mRNA cap-binding protein eIF4E1 is a novel regulator of Toxoplasma gondii latency.

The protozoan parasite Toxoplasma gondii causes serious opportunistic disease due to its ability to persist in patients as latent tissue cysts. The molecular mechanisms coordinating conversion between proliferative parasites (tachyzoites) and latent cysts (bradyzoites) are not fully understood. We previously showed that phosphorylation of eIF2α accompanies bradyzoite formation, suggesting that this clinically relevant process involves regulation of mRNA translation. In this study, we investigated the composition and role of eIF4F multi-subunit complexes in translational control. Using CLIPseq, we find that the cap-binding subunit, eIF4E1, localizes to the 5'-end of all tachyzoite mRNAs, many of which show evidence of stemming from heterogeneous transcriptional start sites. We further show that eIF4E1 operates as the predominant cap-binding protein in two distinct eIF4F complexes. Using genetic and pharmacological approaches, we found that eIF4E1 deficiency triggers efficient spontaneous formation of bradyzoites without stress induction. Consistent with this result, we also show that stress-induced bradyzoites exhibit reduced eIF4E1 expression. Overall, our findings establish a novel role for eIF4F in translational control required for parasite latency and microbial persistence. IMPORTANCE: Toxoplasma gondii is an opportunistic pathogen important to global human and animal health. There are currently no chemotherapies targeting the encysted form of the parasite. Consequently, a better understanding of the mechanisms controlling encystation is required. Here we show that the mRNA cap-binding protein, eIF4E1, regulates the encystation process. Encysted parasites reduce eIF4E1 levels, and depletion of eIF4E1 decreases the translation of ribosome-associated machinery and drives Toxoplasma encystation. Together, these data reveal a new layer of mRNA translational control that regulates parasite encystation and latency.

Translation initiation factor eIF1.2 promotes Toxoplasma stage conversion by regulating levels of key differentiation factors.

The parasite Toxoplasma gondii persists in its hosts by converting from replicating tachyzoites to latent bradyzoites housed in tissue cysts. The molecular mechanisms that mediate T. gondii differentiation remain poorly understood. Through a mutagenesis screen, we identified translation initiation factor eIF1.2 as a critical factor for T. gondii differentiation. A F97L mutation in eIF1.2 or the genetic ablation of eIF1.2 (∆eif1.2) markedly impeded bradyzoite cyst formation in vitro and in vivo. We demonstrated, at single-molecule level, that the eIF1.2 F97L mutation impacts the scanning process of the ribosome preinitiation complex on a model mRNA. RNA sequencing and ribosome profiling experiments unveiled that ∆eif1.2 parasites are defective in upregulating bradyzoite induction factors BFD1 and BFD2 during stress-induced differentiation. Forced expression of BFD1 or BFD2 significantly restored differentiation in ∆eif1.2 parasites. Together, our findings suggest that eIF1.2 functions by regulating the translation of key differentiation factors necessary to establish chronic toxoplasmosis.

Translation initiation factor eIF1.2 promotes Toxoplasma stage conversion by regulating levels of key differentiation factors.

The parasite Toxoplasma gondii persists in its hosts by converting from replicating tachyzoites to latent bradyzoites housed in tissue cysts. The molecular mechanisms that mediate T. gondii differentiation remain poorly understood. Through a mutagenesis screen, we identified translation initiation factor eIF1.2 as a critical factor for T. gondii differentiation. A F97L mutation in eIF1.2 or the genetic ablation of eIF1.2 (Δ eIF1.2 ) markedly impeded bradyzoite cyst formation in vitro and in vivo . We demonstrated, at single-molecule level, that the eIF1.2 F97L mutation impacts the scanning process of the ribosome preinitiation complex on a model mRNA. RNA sequencing and ribosome profiling experiments unveiled that Δ eIF1.2 parasites are defective in the upregulating bradyzoite induction factors BFD1 and BFD2 during stress-induced differentiation. Forced expression of BFD1 or BFD2 significantly restored differentiation in Δ eIF1.2 parasites. Together, our findings suggest that eIF1.2 functions by regulating the translation of key differentiation factors necessary to establish chronic toxoplasmosis.

mRNA cap-binding protein eIF4E1 is a novel regulator of Toxoplasma gondii latency.

