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A Secreted Serine-Threonine Kinase Determines Virulence in the Eukaryotic Pathogen Toxoplasma gondii

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Science  15 Dec 2006:
Vol. 314, Issue 5806, pp. 1776-1780
DOI: 10.1126/science.1133643

Abstract

Toxoplasma gondii strains differ dramatically in virulence despite being genetically very similar. Genetic mapping revealed two closely adjacent quantitative trait loci on parasite chromosome VIIa that control the extreme virulence of the type I lineage. Positional cloning identified the candidate virulence gene ROP18, a highly polymorphic serine-threonine kinase that was secreted into the host cell during parasite invasion. Transfection of the virulent ROP18 allele into a nonpathogenic type III strain increased growth and enhanced mortality by 4 to 5 logs. These attributes of ROP18 required kinase activity, which revealed that secretion of effectors is a major component of parasite virulence.

Toxoplasma gondii is a widespread protozoan parasite that chronically infects ∼25% of the world's human population (1). T. gondii is dominated by three widespread, clonal lineages, which rapidly expanded following a severe genetic bottleneck ∼10,000 years ago (2, 3). Despite ∼98% genetic identity, dramatic differences in virulence exist between T. gondii strains (4). Type I strains cause lethal infection in all strains of laboratory mice even at low inocula [lethal dose (LD100) ≈ 1] (4, 5), whereas types II and III strains are much less virulent [median lethal dose (LD50) ≥ 103] (4). Acute virulence is associated with rapid dissemination, high parasite burdens, and overproduction of T helper cell type 1 cytokines (6, 7). Toxoplasmosis has primarily been associated with type II strains, whereas type III strains are rarely associated with disease (8, 9). Although less prevalent, type I can cause severe congenital infections (10), ocular toxoplasmosis (11, 12), and encephalitis in AIDS patients (13). Acute virulence of T. gondii in the mouse model is genetically determined (14), although the genes involved in this phenotype are unknown.

The extreme linkage disequilibrium of T. gondii populations (4, 9) limits the use of association or population-based studies for identifying virulence genes. Therefore, we used forward genetic mapping to identify genes that determine natural differences in virulence. The recently completed genome map of the 14 chromosomes of the T. gondii ∼65-megabase haploid genome provided a framework for quantitative trait locus (QTL) mapping with 175 informative genetic markers (15, 16). Genomewide QTL mapping was used to analyze the genetic basis of acute virulence in 34 independent progeny from a genetic cross between the virulent type I strain GT-1 and the nonvirulent type III strain CTG (14). These parental strains differ in a number of virulence-related phenotypes including (i) migration under soft agarose, (ii) transmigration across polarized epithelia, (iii) intracellular replication, (iv) acute mortality in the mouse model, and (v) serum response of animals surviving low-dose infection (17).

Toxoplasma gondii and related apicomplexan parasites actively invade mammalian cells by using actin-based motility (18), which also enables passage across polarized epithelia and through tissues. Migration is enhanced in type I strains, and this may contribute to dissemination and, hence, acute virulence (19). In the present study, migration was monitored with two in vitro assays: migration under soft agarose and transmigration across polarized epithelia (17, 19). The progeny from the cross showed correlated responses for migration (MIG) and transmigration (TM) (r = 0.75 linear regression) that ranged from low levels like those of type III to levels that exceeded the type I parent (Fig. 1, A and B, and table S1).

Fig. 1.

Phenotypic analysis of virulence traits in T. gondii. (A) Migration under soft agarose (17) for the parental strains and the progeny in rank order. (B) Transmigration across MDCK monolayers in vitro (17). (C) Cumulative percent mortality in CD1 mice (17). Black boxes, 100% mortality. Serology responses (17) in surviving mice: negative, red; weak positive, yellow; and strong positive, green. See fig. S1 and table S1.

Acute mortality (MOR) of the progeny was tested in outbred CD1 mice challenged with different doses of parasites by intraperitoneal (i.p.) inoculation (17). Although the type III parental strain demonstrated very low cumulative mortality [defined as the number of deaths per number of animals infected (17)], type I was uniformly lethal, even at low inocula (Fig. 1C and table S1). Consistent with a multigenic trait, mortality caused by the progeny clones ranged from low levels to 100% (Fig. 1C and table S1). Animals that survived challenge were subsequently tested for serological responses to parasite antigens by Western blot (14, 17). Serum responses (SER) fell into three distinct categories: negative (not infected), weak positive, and strong positive (Fig. 1C and fig. S1). Intracellular growth (GRO) rates for individual progeny (17) were correlated with virulence (table S1), which is consistent with previous observations of the parental lines (20).

