Laura J. Knoll

Parasite Stage-Specific Recognition of Endogenous Toxoplasma gondii-Derived CD8+ T Cell Epitopes

BACKGROUND BALB/c mice control infection with the obligate intracellular parasite Toxoplasma gondii and develop a latent chronic infection in the brain, as do immunocompetent humans. Interferon-gamma-producing CD8+ T cells provide essential protection against T. gondii infection, but the epitopes recognized have so far remained elusive. METHODS We employed caged major histocompatibility complex molecules to generate approximately 250 H-2L(d) tetramers and to distinguish T. gondii-specific CD8+ T cells in BALB/c mice. RESULTS We identified 2 T. gondii-specific H-2L(d)-restricted T cell epitopes, one from dense granule protein GRA4 and the other from rhoptry protein ROP7. H-2L(d)/GRA4 reactive T cells from multiple organ sources predominated 2 weeks after infection, while the reactivity of the H-2L(d)/ROP7 T cells peaked 6-8 weeks after infection. BALB/c animals infected with T. gondii mutants defective in establishing a chronic infection showed altered levels of antigen-specific T cells, depending on the T. gondii mutant used. CONCLUSIONS Our results shed light on the identity and the parasite stage-specificity of 2 CD8+ T cell epitopes recognized in the acute and chronic phase of infection with T. gondii.

Discovery of parasite virulence genes reveals a unique regulator of chromosome condensation 1 ortholog critical for efficient nuclear trafficking

Eukaryotic parasites are a leading cause of morbidity and mortality worldwide, yet little is known about the genetic basis of their virulence. Here, we present a forward genetic screen to study pathogenesis in the protozoan parasite Toxoplasma gondii. By using modified signature-tagged mutagenesis, the growth of 6,300 T. gondii insertional mutants was compared in cell culture and murine infection to identify genes required specifically in vivo. One of the 39 avirulent mutants is disrupted in a divergent ortholog of the regulator of chromosome condensation 1 (RCC1), which is critical for nuclear trafficking in model systems. Although this RCC1 mutant grows similar to wild type in standard tissue culture conditions, it is growth-impaired under nutrient limitation. Genetic complementation of mutant parasites with the T. gondii RCC1 gene fully restores both virulence in mice and growth under low-nutrient conditions. Further analysis shows that there is a significant defect in nuclear trafficking in the RCC1 mutant. These findings suggest that the rate of nuclear transport is a critical factor affecting growth in low-nutrient conditions in vivo and in vitro. Additionally, we observed that although RCC1 proteins are highly conserved in organisms from humans to yeast, no protozoan parasite encodes a characteristic RCC1. This protein divergence may represent a unique mechanism of nucleocytoplasmic transport. This study illustrates the power of this forward genetics approach to identify atypical virulence mechanisms.

A HT/PEXEL Motif in Toxoplasma Dense Granule Proteins is a Signal for Protein Cleavage but not Export into the Host Cell

Apicomplexan parasites, such as Toxoplasma gondii and Plasmodium, secrete proteins for attachment, invasion and modulation of their host cells. The host targeting (HT), also known as the Plasmodium export element (PEXEL), directs Plasmodium proteins into erythrocytes to remodel the host cell and establish infection. Bioinformatic analysis of Toxoplasma revealed a HT/PEXEL‐like motif at the N‐terminus of several hypothetical unknown and dense granule proteins. Hemagglutinin‐tagged versions of these uncharacterized proteins show co‐localization with dense granule proteins found on the parasitophorous vacuole membrane (PVM). In contrast to Plasmodium, these Toxoplasma HT/PEXEL containing proteins are not exported into the host cell. Site directed mutagenesis of the Toxoplasma HT/PEXEL motif, RxLxD/E, shows that the arginine and leucine residues are permissible for protein cleavage. Mutations within the HT/PEXEL motif that prevent protein cleavage still allow for targeting to the PV but the proteins have a reduced association with the PVM. Addition of a Myc tag before and after the cleavage site shows that processed HT/PEXEL protein has increased PVM association. These findings suggest that while Toxoplasma and Plasmodium share similar HT/PEXEL motifs, Toxoplasma HT/PEXEL containing proteins interact with but do not cross the PVM.