The protozoan parasite Toxoplasma gondii causes serious opportunistic disease due to its ability to persist in patients as latent tissue cysts. The molecular mechanisms coordinating conversion between proliferative parasites (tachyzoites) and dormant cysts (bradyzoites) are not fully understood. We previously showed that phosphorylation of eIF2α accompanies bradyzoite formation, suggesting that this clinically relevant process involves regulation of mRNA translation. In this study, we investigated the composition and role of eIF4F multi-subunit complexes in translational control. Using CLIPseq, we find that the cap-binding subunit, eIF4E1, localizes to the 5'-end of all tachyzoite mRNAs, many of which show evidence of stemming from heterogenous transcriptional start sites. We further show that eIF4E1 operates as the predominant cap-binding protein in two distinct eIF4F complexes. Using genetic and pharmacological approaches, we found that eIF4E1 deficiency triggers efficient spontaneous formation of bradyzoites without stress induction. Consistent with this result, we also show that stress-induced bradyzoites exhibit reduced eIF4E1 expression. Overall, our findings establish a novel role for eIF4F in translational control required for parasite latency and microbial persistence.

A transcriptional network required for bradyzoite development in Toxoplasma gondii is dispensable for recrudescent disease.

Identification of regulators of Toxoplasma gondii bradyzoite development and cyst formation is the most direct way to address the importance of parasite development in long-term persistence and reactivation of this parasite. Here we show that a T. gondii gene (named Regulator of Cystogenesis 1; ROCY1) is sufficient for T. gondii bradyzoite formation in vitro and in vivo. ROCY1 encodes an RNA binding protein that has a preference for 3' regulatory regions of hundreds of T. gondii transcripts, and its RNA-binding domains are required to mediate bradyzoite development. Female mice infected with ΔROCY1 parasites have reduced (>90%) cyst burden. While viable parasites can be cultivated from brain tissue for up to 6 months post-infection, chronic brain-resident ΔROCY1 parasites have reduced oral infectivity compared to wild type. Despite clear defects in bradyzoite formation and oral infectivity, ΔROCY1 parasites were able to reactivate with similar timing and magnitude as wild type parasites for up to 5 months post-infection. Therefore while ROCY1 is a critical regulator of the bradyzoite developmental pathway, it is not required for parasite reactivation, raising new questions about the persisting life stage responsible for causing recrudescent disease.

Model of the Origin of a Ciguatoxic Grouper (Plectropomus leopardus).

