Free-living amoeba are members of microbial communities such as biofilms in terrestrial, fresh, and marine habitats. Although they are known to live in close association with bacteria in many ecosystems such as biofilms, they are considered to be major bacterial predators in many ecosystems. Little is known on the relationship between protozoa and marine bacteria in microbial communities, more precisely on how bacteria are able survive in environmental niches where these bacterial grazers also live. The objective of this work is to study the interaction between the axenized ubiquitous amoeba Acanthamoeba castellanii and four marine bacteria isolated from immersed biofilm, in order to evaluate if they would be all grazed upon by amoeba or if they would be able to survive in the presence of their predator. At a low bacteria-to-amoeba ratio, we show that each bacterium is phagocytized and follows a singular intracellular path within this host cell, which appears to delay or to prevent bacterial digestion. In particular, one of the bacteria was found in the amoeba nucleolar compartment whereas another strain was expelled from the amoeba in vesicles. We then looked at the fate of the bacteria grown in a higher bacteria-to-amoeba ratio, as a preformed mono- or multi-species biofilm in the presence of A. castellanii. We show that all biofilms were subjected to detachment from the surface in the presence of the amoeba or its supernatant. Overall, these results show that bacteria, when facing the same predator, exhibit a variety of escape mechanisms at the cellular and population level, when we could have expected a simple bacterial grazing. Therefore, this study unravels new insights into the survival of environmental bacteria when facing predators that they could encounter in the same microbial communities.

Noncoding circular RNAs are widespread in the tree of life. Particularly, intron-containing circular RNAs which apparently upregulate their parental gene expression. Entamoeba histolytica, the causative agent of dysentery and liver abscesses in humans, codes for several noncoding RNAs, including circular ribosomal RNAs, but no intron containing circular RNAs have been described to date. Divergent RT-PCR and diverse molecular approaches, allowed us to detect bona fide full-length intronic circular RNA (flicRNA) molecules. Self-splicing reactions, RNA polymerase II inhibition with Actinomycin D, and second step of splicing-inhibition with boric acid showed that the production of flicRX13 (one of the flicRNAs found in this work, and our test model) depends on mRNA synthesis and pre-mRNA processing instead of self-splicing. To explore the cues and factors involved in flicRX13 biogenesis in vivo, splicing assays were carried out in amoeba transformants where splicing factors and Dbr1 (intron lariat debranching enzyme 1) were silenced or overexpressed, or where Rabx13 wild-type and mutant 5'ss (splice site) and branch site minigene constructs were overexpressed. Whereas SF1 (splicing factor 1) is not involved, the U2 auxiliary splicing factor, Dbr1, and the GU-rich 5'ss are involved in postsplicing flicRX13 biogenesis, probably by Dbr1 stalling, in a similar fashion to the formation of ciRNAs (circular intronic RNAs), but with distinctive 5'-3'ss ligation points. Different from the reported functions of ciRNAs, the 5'ss GU-rich element of flicRX13 possibly interacts with transcription machinery to silence its own gene in cis. Furthermore, introns of E. histolytica virulence-related genes are also processed as flicRNAs.

Microorganisms in environmental samples are identified by sequential screening, isolation, and culture steps, followed by the verification of physiological characteristics and morphological classification. Isolation and purification of Amoebae from soil samples is extremely complex, laborious, and time-consuming and require considerable expertise for morphological evaluation. PCR testing of soil DNA seems to be an effective means for protozoa habitat screening. In this study, we added Acanthamoeba sp. (MK strain) to soil and developed a method of extracting protozoan DNA from the soil.

Extracellular vesicles (EVs) are small membrane-bound vesicles of growing interest in vetetinary parasitology. The aim of the present report was to provide the first isolation, quantification and protein characterization of EVs from buffalo (Bubalus bubalis) sera infected with Theileria spp.

