Translation control is critical during acute and chronic stages of toxoplasmosis infection

Keywords::

• Apicomplexa
• bradyzoite
• eIF2 kinase
• protozoa
• stress response
• Toxoplasma

Toxoplasma gondii (phylum Apicomplexa) is an obligate intracellular pathogen that relies on a complex life cycle for transmission, pathogenesis and persistence in host organisms. The definitive host is felids, which expel infective oocysts that are stable in the environment for up to a year. Oocysts can infect virtually all warm-blooded vertebrates. Upon infection, the proliferative form of the parasite (tachyzoite) differentiates into a persistent tissue cyst (bradyzoite) that remains infectious and can also be transmitted to new hosts when consumed. In humans, tachyzoites cause acute toxoplasmosis, which can be controlled by a healthy immune system. However, the bradyzoite cysts that form during infection are impervious to the immune response and to current therapies against toxoplasmosis, leading to chronic disease. With a human seroprevalence rate of 30–40% in the USA, it is not uncommon to see life-threatening episodes of reactivated toxoplasmosis in AIDS or other immunocompromised individuals [1,2]. Neonates are also highly susceptible to congenital toxoplasmosis [3]. Current treatments are toxic and do not target chronic toxoplasmosis, underscoring the need to develop new therapies for patients, as well as methods to minimize transmission from cats and livestock to humans [4].

Intracellular parasites like Toxoplasma reside in a setting rich with nutrients and are generally protected from the host immune response. However, during an acute infection, the parasites must circumvent a variety of cellular stresses, including heat shock (fever), oxidative stress (reactive oxygen species produced from immune effector cells) and inflammatory cytokines such as IFN-γ [5]. During progression through the lytic cycle, Toxoplasma proliferates until it lyses its host cell, and the free tachyzoites must cope with the stress of the extracellular environment, which lacks nutrients and other metabolites necessary for the parasite to live and replicate. It is also well established that stress is a potent inducer of tachyzoite to bradyzoite conversion [6]. Given the importance of the stress response during the parasite’s life cycle, it is important to characterize the molecular mechanisms governing these processes in Toxoplasma.

A key regulator of stress response pathways in eukaryotes involves phosphorylation of the α subunit of the translation initiation factor eIF2 [7]. eIF2, in combination with GTP, transports the initiator tRNA to the translation machinery for eventual placement into the P-site of ribosomes. In response to cellular stress, phosphorylation of eIF2α converts this initiation factor from a substrate to an inhibitor of its guanine nucleotide exchange factor, eIF2B. The reduction in eIF2-GTP levels decreases general protein synthesis, allowing the cell sufficient time to reprogram gene expression to remedy stress damage. Accompanying this reduction in general protein synthesis, phosphorylation of eIF2α enhances translation of a subset of mRNAs encoding transcription factors (e.g., yeast GCN4 or mammalian ATF4) that are central for the expression of stress remedy genes [8–10]. Four eIF2 kinases in mammals regulate translation in response to varying stress conditions [7,11–13]. These include GCN2, which directs translational control during nutrient deprivation [14]; PKR, which directs an antiviral defense mechanism [15]; PEK/PERK, which is activated by endoplasmic reticulum (ER) stress [16–18]; and HRI, which is stimulated by heme deficiency and oxidative stress [19,20].

We have identified four eIF2 kinases in Toxoplasma (TgIF2K-A–D) that are suggested to play critical roles in exerting translational control during both acute and chronic infection. The specific stress responses mediated by each TgIF2K are only beginning to be understood. Our findings indicate that TgIF2K-A is localized to the parasite ER and likely mediates the activation of the unfolded protein response analogous to PEK/PERK [21]. Consistent with this idea, TgIF2K-A appears to be regulated through an inhibitory association with the ER-resident chaperone, BiP/GRP78 [21]. TgIF2K-B is parasite specific and likely responds to cytoplasmic stresses based on its cellular distribution [21]. TgIF2K-C and -D are most closely related to GCN2, suggesting a role in alleviation of nutrient stress. In general, the eIF2 kinases in Toxoplasma, as well as other apicomplexan parasites, are very large, possessing unique sequences not conserved in mammalian counterparts. Such features may be important for future drug design if these parasite eIF2 kinases and translation control were shown to be critical for parasite growth, virulence and persistence. Our recent findings argue that this is indeed the case.