Published data were used to model the transfer of ciguatoxins (CTX) across three trophic levels of a marine food chain on the Great Barrier Reef (GBR), Australia, to produce a mildly toxic common coral trout (Plectropomus leopardus), one of the most targeted food fishes on the GBR. Our model generated a 1.6 kg grouper with a flesh concentration of 0.1 µg/kg of Pacific-ciguatoxin-1 (P-CTX-1 = CTX1B) from 1.1 to 4.3 µg of P-CTX-1 equivalents (eq.) entering the food chain from 0.7 to 2.7 million benthic dinoflagellates (Gambierdiscus sp.) producing 1.6 pg/cell of the P-CTX-1 precursor, P-CTX-4B (CTX4B). We simulated the food chain transfer of ciguatoxins via surgeonfishes by modelling Ctenochaetus striatus feeding on turf algae. A C. striatus feeding on ≥1000 Gambierdiscus/cm2 of turf algae accumulates sufficient toxin in <2 days that when preyed on, produces a 1.6 kg common coral trout with a flesh concentration of 0.1 µg/kg P-CTX-1. Our model shows that even transient blooms of highly ciguatoxic Gambierdiscus can generate ciguateric fishes. In contrast, sparse cell densities of ≤10 Gambierdiscus/cm2 are unlikely to pose a significant risk, at least in areas where the P-CTX-1 family of ciguatoxins predominate. The ciguatera risk from intermediate Gambierdiscus densities (~100 cells/cm2) is more difficult to assess, as it requires feeding times for surgeonfish (~4-14 days) that overlap with turnover rates of turf algae that are grazed by herbivorous fishes, at least in regions such as the GBR, where stocks of herbivorous fishes are not impacted by fishing. We use our model to explore how the duration of ciguatoxic Gambierdiscus blooms, the type of ciguatoxins they produce, and fish feeding behaviours can produce differences in relative toxicities between trophic levels. Our simple model indicates thresholds for the design of risk and mitigation strategies for ciguatera and the variables that can be manipulated to explore alternate scenarios for the accumulation and transfer of P-CTX-1 analogues through marine food chains and, potentially, for other ciguatoxins in other regions, as more data become available.

Toxoplasma gondii AP2XII-2 Contributes to Transcriptional Repression for Sexual Commitment.

Toxoplasma gondii is a widespread protozoan parasite that has a significant impact on human and veterinary health. The parasite undergoes a complex life cycle involving multiple hosts and developmental stages. How Toxoplasma transitions between life cycle stages is poorly understood yet central to controlling transmission. Of particular neglect are the factors that contribute to its sexual development, which takes place exclusively in feline intestines. While epigenetic repressors have been shown to play an important role in silencing the spurious gene expression of sexually committed parasites, the specific factors that recruit this generalized machinery to the appropriate genes remain largely unexplored. Here, we establish that a member of the AP2 transcription factor family, AP2XII-2, is targeted to genomic loci associated with sexually committed parasites along with epigenetic regulators of transcriptional silencing, HDAC3 and MORC. Despite its widespread association with gene promoters, AP2XII-2 is required for the silencing of relatively few genes. Using the CUT&Tag (cleavage under targets and tagmentation) methodology, we identify two major genes associated with sexual development downstream of AP2XII-2 control, AP2X-10 and the amino acid hydroxylase AAH1. Our findings show that AP2XII-2 is a key contributor to the gene regulatory pathways modulating Toxoplasma sexual development. IMPORTANCE Toxoplasma gondii is a parasite that undergoes its sexual stage exclusively in feline intestines, making cats a major source of transmission. A better understanding of the proteins controlling the parasite's life cycle stage transitions is needed for the development of new therapies aimed at treating toxoplasmosis and the transmission of the infection. Genes that regulate the sexual stages need to be turned on and off at the appropriate times, activities that are mediated by specific transcription factors that recruit general machinery to silence or activate gene expression. In this study, we identify a transcription factor called AP2XII-2 as being important for the repression of a subset of sexual stage genes, including a sexual stage-specific AP2 factor (AP2X-10) and a protein (AAH1) required to construct the infectious oocysts expelled from infected cats.

Bimodal Cell Size and Fusing Cells Observed in a Clonal Culture of the Ciguatoxin-Producing Benthic Dinoflagellate Gambierdiscus (WC1/1).

