Communication between Toxoplasma gondii and its host: impact on parasite growth, development, immune evasion, and virulence

Toxoplasma gondii is an obligate intracellular protozoan parasite that can infect most warm-blooded animals and cause severe and life-threatening disease in developing fetuses and in immune-compromised patients. Although Toxoplasma was discovered over 100 years ago, we are only now beginning to appreciate the importance of the role that parasite modulation of its host has on parasite growth, bradyzoite development, immune evasion, and virulence. The goal of this review is to highlight these findings, to develop an integrated model for communication between Toxoplasma and its host, and to discuss new questions that arise out of these studies.American Heart Association (Scientist Development Grant 0835099N

Toxoplasma Does Not Secrete the GRA16 and GRA24 Effectors Beyond the Parasitophorous Vacuole Membrane of Tissue Cysts

After invasion, Toxoplasma resides in a parasitophorous vacuole (PV) that is surrounded by the PV membrane (PVM). Once inside the PV, tachyzoites secrete dense granule proteins (GRAs) of which some, such as GRA16 and GRA24, are transported beyond the PVM likely via a putative translocon. However, once tachyzoites convert into bradyzoites within cysts, it is not known if secreted GRAs can traffic beyond the cyst wall membrane. We used the tetracycline inducible system to drive expression of HA epitope tagged GRA16 and GRA24 after inducing stage conversion and show that these proteins are not secreted beyond the cyst wall membrane. This suggests that secretion of GRA beyond the PVM is not important for the tissue cyst stage of Toxoplasma

Serotyping of Toxoplasma gondii Infection Using Peptide Membrane Arrays.

The intracellular parasite Toxoplasma gondii can cause chronic infections in most warm-blooded animals, including humans. In the USA, strains belonging to four different Toxoplasma clonal lineages (types 1, 2, 3, and 12) are commonly isolated, whereas strains not belonging to these lineages are predominant in other continents such as South America. Strain type plays a pivotal role in determining the severity of Toxoplasma infection. Therefore, it is epidemiologically relevant to develop a non-invasive and inexpensive method for determining the strain type in Toxoplasma infections and to correlate the genotype with disease outcome. Serological typing is based on the fact that many host antibodies are raised against immunodominant parasite proteins that are highly polymorphic between strains. However, current serological assays can only reliably distinguish type 2 from non-type 2 infections. To improve these assays, mouse, rabbit, and human infection serum were reacted against 950 peptides from 62 different polymorphic Toxoplasma proteins by using cellulose membrane peptide arrays. This allowed us to identify the most antigenic peptides and to pinpoint the most relevant polymorphisms that determine strain specificity. Our results confirm the utility of previously described peptides and identify novel peptides that improve and increase the specificity of the assay. In addition, a large number of novel proteins showed potential to be used for Toxoplasma diagnosis. Among these, peptides derived from several rhoptry, dense granule, and surface proteins represented promising candidates that may be used in future experiments to improve Toxoplasma serotyping. Moreover, a redesigned version of the published GRA7 typing peptide performed better and specifically distinguished type 3 from non-type 3 infections in sera from mice, rabbits, and humans

The genetic basis for individual differences in mRNA splicing and APOBEC1 editing activity in murine macrophages

Alternative splicing and mRNA editing are known to contribute to transcriptome diversity. Although alternative splicing is pervasive and known to contribute to a variety of pathologies, including cancer, the genetic context for individual differences in isoform usage is still evolving. Similarly, although mRNA editing is ubiquitous and associated with important biological processes such as intracellular viral replication and cancer development, individual variations in and the genetic transmissibility of mRNA editing are equivocal. Here, we have used linkage analysis to show that both mRNA editing and alternative splicing are regulated by the macrophage genetic background and environmental cues. We show that distinct loci, potentially harboring variable splice factors, regulate the splicing of multiple transcripts. Additionally, we show that individual genetic variability at the Apobec1 locus results in differential rates of C-to-U editing in murine macrophages; with mouse strains expressing mostly a truncated isoform of Apobec1 exhibiting lower rates of editing. As a proof of concept, we have used linkage analysis to identify 36 high confidence novel edited sites. These results provide a novel and complementary method that can be used to identify C-to-U editing sites in individuals segregating at specific loci and show that, beyond individual DNA sequence and structural changes, differential isoform usage and mRNA editing can contribute to intra-species genomic and phenotypic diversity

The Toxoplasma secreted effector TgWIP modulates dendritic cell motility by activating host tyrosine phosphatases Shp1 and Shp2

The obligate intracellular parasite Toxoplasma gondii causes life-threatening toxoplasmosis to immunocompromised individuals. The pathogenesis of Toxoplasma relies on its swift dissemination to the central nervous system through a 'Trojan Horse' mechanism using infected leukocytes as carriers. Previous work found TgWIP, a protein secreted from Toxoplasma, played a role in altering the actin cytoskeleton and promoting cell migration in infected dendritic cells (DCs). However, the mechanism behind these changes was unknown. Here, we report that TgWIP harbors two SH2-binding motifs that interact with tyrosine phosphatases Shp1 and Shp2, leading to phosphatase activation. DCs infected with Toxoplasma exhibited hypermigration, accompanying enhanced F-actin stress fibers and increased membrane protrusions such as filopodia and pseudopodia. By contrast, these phenotypes were abrogated in DCs infected with Toxoplasma expressing a mutant TgWIP lacking the SH2-binding motifs. We further demonstrated that the Rho-associated kinase (Rock) is involved in the induction of these phenotypes, in a TgWIP-Shp1/2 dependent manner. Collectively, the data uncover a molecular mechanism by which TgWIP modulates the migration dynamics of infected DCs in vitro

Identification of three novel Toxoplasma gondii rhoptry proteins

The rhoptries are key secretory organelles from apicomplexan parasites that contain proteins involved in invasion and modulation of the host cell. Some rhoptry proteins are restricted to the posterior bulb (ROPs) and others to the anterior neck (RONs). As many rhoptry proteins have been shown to be key players in Toxoplasma invasion and virulence, it is important to identify, understand and characterise the biological function of the components of the rhoptries. In this report, we identified putative novel rhoptry genes by identifying Toxoplasma genes with similar cyclical expression profiles as known rhoptry protein encoding genes. Using this approach we identified two new rhoptry bulb (ROP47 and ROP48) and one new rhoptry neck protein (RON12). ROP47 is secreted and traffics to the host cell nucleus, RON12 was not detected at the moving junction during invasion. Deletion of ROP47 or ROP48 in a type II strain did not show major influence in in vitro growth or virulence in mice.United States. National Institutes of Health (R01-AI080621