A patatin-like protein protects Toxoplasma gondii from degradation in activated macrophages

The apicomplexan parasite Toxoplasma gondii is able to suppress nitric oxide production in activated macrophages. A screen of over 6000 T. gondii insertional mutants identified two clones, which were consistently unable to suppress nitric oxide production from activated macrophages. One strain, called 89B7, grew at the same rate as wild‐type parasites in naïve macrophages, but unlike wild type, the mutant was degraded in activated macrophages. This degradation was marked by a reduction in the number of parasites within vacuoles over time, the loss of GRA4 and SAG1 protein staining by immunofluorescence assay, and the vesiculation and breakdown of the internal parasite ultrastructure by electron microscopy. The mutagenesis plasmid in the 89B7 clone disrupts the promoter of a 3.4 kb mRNA that encodes a predicted 68 kDa protein with a cleavable signal peptide and a patatin‐like phospholipase domain. Genetic complementation with the genomic locus of this patatin‐like protein restores the parasites ability to suppress nitric oxide and replicate in activated macrophages. A haemagglutinin‐tagged version of this patatin‐like protein shows punctate localization into atypical T. gondii structures within the parasite. This is the first study that defines a specific gene product that is needed for parasite survival in activated but not naïve macrophages.

The ins and outs of nuclear trafficking: unusual aspects in apicomplexan parasites.

Apicomplexa is a phylum within the kingdom Protista that contains some of the most significant threats to public health. One of the members of this phylum, Toxoplasma gondii, is amenable to molecular genetic analyses allowing for the identification of factors critical for colonization and disease. A pathway found to be important for T. gondii pathogenesis is the Ran network of nuclear trafficking. Bioinformatics analysis of apicomplexan genomes shows that while Ran is well conserved, the key regulators of Ran--Regulator of Chromosome Condensation 1 and Ran GTPase activating protein--are either highly divergent or absent. Likewise, several import and export receptor molecules that are crucial for nuclear transport are either not present or have experienced genetic drift such that they are no longer recognizable by bioinformatics tools. In this minireview we describe the basics of nuclear trafficking and compare components within apicomplexans to defined systems in humans and yeast. A detailed analysis of the nuclear trafficking network in these eukaryotes is required to understand how this potentially unique cellular biological pathway contributes to host-parasite interactions.

Isolation of Toxoplasma gondii development mutants identifies a potential proteophosphogylcan that enhances cyst wall formation.

Within warm-blooded animals, Toxoplasma gondii switches from an actively replicating form called a tachyzoite into a slow growing encysted form called a bradyzoite. To uncover the genes involved in bradyzoite development, we screened over 8000 T. gondii insertional mutants by immunofluorescence microscopy. We identified nine bradyzoite development mutants that were defective in both cyst wall formation and expression of a bradyzoite specific heat shock protein. One of these mutants, named 42F5, contained an insertion into the predicted gene TGME49_097520. The disrupted protein is serine/proline-rich with homology to proteophosphoglycans from Leishmania. T. gondii proteophosphoglycan (GU182879) expressed from the native promoter was undetectable in tachyzoites, but bradyzoites show punctate spots within the parasite and staining around the parasitophorous vacuole. Complementation of the 42F5 mutant with GU182879 expressed from either the alpha-tubulin or native promoter restores cyst wall formation. Overall, GU182879 is upregulated in bradyzoites and enhances cyst wall component expression and assembly.

Highly Polymorphic Family of Glycosylphosphatidylinositol-Anchored Surface Antigens with Evidence of Developmental Regulation in Toxoplasma gondii