Cells in a clonal culture of the WC1/1 strain of Gambierdiscus that produced ciguatoxin and maitotoxin-3 were observed to spontaneously fuse during the light phase of culture growth. Cells in the process of fusion were indistinguishable from other cells under the light microscope, except that at least one (often both) of the fusing cells displayed an extendible, finger-like protrusion (presumed peduncle) arising from near the sulcul region. Fusion started with one of the cells turning 90° to place the planes of the girdles approximately at right angles to each other, and movement of the transverse flagella ceased in both cells, or in the cell seen in girdle (lateral) view. The cell in girdle view appeared to fuse into the theca of the other cell. The cell that had turned 90° often rounded up and become egg shaped (obovoid) during early fusion. Fusion can be quick (<10 min) or can take more than an hour. We saw no evidence of the theca being shed during fusion. Measurement of the dorsoventral and transdiameters revealed a wide range for cell sizes that were distributed as a bimodal population in the clonal culture. This bimodal cell population structure was maintained in clonal cultures reisolated from a small or large cell from the original WC1/1 culture. Cellular production of ciguatoxins by the WC1/1 clone increased during the first two years in culture with a corresponding decrease in production of maitotoxin-3, but this inverse relationship was not maintained over the following ~1.5 years.

Origin of Ciguateric Fish: Quantitative Modelling of the Flow of Ciguatoxin through a Marine Food Chain.

To begin to understand the impact of food chain dynamics on ciguatera risk, we used published data to model the transfer of ciguatoxins across four trophic levels of a marine food chain in Platypus Bay, Australia. The data to support this first attempt to conceptualize the scale of each trophic transfer step was limited, resulting in broad estimates. The hypothetical scenario we explored generated a low-toxicity 10 kg Spanish mackerel (Scomberomorus commerson) with a flesh concentration of 0.1 µg/kg of Pacific-ciguatoxin-1 (P-CTX-1, also known as CTX1B) from 19.5-78.1 µg of P-CTX-1 equivalents (eq.) that enter the marine food chain from a population of 12-49 million benthic dinoflagellates (Gambierdiscus sp.) producing 1.6 × 10-12 g/cell of the P-CTX-1 precursor, P-CTX-4B. This number of Gambierdiscus could be epiphytic on 22-88 kg of the benthic macroalgae (Cladophora) that carpets the bottom of much of Platypus Bay, with the toxin transferred to an estimated 40,000-160,000 alpheid shrimps in the second trophic level. This large number of shrimps appears unrealistic, but toxic shrimps would likely be consumed by a school of small, blotched javelin fish (Pomadasys maculatus) at the third trophic level, reducing the number of shrimps consumed by each fish. The Spanish mackerel would accumulate a flesh concentration of 0.1 µg/kg P-CTX-1 eq. by preying upon the school of blotched javelin and consuming 3.6-14.4 µg of P-CTX-1 eq. However, published data indicate this burden of toxin could be accumulated by a 10 kg Spanish mackerel from as few as one to three blotched javelin fish, suggesting that much greater amounts of toxin than modelled here must at certain times be produced and transferred through Platypus Bay food chains. This modelling highlights the need for better quantitative estimates of ciguatoxin production, biotransformation, and depuration through marine food chains to improve our understanding and management of ciguatera risk.

Analysis of Translational Control in the Integrated Stress Response by Polysome Profiling.

Translational control provides a strategy for rapid optimization of gene expression and restoration of protein homeostasis in response to cellular stresses. An important mechanism for translational control involves phosphorylation of eIF2, which invokes the integrated stress response (ISR). In the ISR, initiation of bulk protein synthesis is lowered coincident with enhanced translation efficiency of select gene transcripts that serve critical functions in stress adaptation. In this chapter, we focus on polysome profiling as a tool for establishing and characterizing translation control induced by eIF2 phosphorylation during environmental stresses. We describe in detail the experimental strategies of polysome profiling for detecting bulk repression of the translational machinery and quantifying translational control of key stress-induced gene transcripts. These experimental strategies can be adjusted to measure individual gene transcripts or genome-wide analyses and can be adapted to measure changes in the levels of ribosome subunits and associated factors invoked by various cellular cues in the ISR.

Critical Review and Conceptual and Quantitative Models for the Transfer and Depuration of Ciguatoxins in Fishes.