We first established that a variety of stress conditions, including those that trigger bradyzoite development, induce the phosphorylation of the Toxoplasma eIF2α orthologue (TgIF2α) on the conserved serine-71 [21,22]. Furthermore, mature bradyzoite cysts induced in vitro had significantly elevated levels of phosphorylated TgIF2α when compared with growing tachyzoites. These data indicated that translation control mediated by TgIF2α phosphorylation may be important for the development and maintenance of bradyzoite cyst forms. Consistent with this idea, we found that salubrinal, a pharmacological inhibitor of TgIF2α dephosphorylation, leads to increased levels of phosphorylated TgIF2α, activation of bradyzoite gene expression and cyst wall formation [21]. These findings implicate translation control to be an important factor involved in the establishment of chronic toxoplasmosis [22].

To assess the impact of TgIF2α phosphorylation on tachyzoites and acute infection, we generated a type I (RH) strain mutant parasite harboring a single point mutation in TgIF2α that prevents phosphorylation at serine-71. Tachyzoites containing mutated TgIF2α (S71A) possess rates of invasion, replication and egress comparable to wild-type, yet exhibit reduced viability in vitro after exposure to the extracellular environment [23]. In support of this finding, we also showed that TgIF2α is increasingly phosphorylated in extracellular wild-type parasites in a time-dependent fashion. The TgIF2α-S71A mutant strain is also significantly less virulent in vivo, indicating that the phosphorylation of TgIF2α plays a critical role during the lytic cycle by ameliorating the stress of the extracellular environment while the parasite searches for a new host cell [23]. These studies support the idea that tachyzoites experience stress when deprived of their host cells and establish that translation control via TgIF2α phosphorylation is instrumental to promoting survival of the parasite while it locates a new host cell.

A role for eIF2α phosphorylation in parasite stage conversion and virulence has recently been demonstrated in the related apicomplexan parasite Plasmodium, the causative agent of malaria. To date, three Plasmodium eIF2 kinases have been characterized: PbeIK2, PfPK4 and PfeIK1, which respond to heat shock, heme deprivation and amino acid starvation, respectively [24–26]. Importantly, Zhang et al. found that PbeIK2 is a critical regulator of sporozoite latency in the mosquito salivary glands [26]. Disruption of the PbeIK2 genomic locus results in a loss of translation control, which causes the latent sporozoites to prematurely develop into liver-stage forms, rendering the parasite noninfectious in mice [26].

A key question that remains involves the molecular mechanism of preferential translation during TgIF2α phosphorylation. In other species, the presence of upstream open reading frames (uORFs) within the 5´-UTR allows for the preferential translation of a subset of mRNAs in response to eIF2α phosphorylation through a delayed ribosome re-initiation mechanism [7]. These mRNAs tend to encode a transcriptional activator that induces the expression of stress remedy genes. In Plasmodium, an uORF was identified within the 5´-UTR of the hypervariable surface antigen VAR2CSA that restricts its translation to specific cellular environments [27], suggesting that uORF-mediated translation control occurs in Apicomplexa. In addition, while there are no GCN4 or ATF4 homologues in Apicomplexa, a lineage-specific series of plant-like transcription factors harboring AP2-like domains has recently been described [28]. Whether these AP2-domain factors are subject to translation control during stress-induced TgIF2α phosphorylation is an important question for future study.

In summary, translation control through eIF2 phosphorylation is a stress response that arose very early in the evolution of eukaryotic cells. These studies support the idea that eIF2 kinases phosphorylate TgIF2α to elicit translation control as a means to cope with the variety of stresses or environmental changes encountered during a parasite’s life cycle. Our experimental data supports the model and indicate that translation control through TgIF2α phosphorylation is critical during the lytic cycle of tachyzoites and in the development and maintenance of bradyzoite tissue cysts. These findings suggest that interference with TgIF2α phosphorylation may be an innovative new approach that targets both acute and chronic forms of toxoplasmosis. Moreover, mutant parasites defective in the eIF2 kinase pathway may be useful as novel vaccines for pets or livestock that bestow immunity but fail to develop into infectious transmissible forms.

Financial & competing interests disclosure

Support for this research was provided through grants from the American Heart Association (0920034G to Bradley R Joyce) and the NIH (AI084031 to Ronald C Wek and William J Sullivan Jr and GM49164 to Ronald C Wek). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.