A latent ability to persist: differentiation in Toxoplasma gondii

A critical factor in the transmission and pathogenesis of Toxoplasma gondii is the ability to convert from an acute disease-causing, proliferative stage (tachyzoite), to a chronic, dormant stage (bradyzoite). The conversion of the tachyzoite-containing parasitophorous vacuole membrane into the less permeable bradyzoite cyst wall allows the parasite to persist for years within the host to maximize transmissibility to both primary (felids) and secondary (virtually all other warm-blooded vertebrates) hosts. This review presents our current understanding of the latent stage, including the factors that are important in bradyzoite induction and maintenance. Also discussed are the recent studies that have begun to unravel the mechanisms behind stage switching

Transcriptional repression by ApiAP2 factors is central to chronic toxoplasmosis

Tachyzoite to bradyzoite development in Toxoplasma is marked by major changes in gene expression resulting in a parasite that expresses a new repertoire of surface antigens hidden inside a modified parasitophorous vacuole called the tissue cyst. The factors that control this important life cycle transition are not well understood. Here we describe an important transcriptional repressor mechanism controlling bradyzoite differentiation that operates in the tachyzoite stage. The ApiAP2 factor, AP2IV-4, is a nuclear factor dynamically expressed in late S phase through mitosis/cytokinesis of the tachyzoite cell cycle. Remarkably, deletion of the AP2IV-4 locus resulted in the expression of a subset of bradyzoite-specific proteins in replicating tachyzoites that included tissue cyst wall components BPK1, MCP4, CST1 and the surface antigen SRS9. In the murine animal model, the mis-timing of bradyzoite antigens in tachyzoites lacking AP2IV-4 caused a potent inflammatory monocyte immune response that effectively eliminated this parasite and prevented tissue cyst formation in mouse brain tissue. Altogether, these results indicate that suppression of bradyzoite antigens by AP2IV-4 during acute infection is required for Toxoplasma to successfully establish a chronic infection in the immune-competent host

Characterization of Plasmodium Atg3-Atg8 Interaction Inhibitors Identifies Novel Alternative Mechanisms of Action in Toxoplasma gondii

Protozoan parasites, including the apicomplexan pathogens Plasmodium falciparum (which causes malaria) and Toxoplasma gondii (which causes toxoplasmosis), infect millions of people worldwide and represent major human disease burdens. Despite their prevalence, therapeutic strategies to treat infections caused by these parasites remain limited and are threatened by the emergence of drug resistance, highlighting the need for the identification of novel drug targets. Recently, homologues of the core autophagy proteins, including Atg8 and Atg3, were identified in many protozoan parasites. Importantly, components of the Atg8 conjugation system that facilitate the lipidation of Atg8 are required for both canonical and parasite-specific functions and are essential for parasite viability. Structural characterization of the P. falciparum Atg3-Atg8 (PfAtg3-Atg8) interaction has led to the identification of compounds that block this interaction. Additionally, many of these compounds inhibit P. falciparum growth in vitro, demonstrating the viability of this pathway as a drug target. Given the essential role of the Atg8 lipidation pathway in Toxoplasma, we sought to determine whether three PfAtg3-Atg8 interaction inhibitors identified in the Medicines for Malaria Venture Malaria Box exerted a similar inhibitory effect in Toxoplasma While all three inhibitors blocked Toxoplasma replication in vitro at submicromolar concentrations, they did not inhibit T. gondii Atg8 (TgAtg8) lipidation. Rather, high concentrations of two of these compounds induced TgAtg8 lipidation and fragmentation of the parasite mitochondrion, similar to the effects seen following starvation and monensin-induced autophagy. Additionally, we report that one of the PfAtg3-Atg8 interaction inhibitors induces Toxoplasma egress and provide evidence that this is mediated by an increase in intracellular calcium in response to drug treatment

Efficacy of Guanabenz Combination Therapy against Chronic Toxoplasmosis across Multiple Mouse Strains

Toxoplasma gondii, an obligate intracellular parasite that can cause life-threatening acute disease, differentiates into a quiescent cyst stage to establish lifelong chronic infections in animal hosts, including humans. This tissue cyst reservoir, which can reactivate into an acute infection, is currently refractory to clinically available therapeutics. Recently, we and others have discovered drugs capable of significantly reducing the brain cyst burden in latently infected mice, but not to undetectable levels. In this study, we examined the use of novel combination therapies possessing multiple mechanisms of action in mouse models of latent toxoplasmosis. Our drug regimens included combinations of pyrimethamine, clindamycin, guanabenz, and endochin-like quinolones (ELQs) and were administered to two different mouse strains in an attempt to eradicate brain tissue cysts. We observed mouse strain-dependent effects with these drug treatments: pyrimethamine-guanabenz showed synergistic efficacy in C57BL/6 mice yet did not improve upon guanabenz monotherapy in BALB/c mice. Contrary to promising in vitro results demonstrating toxicity to bradyzoites, we observed an antagonistic effect between guanabenz and ELQ-334 in vivo While we were unable to completely eliminate the brain cyst burden, we found that a combination treatment with ELQ-334 and pyrimethamine impressively reduced the brain cyst burden by 95% in C57BL/6 mice, which approached the limit of detection. These analyses highlight the importance of evaluating anti-infective drugs in multiple mouse strains and will help inform further preclinical studies of cocktail therapies designed to treat chronic toxoplasmosis