We review and develop conceptual models for the bio-transfer of ciguatoxins in food chains for Platypus Bay and the Great Barrier Reef on the east coast of Australia. Platypus Bay is unique in repeatedly producing ciguateric fishes in Australia, with ciguatoxins produced by benthic dinoflagellates (Gambierdiscus spp.) growing epiphytically on free-living, benthic macroalgae. The Gambierdiscus are consumed by invertebrates living within the macroalgae, which are preyed upon by small carnivorous fishes, which are then preyed upon by Spanish mackerel (Scomberomorus commerson). We hypothesise that Gambierdiscus and/or Fukuyoa species growing on turf algae are the main source of ciguatoxins entering marine food chains to cause ciguatera on the Great Barrier Reef. The abundance of surgeonfish that feed on turf algae may act as a feedback mechanism controlling the flow of ciguatoxins through this marine food chain. If this hypothesis is broadly applicable, then a reduction in herbivory from overharvesting of herbivores could lead to increases in ciguatera by concentrating ciguatoxins through the remaining, smaller population of herbivores. Modelling the dilution of ciguatoxins by somatic growth in Spanish mackerel and coral trout (Plectropomus leopardus) revealed that growth could not significantly reduce the toxicity of fish flesh, except in young fast-growing fishes or legal-sized fishes contaminated with low levels of ciguatoxins. If Spanish mackerel along the east coast of Australia can depurate ciguatoxins, it is most likely with a half-life of ≤1-year. Our review and conceptual models can aid management and research of ciguatera in Australia, and globally.

m6A RNA methylation facilitates pre-mRNA 3'-end formation and is essential for viability of Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite that can cause serious opportunistic disease in the immunocompromised or through congenital infection. To progress through its life cycle, Toxoplasma relies on multiple layers of gene regulation that includes an array of transcription and epigenetic factors. Over the last decade, the modification of mRNA has emerged as another important layer of gene regulation called epitranscriptomics. Here, we report that epitranscriptomics machinery exists in Toxoplasma, namely the methylation of adenosines (m6A) in mRNA transcripts. We identified novel components of the m6A methyltransferase complex and determined the distribution of m6A marks within the parasite transcriptome. m6A mapping revealed the modification to be preferentially located near the 3'-boundary of mRNAs. Knockdown of the m6A writer components METTL3 and WTAP resulted in diminished m6A marks and a complete arrest of parasite replication. Furthermore, we examined the two proteins in Toxoplasma that possess YTH domains, which bind m6A marks, and showed them to be integral members of the cleavage and polyadenylation machinery that catalyzes the 3'-end processing of pre-mRNAs. Loss of METTL3, WTAP, or YTH1 led to a defect in transcript 3'-end formation. Together, these findings establish that the m6A epitranscriptome is essential for parasite viability by contributing to the processing of mRNA 3'-ends.

Adaptive, caste-specific changes to recombination rates in a thelytokous honeybee population.

The ability to clone oneself has clear benefits-no need for mate hunting or dilution of one's genome in offspring. It is therefore unsurprising that some populations of haplo-diploid social insects have evolved thelytokous parthenogenesis-the virgin birth of a female. But thelytokous parthenogenesis has a downside: the loss of heterozygosity (LoH) as a consequence of genetic recombination. LoH in haplo-diploid insects can be highly deleterious because female sex determination often relies on heterozygosity at sex-determining loci. The two female castes of the Cape honeybee, Apis mellifera capensis, differ in their mode of reproduction. While workers always reproduce thelytokously, queens always mate and reproduce sexually. For workers, it is important to reduce the frequency of recombination so as to not produce offspring that are homozygous. Here, we ask whether recombination rates differ between Cape workers and Cape queens that we experimentally manipulated to reproduce thelytokously. We tested our hypothesis that Cape workers have evolved mechanisms that restrain genetic recombination, whereas queens have no need for such mechanisms because they reproduce sexually. Using a combination of microsatellite genotyping and whole-genome sequencing we find that a reduction in recombination is confined to workers only.

Discordant regulation of eIF2 kinase GCN2 and mTORC1 during nutrient stress.