Food and waterborne protozoan pathogens can cause serious disease in people. Three common species Cryptosporidium parvum, Giardia enterica and Toxoplasma gondii can contaminate diverse shellfish species, including commercial oysters. Current methods of protozoan detection in shellfish are not standardized, and few are able to simultaneously identify multiple species. Here, we present a novel metabarcoding assay targeting the 18S rRNA gene followed by next generation sequencing (NGS) for simultaneous detection of Cryptosporidium spp., Giardia spp. and T. gondii spiked into oyster samples. We further developed a bioinformatic pipeline to process and analyze 18S rRNA data for protozoa classification. The ability of the NGS assay to detect protozoa was later compared with conventional PCR. Results demonstrated that background amplification of oyster and other eukaryotic DNA competed with that of protozoa for obtained sequence reads. Sequences of target protozoans were obtained across all spiking levels; however, low numbers of target sequences in negative controls imply that a threshold for true positives must be defined for assay interpretation. While this study focused on three target parasites, the ability of this approach to detect numerous known and potentially unknown protozoan pathogens make it a promising screening tool for monitoring protozoan contamination in food and water.

Toxoplasma gondii is a widespread apicomplexan parasite that can cause severe disease in its human hosts. The ability of T. gondii and other apicomplexan parasites to invade into, egress from, and move between cells of the hosts they infect is critical to parasite virulence and disease progression. An unusual and highly conserved parasite myosin motor (TgMyoA) plays a central role in T. gondii motility. The goal of this work was to determine whether the parasite's motility and lytic cycle can be disrupted through pharmacological inhibition of TgMyoA, as an approach to altering disease progression in vivo. To this end, we first sought to identify inhibitors of TgMyoA by screening a collection of 50,000 structurally diverse small molecules for inhibitors of the recombinant motor's actin-activated ATPase activity. The top hit to emerge from the screen, KNX-002, inhibited TgMyoA with little to no effect on any of the vertebrate myosins tested. KNX-002 was also active against parasites, inhibiting parasite motility and growth in culture in a dose-dependent manner. We used chemical mutagenesis, selection in KNX-002, and targeted sequencing to identify a mutation in TgMyoA (T130A) that renders the recombinant motor less sensitive to compound. Compared to wild-type parasites, parasites expressing the T130A mutation showed reduced sensitivity to KNX-002 in motility and growth assays, confirming TgMyoA as a biologically relevant target of KNX-002. Finally, we present evidence that KNX-002 can slow disease progression in mice infected with wild-type parasites, but not parasites expressing the resistance-conferring TgMyoA T130A mutation. Taken together, these data demonstrate the specificity of KNX-002 for TgMyoA, both in vitro and in vivo, and validate TgMyoA as a druggable target in infections with T. gondii. Since TgMyoA is essential for virulence, conserved in apicomplexan parasites, and distinctly different from the myosins found in humans, pharmacological inhibition of MyoA offers a promising new approach to treating the devastating diseases caused by T. gondii and other apicomplexan parasites.

Lyme disease is the most prominent tick-borne disease in the United States. Co-infections with the tick-transmitted pathogens Babesia microti and Borrelia burgdorferi sensu stricto are becoming a serious health problem. B. burgdorferi is an extracellular spirochete that causes Lyme disease while B. microti is a protozoan that infects erythrocytes and causes babesiosis. Testing of donated blood for Babesia species is not currently mandatory due to unavailability of an FDA approved test. Transmission of this protozoan by blood transfusion often results in high morbidity and mortality in recipients. Infection of C3H/HeJ mice with B. burgdorferi and B. microti individually results in inflammatory Lyme disease and display of human babesiosis-like symptoms, respectively. Here we use this mouse model to provide a detailed investigation of the reciprocal influence of the two pathogens on each other during co-infection. We show that B. burgdorferi infection attenuates parasitemia in mice while B. microti subverts the splenic immune response, such that a marked decrease in splenic B and T cells, reduction in antibody levels and diminished functional humoral immunity, as determined by spirochete opsonophagocytosis, are observed in co-infected mice compared to only B. burgdorferi infected mice. Furthermore, immunosuppression by B. microti in co-infected mice showed an association with enhanced Lyme disease manifestations. This study demonstrates the effect of only simultaneous infection by B. burgdorferi and B. microti on each pathogen, immune response and on disease manifestations with respect to infection by the spirochete and the parasite. In our future studies, we will examine the overall effects of sequential infection by these pathogens on host immune responses and disease outcomes.