Genomewide scans were used to locate QTLs in the parasite genome that control these quantitative traits (17). Remarkably, a single strong primary association was detected for each of the MIG, TM, MOR, and SER phenotypes, which were clustered at the same region of parasite chromosome VIIa (chr VIIa) (P < 0.001) (Fig. 2A). A major component of GRO also mapped to the same segment of chr VIIa, although with lesser statistical significance (Fig. 2A). Secondary associations for MOR and SER were also noted on chr Ia, and for GRO on chr XI and XII (Fig. 2A) (17). Analyses of two-locus interactions for SER and MOR showed a significant interaction only for the QTLs on VIIa and Ia (Fig. 2A). When we controlled for the major QTL for MOR/SER statistically (17), secondary scans did not reveal any other major peaks contributing to these phenotypes. The extremely strong support for the highly clustered QTLs on chr VIIa indicates that a few closely linked genes determine virulence.

Fig. 2.

Genomewide QTL scans for virulence phenotypes. (A) Migration, transmigration, mortality, and serum response mapped to chr VIIa. Growth mapped to chr VIIa and chr XI; the chr XII peak is attributable to effects of drug-resistance mutations (17). Markers are equally spaced, except chr VIIa, which is plotted by physical scale. Chromosome names listed at the top. Dotted lines indicate significance levels; red lines, log likelihood plots; blue lines, 95% confidence intervals (17). (B) Fine mapping of QTLs on chr VIIa and positioning on the genome scaffolds. Mortality (MOR) and serum response (SER) showed an overlapping peak at CS3 to ST3921. MIG and TM mapped to a broad region with a peak at ST4000 to ST1170. Markers (black bars) are listed below the sequenced genome scaffolds (gray rectangles with TGG scaffold numbers) and their corresponding positions are shown in centimorgans on the x axis.

Fine mapping of the QTLs on chr VIIa was aided by isolation of additional recombinant progeny (17). The primary MOR/SER QTL mapped to a region spanning the CS3 to ST3921 markers with a peak at marker ST3921 (Fig. 2B and table S2). MIG and TM mapped to a broad region of chr VIIa between genetic markers CS12 and CS11 (Fig. 2B), with a peak at markers ST4000 and ST1107 that lie within 28 kilobases (kb) of each other (table S2). Smaller secondary peaks were also observed for MIG and TM, and these partially overlapped with the major SER/MOR QTL (Fig. 2B). These findings indicate that a broad QTL on chr VIIa contributes to MIG/TM, and a second sharper QTL controls MOR/SER (Fig. 2B).

Pinning the QTLs to the sequenced genome scaffolds identified corresponding genomic regions and candidate genes that may control these phenotypes (Fig. 2B and table S2). The MIG/TM QTL contains several excellent candidate genes that may mediate enhanced migration, including two formin homology domain proteins, a calcium-dependent protein kinase (CDPK), and a unique myosin VI–like gene known as MyoK (table S2). The traits MOR/SER map to a distinct QTL of 140 kb containing 21 genes (Fig. 2B and table S2), representing a 10-fold improvement in resolution over previous mapping of this QTL (14). Analysis of the 21 genes in this region with the extensive expressed sequence tag (EST) database for T. gondii (21, 22) revealed that only ROP18 contained abundant type I polymorphisms. ROP18 is a member of a family of parasite proteins that are secreted from apical organelles called rhoptries (23). The type III and type I alleles of ROP18 differed at 78 positions in a total of 541 residues (∼14%) (fig. S2A), compared with the typical ∼1% divergence between lineages (2, 3). Microarray and quantitative real-time fluorescence polymerase chain reaction (QPCR) analyses indicated that only ROP18 showed significant differences in expression; type III was almost undetectable relative to type I (fig. S2, B and C). The high polymorphism and dramatic difference in expression indicate that ROP18 is the basis for the dramatic difference in acute virulence.

To evaluate the contribution of ROP18 to virulence, we expressed an epitope-tagged copy of the type I allele (ROP18-I) in the wild-type III strain CTG, which effectively provides a null background. Two transformants expressing Ty epitope–tagged ROP18-I were selected for comparison with a control expressing only the selectable marker (Fig. 3A). QPCR revealed that ROP18-I was expressed in the transgenic lines at levels comparable to those of the wild-type I strain (fig. S2C). Ty epitope–tagged ROP18-I was found in early parasitophorous vacuoles (PVs), where it overlapped with the rhoptry marker ROP1, and in vesicular structures in the host cell cytosol (Fig. 3B, arrows). Cryoimmuno–electron microscopy confirmed that ROP18 was found in rhoptries and released into the PV (Fig. 3C and fig. S3). In the presence of cytochalasin D, which allows apical secretion but blocks parasite entry (17, 24), ROP18-I was secreted abundantly into the host cell cytosol (fig. S4), as are other rhoptry proteins (24). Previous studies have indicated that ROP2 (25) and ROP5 (26) are exposed to the host cell cytosol, which suggests that after secretion into the host cell and targeting to the PV, ROP18 is also in direct contact with the host cell.

Fig. 3.