Appropriate regulation of the Integrated stress response (ISR) and mTORC1 signaling are central for cell adaptation to starvation for amino acids. Halofuginone (HF) is a potent inhibitor of aminoacylation of tRNAPro with broad biomedical applications. Here, we show that in addition to translational control directed by activation of the ISR by general control nonderepressible 2 (GCN2), HF increased free amino acids and directed translation of genes involved in protein biogenesis via sustained mTORC1 signaling. Deletion of GCN2 reduced cell survival to HF whereas pharmacological inhibition of mTORC1 afforded protection. HF treatment of mice synchronously activated the GCN2-mediated ISR and mTORC1 in liver whereas Gcn2-null mice allowed greater mTORC1 activation to HF, resulting in liver steatosis and cell death. We conclude that HF causes an amino acid imbalance that uniquely activates both GCN2 and mTORC1. Loss of GCN2 during HF creates a disconnect between metabolic state and need, triggering proteostasis collapse.

Effects of inorganic nitrate supplementation on cardiovascular function and exercise tolerance in heart failure.

Heart failure (HF) results in a myriad of central and peripheral abnormalities that impair the ability to sustain skeletal muscle contractions and, therefore, limit tolerance to exercise. Chief among these abnormalities is the lowered maximal oxygen uptake, which is brought about by reduced cardiac output and exacerbated by O2 delivery-utilization mismatch within the active skeletal muscle. Impaired nitric oxide (NO) bioavailability is considered to play a vital role in the vascular dysfunction of both reduced and preserved ejection fraction HF (HFrEF and HFpEF, respectively), leading to the pursuit of therapies aimed at restoring NO levels in these patient populations. Considering the complementary role of the nitrate-nitrite-NO pathway in the regulation of enzymatic NO signaling, this review explores the potential utility of inorganic nitrate interventions to increase NO bioavailability in the HFrEF and HFpEF patient population. Although many preclinical investigations have suggested that enhanced reduction of nitrite to NO in low Po2 and pH environments may make a nitrate-based therapy especially efficacious in patients with HF, inconsistent results have been found thus far in clinical settings. This brief review provides a summary of the effectiveness (or lack thereof) of inorganic nitrate interventions on exercise tolerance in patients with HFrEF and HFpEF. Focus is also given to practical considerations and current gaps in the literature to facilitate the development of effective nitrate-based interventions to improve exercise tolerance in patients with HF.

The Secreted Acid Phosphatase Domain-Containing GRA44 from Toxoplasma gondii Is Required for c-Myc Induction in Infected Cells.

During host cell invasion, the eukaryotic pathogen Toxoplasma gondii forms a parasitophorous vacuole to safely reside within the cell, while it is partitioned from host cell defense mechanisms. From within this safe niche, parasites sabotage multiple host cell systems, including gene expression, apoptosis, and intracellular immune recognition, by secreting a large arsenal of effector proteins. Many parasite proteins studied for active host cell manipulative interactions have been kinases. The translocation of effectors from the parasitophorous vacuole into the host cell is mediated by a putative translocon complex, which includes the proteins MYR1, MYR2, and MYR3. Whether other proteins are involved in the structure or regulation of this putative translocon is not known. We have discovered that the secreted protein GRA44, which contains a putative acid phosphatase domain, interacts with members of this complex and is required for host cell effects downstream of effector secretion. We have determined that GRA44 is processed in a region with homology to sequences targeted by protozoan proteases of the secretory pathway and that both major cleavage fragments are secreted into the parasitophorous vacuole. Immunoprecipitation experiments showed that GRA44 interacts with a large number of secreted proteins, including MYR1. Importantly, conditional knockdown of GRA44 resulted in a lack of host cell c-Myc upregulation, which mimics the phenotype seen when members of the translocon complex are genetically disrupted. Thus, the putative acid phosphatase GRA44 is crucial for host cell alterations during Toxoplasma infection and is associated with the translocon complex which Toxoplasma relies upon for success as an intracellular pathogen.IMPORTANCE Approximately one-third of humans are infected with the parasite Toxoplasma gondiiToxoplasma infections can lead to severe disease in those with a compromised or suppressed immune system. Additionally, infections during pregnancy present a significant health risk to the developing fetus. Drugs that target this parasite are limited, have significant side effects, and do not target all disease stages. Thus, a thorough understanding of how the parasite propagates within a host is critical in the discovery of novel therapeutic targets. Toxoplasma replication requires that it enter the cells of the infected organism. In order to survive the environment inside a cell, Toxoplasma secretes a large repertoire of proteins, which hijack a number of important cellular functions. How these Toxoplasma proteins move from the parasite into the host cell is not well understood. Our work shows that the putative phosphatase GRA44 is part of a protein complex responsible for this process.