Trichomonas vaginalis is an extracellular flagellated protozoan parasite that causes trichomoniasis, one of the most common non-viral sexually transmitted diseases. To survive and to maintain infection, T. vaginalis adapts to a hostile host environment by regulating gene expression. However, the mechanisms of transcriptional regulation are poorly understood for this parasite. Histone modification has a marked effect on chromatin structure and directs the recruitment of transcriptional machinery, thereby regulating essential cellular processes. In this study, we aimed to outline modes of chromatin-mediated gene regulation in T. vaginalis. Inhibition of histone deacetylase (HDAC) alters global transcriptional responses and induces hyperacetylation of histones and hypermethylation of H3K4. Analysis of the genome of T. vaginalis revealed that a number of enzymes regulate histone modification, suggesting that epigenetic mechanisms are important to controlling gene expression in this organism. Additionally, we describe the genome-wide localization of two histone H3 modifications (H3K4me3 and H3K27Ac), which we found to be positively associated with active gene expression in both steady and dynamic transcriptional states. These results provide the first direct evidence that histone modifications play an essential role in transcriptional regulation of T. vaginalis, and may help guide future epigenetic research into therapeutic intervention strategies against this parasite.

Members of the family Corvidae are ecologically flexible omnivorous birds, particularly adaptive to urban habitats, and living in proximity to humans; these birds may serve as definitive hosts (DH) for Sarcocystis spp., but research about this is lacking. In the present study, intestinal samples from 91 corvids collected in Lithuania were molecularly tested by species-specific PCR targeting the ITS1 and cox1 genes and subsequently sequenced for the presence of Sarcocystis spp. Under a light microscope, oocysts of Sarcocystis spp. were observed in 43 samples (47.3%), while molecular methods, detected Sarcocystis spp. in 77 birds (84.6%). Eleven Sarcocystis spp. (S. columbae, S. cornixi, potentially pathogenic S. halieti, S. kutkienae, S. lari, S. turdusi, S. wobeseri, S. arctica, S. lutrae, S. ovalis, and S. oviformis) were identified in the intestinal samples from six corvid species from Lithuania. Infections with multiple Sarcocystis spp. were detected in 79.2% of the infected corvid birds. Three of the identified Sarcocystis spp. use corvids as intermediate hosts (IH); therefore, corvids may serve as IH and DH of the same Sarcocystis species. Based on molecular results and on corvid diet, omnivorous corvids may play an important role in transmitting Sarcocystis spp.

Intestinal parasitic infections are common in rural areas with poor infrastructure and low socioeconomic status. The aim of this study was to estimate the prevalence of selected parasitic infections in marginalized rural areas in the northern part of the Palestinian West Bank Region, using conventional and PCR-based methods, and also to assess risk predictors of infection.

Climacostol (5-[(2Z)-non-2-en-1-yl]benzene-1,3-diol) is a resorcinol produced by the protozoan Climacostomum virens for defence against predators. It exerts a potent antimicrobial activity against bacterial and fungal pathogens, inhibits the growth of several human and rodent tumour cells, and is now available by chemical synthesis. In this study, we chemically synthesized two novel analogues of climacostol, namely, 2-methyl-5 [(2Z)-non-2-en-1-yl]benzene-1,3-diol (AN1) and 5-[(2Z)-non-2-en-1-yl]benzene-1,2,3-triol (AN2), with the aim to increase the activity of the native toxin, evaluating their effects on prokaryotic and free-living protists and on mammalian tumour cells. The results demonstrated that the analogue bearing a methyl group (AN1) in the aromatic ring exhibited appreciably higher toxicity against pathogen microbes and protists than climacostol. On the other hand, the analogue bearing an additional hydroxyl group (AN2) in the aromatic ring revealed its ability to induce programmed cell death in protistan cells. Overall, the data collected demonstrate that the introduction of a methyl or a hydroxyl moiety to the aromatic ring of climacostol can effectively modulate its potency and its mechanism of action.

Representatives of the genus Sarcocystis are unicellular parasites having a two-host life cycle and infecting mammals, birds, and reptiles. Until now, Sarcocystis spp. have been mainly investigated in definitive and intermediate hosts. Only a few studies have been conducted on the detection of Sarcocystis parasites in water samples. The aim of this research was to examine whether the prevalence of Sarcocystis spp. parasitizing farm animals varies in different types of water bodies. Water samples (n = 150) were collected from the entire territory of Lithuania, dividing water bodies into five groups (lakes, rivers, ponds/canals, swamps, and the inshore zone of the territorial Baltic Sea area). One-liter samples were filtered and subsequently analyzed using nested PCR. At least one of the analyzed Sarcocystis spp. (S. arieticanis, S. bertrami, S. bovifelis, S. capracanis, S. cruzi, S. hirsuta, S. miescheriana, and S. tenella) was determined in all examined samples from water bodies. No significant difference in Sarcocystis spp. prevalence between different types of water sources was detected. Our research proved that selecting appropriate primers is important for the accurate identification of parasites in samples collected from water bodies.