Localization and homology modeling of ROP18 in T. gondii. (A) Diagram of ROP18-I Ty construct. Western blot analysis of ROP18-I transformant clones (K1, V1) versus control transformant (Ctl) (17). (B) Immunofluorescence localization of ROP18-I in transformant (V1 clone) vs. control (17). ROP18-I (green) was secreted into early PVs and into vesicles in the cytosol (arrows). In late PVs, ROP18-I accumulated in the rhoptries of mature daughter cells. Rhoptry marker ROP1 (red), nuclei stained with DAPI (4′,6′-diamidino-2-phenylindole) (blue). Scale bars, 5 μm. (C) Immuno–electron microscopy localization of ROP18-I in rhoptries (R) but not micronemes (M) or the nucleus (N) (17). ROP18-I was secreted into the PV (arrowheads in right image). Scale bars, 200 nm. (D) Homology model of ROP18-I (blue) based on Tao2 kinase (yellow) (17). (E) Conserved active site residues in PKCα and ROP18-I kinase domains.

To evaluate the contribution of ROP18 to growth, we measured the intracellular replication of ROP18-I transformants in vitro. Expression of ROP18-I in the slower-growing type III CTG strain led to a twofold increase in the average number of parasites per vacuole (Fig. 4A). When extrapolated over an 8- to 10-day period, this difference could account for the much higher tissue burdens following in vivo infection with virulent type I strains (6, 7). Consistent with this, challenge of outbred CD1 mice with CTG transformants expressing ROP18-I revealed a 4 to 5 log increase in virulence when compared with the wild-type III CTG strain (Fig. 4B). Collectively, these results establish that expression of the type I allele of ROP18 in the non-virulent type III background is sufficient for conferring acute virulence of T. gondii. In an accompanying report, Saeij et al. demonstrate that ROP18 also contributes to differences in pathogenesis between the less-virulent type II and III strains.

Fig. 4.

Enhanced intracellular growth and virulence of ROP18-I transformants in mice. (A) ROP18-I transformants grew more rapidly, which led to larger vacuole sizes (*) and an average of two times as many parasites per vacuole at 40 hours post infection. (B) Mouse survival curves for parental and ROP18-I transgenic lines. Transformants expressing ROP18-I (clones K1 and V1) showed a ∼4 to 5 log increase in virulence compared with the type III strain CTG, and death occurred with kinetics similar to those of the virulent type I strain GT-1. ROP18-I kinase mutants (Asp394Ala) (clones H1, L1) remained avirulent. Serological responses in survivors: negative (–), weak positive (+), strong positive (++).

ROP18 is related to the ROP2 subfamily of rhoptry proteins that share a conserved serine-threonine protein kinase domain (27). All of the conserved residues important for serine-threonine kinase activity are present in ROP18 and homology modeling revealed that ROP18 contains a typical kinase domain fold, complete with a highly conserved catalytic active site (17), similar to the serine-threonine kinases Tao2 and protein kinase Cα (PKCα) (Fig. 3, D and E). To test the role of kinase activity in the phenotype conferred by ROP18, a point mutation disrupting kinase activity (Asp394Ala) (28) was expressed as a stable transgene in T. gondii (fig. S5). Type III transformants carrying the Asp394Ala mutation of ROP18-I did not show enhanced growth and remained avirulent (Fig. 4B), which demonstrated that ROP18 requires kinase activity for mediating enhanced virulence. Although all three alleles of ROP18 contain the highly conserved active-site residues, the type III allele contains significant changes that may alter substrate binding, regulation, or activity (fig. S2A).

Collectively, our data suggest that, after secretion into the host cell, ROP18 kinase acts on a cellular target to enhance parasite infection. Consequently, ROP18 is a prime candidate for mediating previously reported alterations in host cell signaling (29), prevention of apoptosis (30), and altered gene expression (31), in T. gondii– infected cells. Although ROP18 is the major factor controlling acute mortality, ROP18-I transformants did not show enhanced migration (fig. S6), which indicates that other nearby genes mediate this phenotype. The close linkage of these QTLs on chr VIIa is consistent with co-adaptation. In the current highly clonal T. gondii population structure (2, 3), these coinherited QTLs may underpin the shared acute virulence of type I strains and may also contribute to pathogenesis in strains with mixed or exotic genotypes, which have been implicated in severe ocular toxoplasmosis (11, 12).

Our findings identify a major role for ROP18 in mediating pathogenesis in T. gondii and suggest that both expression level and allelic differences contribute to acute virulence. Conserved serine-threonine kinase domains are found in a variety of other rhoptry proteins in T. gondii (27) and also in secretory proteins in Plasmodium (32), which implicates secretion of effector proteins into the host cell as a general mechanism of virulence in eukaryotic pathogens.

Supporting Online Material

www.sciencemag.org/cgi/content/full/314/5806/1776/DC1

Materials and Methods

Figs. S1 to S6

Tables S1 to S3

References and Notes

References and Notes

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