Simultaneous Ribosome Profiling of Human Host Cells Infected with Toxoplasma gondii.

Toxoplasma gondii is a ubiquitous obligate intracellular parasite that infects the nucleated cells of warm-blooded animals. From within the parasitophorous vacuole in which they reside, Toxoplasma tachyzoites secrete an arsenal of effector proteins that can reprogram host gene expression to facilitate parasite survival and replication. Gaining a better understanding of how host gene expression is altered upon infection is central for understanding parasite strategies for host invasion and for developing new parasite therapies. Here, we applied ribosome profiling coupled with mRNA measurements to concurrently study gene expression in the parasite and in host human foreskin fibroblasts. By examining the parasite transcriptome and translatome, we identified potential upstream open reading frames that may permit the stress-induced preferential translation of parasite mRNAs. We also determined that tachyzoites reduce host death-associated pathways and increase survival, proliferation, and motility in both quiescent and proliferative host cell models of infection. Additionally, proliferative cells alter their gene expression in ways that are consistent with massive transcriptional rewiring, while quiescent cells were best characterized by reentry into the cell cycle. We also identified a translational control regimen consistent with mechanistic target of rapamycin (mTOR) activation in quiescent cells and, to a lesser degree, in proliferative cells. This study illustrates the utility of the method for dissection of gene expression programs simultaneously in the parasite and host.IMPORTANCEToxoplasma gondii is a single-celled parasite that has infected up to one-third of the world's population. Significant overhauls in gene expression in both the parasite and the host cell accompany parasite invasion, and a better understanding of these changes may lead to the development of new therapeutic agents. In this study, we employed ribosome profiling to determine the changes that occur at the levels of transcription and translation in both the parasite and the infected host cell at the same time. We discovered features of Toxoplasma mRNAs that suggest a means for controlling parasite gene expression under stressful conditions. We also show that differences in host gene expression occur depending on whether they are confluent or not. Our findings demonstrate the feasibility of using ribosomal profiling to interrogate the host-parasite dynamic under a variety of conditions.

Convergent eusocial evolution is based on a shared reproductive groundplan plus lineage-specific plastic genes.

Eusociality has convergently evolved multiple times, but the genomic basis of caste-based division of labor and degree to which independent origins of eusociality have utilized common genes remain largely unknown. Here we characterize caste-specific transcriptomic profiles across development and adult body segments from pharaoh ants (Monomorium pharaonis) and honey bees (Apis mellifera), representing two independent origins of eusociality. We identify a substantial shared core of genes upregulated in the abdomens of queen ants and honey bees that also tends to be upregulated in mated female flies, suggesting that these genes are part of a conserved insect reproductive groundplan. Outside of this shared groundplan, few genes are differentially expressed in common. Instead, the majority of the thousands of caste-associated genes are plastically expressed, rapidly evolving, and relatively evolutionarily young. These results emphasize that the recruitment of both highly conserved and lineage-specific genes underlie the convergent evolution of novel traits such as eusociality.

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.