Translational Control in the Latency of Apicomplexan Parasites

Apicomplexan parasites Toxoplasma gondii and Plasmodium spp. use latent stages to persist in the host, facilitate transmission, and thwart treatment of infected patients. Therefore, it is important to understand the processes driving parasite differentiation to and from quiescent stages. Here, we discuss how a family of protein kinases that phosphorylate the eukaryotic initiation factor-2 (eIF2) function in translational control and drive differentiation. This translational control culminates in reprogramming of the transcriptome to facilitate parasite transition towards latency. We also discuss how eIF2 phosphorylation contributes to the maintenance of latency and provides a crucial role in the timing of reactivation of latent parasites towards proliferative stages

Host sensing and signal transduction during Toxoplasma stage conversion

The intracellular parasite Toxoplasma gondii infects nucleated cells in virtually all warm-blooded vertebrates, including one-third of the human population. While immunocompetent hosts do not typically show symptoms of acute infection, parasites are retained in latent tissue cysts that can be reactivated upon immune suppression, potentially damaging key organ systems. Toxoplasma has a multistage life cycle that is intimately linked to environmental stresses and host signals. As this protozoan pathogen is transmitted between multiple hosts and tissues, it evaluates these external signals to appropriately differentiate into distinct life cycle stages, such as the transition from its replicative stage (tachyzoite) to the latent stage (bradyzoite) that persists as tissue cysts. Additionally, in the gut of its definitive host, felines, Toxoplasma converts into gametocytes that produce infectious oocysts (sporozoites) that are expelled into the environment. In this review, we highlight recent advances that have illuminated the interfaces between Toxoplasma and host and how these interactions control parasite stage conversion. Mechanisms underlying these stage transitions are important targets for therapeutic intervention aimed at thwarting parasite transmission and pathogenesis

Elp3 and RlmN: A tale of two mitochondrial tail-anchored radical SAM enzymes in Toxoplasma gondii

Radical S-adenosylmethionine (rSAM) enzymes use a 5'-deoxyadensyl 5'-radical to methylate a wide array of diverse substrates including proteins, lipids and nucleic acids. One such enzyme, Elongator protein-3 (TgElp3), is an essential protein in Toxoplasma gondii, a protozoan parasite that can cause life-threatening opportunistic disease. Unlike Elp3 homologues which are present in all domains of life, TgElp3 localizes to the outer mitochondrial membrane (OMM) via a tail-anchored trafficking mechanism in Toxoplasma. Intriguingly, we identified a second tail-anchored rSAM domain containing protein (TgRlmN) that also localizes to the OMM. The transmembrane domain (TMD) on Toxoplasma Elp3 and RlmN homologues is required for OMM localization and has not been seen beyond the chromalveolates. Both TgElp3 and TgRlmN contain the canonical rSAM amino acid sequence motif (CxxxCxxC) necessary to form the 4Fe-4S cluster required for tRNA modifications. In E. coli, RlmN is responsible for the 2-methlyadenosine (m2A) synthesis at purine 37 in tRNA while in S. cerevisiae, Elp3 is necessary for the formation of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at the wobble tRNA position. To investigate why these two rSAM enzymes localize to the mitochondrion in Toxoplasma, and whether or not TgRlmN and TgElp3 possess tRNA methyltransferase activity, a series of mutational and biochemical studies were performed. Overexpression of either TgElp3 or TgRlmN resulted in a significant parasite replication defect, but overexpression was tolerated if either the TMD or rSAM domain was mutated. Furthermore, we show the first evidence that Toxoplasma tRNAGlu contains the mcm5s2U modification, which is the putative downstream product generated by TgElp3 activity

Guanabenz repurposed as an antiparasitic with activity against acute and latent toxoplasmosis

Toxoplasma gondii is a protozoan parasite that persists as a chronic infection. Toxoplasma evades immunity by forming tissue cysts, which reactivate to cause life-threatening disease during immune suppression. There is an urgent need to identify drugs capable of targeting these latent tissue cysts, which tend to form in the brain. We previously showed that translational control is critical during infections with both replicative and latent forms of Toxoplasma. Here we report that guanabenz, an FDA-approved drug that interferes with translational control, has antiparasitic activity against replicative stages of Toxoplasma and the related apicomplexan parasite Plasmodium falciparum (a malaria agent). We also found that inhibition of translational control interfered with tissue cyst biology in vitro. Toxoplasma bradyzoites present in these abnormal cysts were diminished and misconfigured, surrounded by empty space not seen in normal cysts. These findings prompted analysis of the efficacy of guanabenz in vivo by using established mouse models of acute and chronic toxoplasmosis. In addition to protecting mice from lethal doses of Toxoplasma, guanabenz has a remarkable ability to reduce the number of brain cysts in chronically infected mice. Our findings suggest that guanabenz can be repurposed into an effective antiparasitic with a unique ability to reduce tissue cysts in the brain