The population genetic structure of Toxoplasma gondii was determined by multilocus restriction fragment length polymorphism analysis at 6 loci in 106 independent isolates from humans and animals. Phylogenetic and statistical analyses indicated a highly unusual population structure consisting of 3 widespread clonal lineages. Extensively mixed genotypes were only apparent in 4 strains, which indicated that, while not separate species, sexual recombination between the 3 lineages is exceedingly rare in natural populations. T. gondii is a major cause of subclinical human infection and an important opportunistic pathogen that causes severe disease in immunocompromised patients. While strains from all 3 lineages were isolated from humans, the majority of human toxoplasmosis cases were associated with strains of a type II genotype. The correlation of specific clonal lineages with human toxoplasmosis has important implications for development of vaccines, drug treatments, and diagnostic protocols.

The protozoan Toxoplasma gondii is a prevalent parasite in wild and domestic animals worldwide, being transmitted through the food chain by carnivorous feeding and scavenging. Toxoplasma normally divides asexually to yield a haploid form that can infect virtually any vertebrate but it also has a well defined sexual cycle that occurs exclusively in cats. Toxoplasma has become important as an often fatal opportunistic pathogen in patients with AIDS, although the 15-85% of adult human populations that are chronically infected with T. gondii are typically asymptomatic. Infections in immunocompromised hosts have variable outcomes. For example, only 30 to 50% of AIDS patients that are chronically infected with the parasite develop toxoplasmic encephalitis and only about half of acute maternal infections result in congenital disease of the newborn. T. gondii strains differ in their virulence in animals, but the extent to which different strains are related has not been determined. Here we analyse 28 strains from a variety of hosts on five continents and find that the ten virulent strains have an essentially identical genotype, whereas the nonvirulent strains are moderately polymorphic. These data strongly suggest that virulent strains of T. gondii originated from a single lineage which has remained genetically homogeneous despite being globally widespread, and despite the ability of this organism to reproduce sexually.

Toxoplasma gondii has become a model for studying the phylum Apicomplexa, in part due to the availability of excellent genetic tools. Although reverse genetic tools are available in a few widely utilized laboratory strains, they rely on special genetic backgrounds that are not easily implemented in natural isolates. Recent progress in modifying CRISPR (clustered regularly interspaced short palindromic repeats), a system of DNA recognition used as a defense mechanism in bacteria and archaea, has led to extremely efficient gene disruption in a variety of organisms. Here we utilized a CRISPR/CAS9-based system with single guide RNAs to disrupt genes in T. gondii. CRISPR/CAS9 provided an extremely efficient system for targeted gene disruption and for site-specific insertion of selectable markers through homologous recombination. CRISPR/CAS9 also facilitated site-specific insertion in the absence of homology, thus increasing the utility of this approach over existing technology. We then tested whether CRISPR/CAS9 would enable efficient transformation of a natural isolate. Using CRISPR/CAS9, we were able to rapidly generate both rop18 knockouts and complemented lines in the type I GT1 strain, which has been used for forward genetic crosses but which remains refractory to reverse genetic approaches. Assessment of their phenotypes in vivo revealed that ROP18 contributed a greater proportion to acute pathogenesis in GT1 than in the laboratory type I RH strain. Thus, CRISPR/CAS9 extends reverse genetic techniques to diverse isolates of T. gondii, allowing exploration of a much wider spectrum of biological diversity.

Preface Toxoplasma gondii is a common parasite of animals and humans that can cause serious opportunistic infections. However, the majority of infections are asymptomatic possibly because the organism has co-evolved with its many vertebrate hosts and has developed multiple strategies to persist asymptomatically for the lifetime of the host. Over the past two decades, infection studies in the mouse, combined with forward genetic approaches aimed at unraveling the molecular basis of infection, have revealed that T. gondii virulence is mediated, in part, by secretion of effector proteins into the host cell during invasion. Here, we review recent advances that illustrate how these virulence factors disarm innate immunity and promote survival of the parasite.

The Apicomplexa are a phylum of diverse obligate intracellular parasites including Plasmodium spp., the cause of malaria; Toxoplasma gondii and Cryptosporidium parvum, opportunistic pathogens of immunocompromised individuals; and Eimeria spp. and Theileria spp., parasites of considerable agricultural importance. These protozoan parasites share distinctive morphological features, cytoskeletal organization, and modes of replication, motility, and invasion. This review summarizes our current understanding of the cytoskeletal elements, the properties of cytoskeletal proteins, and the role of the cytoskeleton in polarity, motility, invasion, and replication. We discuss the unusual properties of actin and myosin in the Apicomplexa, the highly stereotyped microtubule populations in apicomplexans, and a network of recently discovered novel intermediate filament-like elements in these parasites

Virulence in Toxoplasma gondii is strongly influenced by the genotype of the parasite. Type I strains uniformly cause rapid death in mice regardless of the host genotype or the challenge dose. In contrast, the outcome of infections with type II strains is highly dependent on the challenge dose and the genotype of the host. To understand the basis of acute virulence in toxoplasmosis, we compared low and high doses of the RH strain (type I) and the ME49/PTG strain (type II) of T. gondii in outbred mice. Differences in virulence were reflected in only modestly different growth rates in vivo, and both strains disseminated widely to different tissues. The key difference in the virulent RH strain was the ability to reach high tissue burdens rapidly following a low dose challenge. Lethal infections caused by type I (RH) or type II (PTG) strain infections were accompanied by extremely elevated levels of Th1 cytokines in the serum, including IFN-γ, TNF-α, IL-12, and IL-18. Extensive liver damage and lymphoid degeneration accompanied the elevated levels of cytokines produced during lethal infection. Increased time of survival following lethal infection with the RH strain was provided by neutralization of IL-18, but not TNF-α or IFN-γ. Nonlethal infections with a low dose of type II PTG strain parasites were characterized by a modest induction of Th1 cytokines that led to control of infection and minimal damage to host tissues. Our findings establish that overstimulation of immune responses that are normally necessary for protection is an important feature of acute toxoplasmosis.

Summary CD8α+ dendritic cells (DCs) are important in vivo for cross-presentation of antigens derived from intracellular pathogens and tumors. Additionally, secretion of interleukin-12 (IL-12) by CD8α+ DCs suggests a role for these cells in response to Toxoplasma gondii antigens, although it remains unclear whether these cells are required for protection against T. gondii infection. Towards this goal, we examined T. gondii infection of Batf3−/− mice, which selectively lack only lymphoid-resident CD8α+ DCs and related peripheral CD103+ DCs. Batf3−/− mice were extremely susceptible to T. gondii infection, with decreased production of IL-12 and interferon-γ. IL-12 administration restored resistance in Batf3−/− mice, and mice in which IL-12 production was ablated only from CD8α+ DCs failed to control infection. These results reveal that the function of CD8α+ DCs extends beyond a role in cross-presentation and includes a critical role for activation of innate immunity through IL-12 production during T. gondii infection.

Host cell invasion by apicomplexan parasites requires coordinated interactions between cell surface adhesins and the parasite cytoskeleton. We have identified a complex of parasite proteins, including the actin binding protein aldolase, which specifically interacts with the C-terminal domains of several parasite adhesins belonging to the thrombospondin-related anonymous protein (TRAP) family. Binding of aldolase to the adhesin was disrupted by mutation of a critical tryptophan in the C domain, a residue that was previously shown to be essential for parasite motility. Our findings reveal a potential role for aldolase in connecting TRAP family adhesins with the cytoskeleton, and provide a model linking adhesion with motility in apicomplexan parasites.