ABSTRACT The life cycle of the apicomplexan parasite Toxoplasma gondii requires that an infectious cyst develop and be maintained throughout the life of the host. The molecules displayed on the parasite surface are important in controlling the immune response to the parasite. T. gondii has a superfamily of glycosylphosphatidylinositol (GPI)-anchored surface antigens, termed the surface antigen (SAG) and SAG-related surface antigens, that are developmentally regulated during infection. Using a clustering algorithm, we identified a new family of 31 surface proteins that are predicted to be GPI anchored but are unrelated to the SAG proteins, and thus we named these proteins SAG-unrelated surface antigens (SUSA). Analysis of the single nucleotide polymorphism density showed that the members of this family are the most polymorphic genes within the T. gondii genome. Immunofluorescence of SUSA1 and SUSA2, two members of the family, revealed that they are found on the parasite surface. We confirmed that SUSA1 and SUSA2 are GPI anchored by phospholipase cleavage. Analysis of expressed sequence tags (ESTs) revealed that SUSA1 had 22 of 23 ESTs from chronic infection. Analysis of mRNA and protein confirmed that SUSA1 is highly expressed in the chronic form of the parasite. Sera from mice with chronic T. gondii infection reacted to SUSA1, indicating that SUSA1 interacts with the host immune system during infection. This group of proteins likely represents a new family of polymorphic GPI-anchored surface antigens that are recognized by the hosts immune system and whose expression is regulated during infection.

The role of specific Toxoplasma gondii molecules in manipulation of innate immunity

Infection with the parasite Toxoplasma gondii stimulates an innate immune response in the host. T. gondii also induces alterations in infected monocytes and dendritic cells that probably contribute to its ability to disseminate and ultimately to establish persistent infection. Recent progress has linked specific parasite molecules to immune stimulation or the ability of the parasite to subvert intracellular signaling pathways in infected cells to evade immunity.

Long-Term Relationships: the Complicated Interplay between the Host and the Developmental Stages of Toxoplasma gondii during Acute and Chronic Infections

SUMMARY Toxoplasma gondii represents one of the most common parasitic infections in the world. The asexual cycle can occur within any warm-blooded animal, but the sexual cycle is restricted to the feline intestinal epithelium. T. gondii is acquired through consumption of tissue cysts in undercooked meat as well as food and water contaminated with oocysts. Once ingested, it differentiates into a rapidly replicating asexual form and disseminates throughout the body during acute infection. After stimulation of the host immune response, T. gondii differentiates into a slow-growing, asexual cyst form that is the hallmark of chronic infection. One-third of the human population is chronically infected with T. gondii cysts, which can reactivate and are especially dangerous to individuals with reduced immune surveillance. Serious complications can also occur in healthy individuals if infected with certain T. gondii strains or if infection is acquired congenitally. No drugs are available to clear the cyst form during the chronic stages of infection. This therapeutic gap is due in part to an incomplete understanding of both host and pathogen responses during the progression of T. gondii infection. While many individual aspects of T. gondii infection are well understood, viewing the interconnections between host and parasite during acute and chronic infection may lead to better approaches for future treatment. The aim of this review is to provide an overview of what is known and unknown about the complex relationship between the host and parasite during the progression of T. gondii infection, with the ultimate goal of bridging these events.

A Patatin-Like Protein Protects Toxoplasma gondii from Degradation in a Nitric Oxide-Dependent Manner

ABSTRACT Toxoplasma gondii is an obligate intracellular parasite that uses immune cells to disseminate throughout its host. T. gondii can persist and even slowly replicate in activated host macrophages by reducing the antimicrobial effects of molecules such as nitric oxide (NO). A T. gondii patatin-like protein called TgPL1 was previously shown to be important for survival in activated macrophages. Here we show that a T. gondii mutant with a deletion of the TgPL1 gene (ΔTgPL1) is degraded in activated macrophages. This degradation phenotype is abolished by the removal of NO by the use of an inducible NO synthase (iNOS) inhibitor or iNOS-deficient macrophages. The exogenous addition of NO to macrophages results in reduced parasite growth but not the degradation of ΔTgPL1 parasites. These results suggest that NO is necessary but not sufficient for the degradation of ΔTgPL1 parasites in activated macrophages. While some patatin-like proteins have phospholipase A2 (PLA2) activity, recombinant TgPL1 purified from Escherichia coli does not have phospholipase activity. This result was not surprising, as TgPL1 contains a G-to-S change at the predicted catalytic serine residue. An epitope-tagged version of TgPL1 partially colocalized with a dense granule protein in the parasitophorous vacuole space. These results may suggest that TgPL1 moves to the parasitophorous vacuole to protect parasites from nitric oxide by an undetermined mechanism.