anti-GRA5 (shown in red). At least 500 PVs were scored to determine significance. Significant P values were calculated with a Student’s t test. The values for all strains shown in red were not significantly different from the value for the Δku80 parent. The values for all strains shown in green were significantly different from the value for the Δku80 parent strain. The P values for the knockout strains shown in green are listed in parentheses after the genotype: Δrop18 (P < 0.0001), Δrop18::ROP18^KD (P < 0.0001), Δrop18::ROP18^RAH2(ATF) (P < 0.0001), Δrop5 (P < 0.0013), Δrop5::ROP5A (P < 0.0001), and Δrop5::ROP5C (P < 0.0006).

FIG 6

Type II parasites deficient in ROP5 and ROP18 are less virulent than parasites deficient in ROP17, ROP35, or ROP38/29/19. The Δrop5, Δrop17, Δrop18, Δrop35, and Δrop38/29/19 mutants and complemented strains were evaluated for virulence lethality in mice. (A) C57BL/6 mice were infected i.p. with 2 × 10⁵ tachyzoites of the Δrop5, Δrop17, or Δrop18 knockout strain or the Δku80 parent strain. Data shown are combined from two independent experiments, each with four mice per group. The P values were calculated by a log rank Mantel-Cox test, and a P of <0.05 was considered significant. (B) C57BL/6 IFN-γ^−/− knockout mice were infected i.p. with 2 × 10² tachyzoites of the Δrop5, Δrop17, or Δrop18 strain or the Δku80 parental strain. Data shown are from a single experiment with four mice per group. The P values were calculated by a log rank Mantel-Cox test, and a P of <0.05 was considered significant. ns, not significant. (C) C57BL/6 mice were infected i.p. with 2 × 10⁶ tachyzoites of the Δrop5, Δrop17, or Δrop18 strain or the Δku80 parental strain or with 2 × 10⁶ tachyzoites of the Δrop5::ROP5A or Δrop5::ROP5C [shown together as Δrop5::ROP5A/C], Δrop17::ROP17^FLHA, Δrop18::ROP18, Δrop18::ROP18^KD, or Δrop18::ROP18^RAH2(ATF) complemented strain. Data shown are combined from two independent experiments, each with four mice per group. The P values were calculated by a log rank Mantel-Cox test, and a P of <0.05 was considered significant. (D) C57BL/6 mice were infected i.p. with 2 × 10⁷ tachyzoites of the Δrop5, Δrop17, or Δrop18 strain or the Δku80 parental strain. Data shown are combined from two independent experiments, each with four mice per group. The P values were calculated by a log rank Mantel-Cox test, and a P of <0.05 was considered significant. (E) C57BL/6 mice were infected i.p. with 2 × 10⁵ tachyzoites of the Δrop35 or Δrop38/29/19 strain or the Δku80 parent strain. Data shown are combined from two independent experiments, each with four mice per group.