PTEN is a lipid phosphatase that is highly conserved and involved in a broad range of biological processes including cytoskeletal reorganization, endocytosis, signal transduction, and cell migration in all eukaryotes. Although regulation of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] signaling via PTEN has been well established in model organisms and mammals, it remains elusive in the parasitic protist E. histolytica, which heavily relies on PtdIns phosphate(s)-dependent membrane traffic, migration, and phago- and trogocytosis for its pathogenesis. In this study, we characterized the major PTEN from E. histolytica, EhPTEN1, which shows the highest expression at the transcript level in the trophozoite stage among 6 possible PTENs, to understand the significance of PtdIns(3,4,5)P3 signaling in this parasite. Live imaging of GFP-EhPTEN1 expressing amebic trophozoites showed localization mainly in the cytosol with a higher concentration at pseudopods and the extending edge of the phago- and trogocytic cups. Furthermore, quantitative analysis of phago- and trogocytosis using a confocal image cytometer showed that overexpression of EhPTEN1 caused reduction in trogo- and phagocytosis while transcriptional gene silencing of EhPTEN1 gene caused opposite phenotypes. These data suggest that EhPTEN1 has an inhibitory role in these biological processes. Conversely, EhPTEN1 acts as a positive regulator for fluid-phase and receptor-mediated endocytosis in E. histolytica trophozoites. Moreover, we showed that EhPTEN1 was required for optimal growth and migration of this parasite. Finally, the phosphatase activity of EhPTEN1 towards PtdIns(3,4,5)P3 was demonstrated, suggesting that the biological roles of EhPTEN1 are likely linked to its catalytic function. Taken together, these results indicate that EhPTEN1 differentially regulates multiple cellular activities essential for proliferation and pathogenesis of the organism, via PtdIns(3,4,5)P3 signaling. Elucidation of biological roles of PTEN and PtdIns(3,4,5)P3 signaling at the molecular levels promotes our understanding of the pathogenesis of this parasite.

Trypanosomatids (order Kinetoplastida), including the human pathogens Trypanosoma cruzi (agent of Chagas disease), Trypanosoma brucei, (African sleeping sickness), and Leishmania (leishmaniasis), affect millions of people and animals globally. T. cruzi is considered one of the least studied and most poorly understood tropical disease-causing parasites, in part because of the relative lack of facile genetic engineering tools. This situation has improved recently through the application of clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) technology, but a number of limitations remain, including the toxicity of continuous Cas9 expression and the long drug marker selection times. In this study, we show that the delivery of ribonucleoprotein (RNP) complexes composed of recombinant Cas9 from Staphylococcus aureus (SaCas9), but not from the more routinely used Streptococcus pyogenes Cas9 (SpCas9), and in vitro-transcribed single guide RNAs (sgRNAs) results in rapid gene edits in T. cruzi and other kinetoplastids at frequencies approaching 100%. The highly efficient genome editing via SaCas9/sgRNA RNPs was obtained for both reporter and endogenous genes and observed in multiple parasite life cycle stages in various strains of T. cruzi, as well as in T. brucei and Leishmania major RNP complex delivery was also used to successfully tag proteins at endogenous loci and to assess the biological functions of essential genes. Thus, the use of SaCas9 RNP complexes for gene editing in kinetoplastids provides a simple, rapid, and cloning- and selection-free method to assess gene function in these important human pathogens.IMPORTANCE Protozoan parasites remain some of the highest-impact human and animal pathogens, with very limited treatment and prevention options. The development of improved therapeutics and vaccines depends on a better understanding of the unique biology of these organisms, and understanding their biology, in turn, requires the ability to track and manipulate the products of genes. In this work, we describe new methods that are available to essentially any laboratory and applicable to any parasite isolate for easily and rapidly editing the genomes of kinetoplastid parasites. We demonstrate that these methods provide the means to quickly assess function, including that of the products of essential genes and potential targets of drugs, and to tag gene products at their endogenous loci. This is all achieved without gene cloning or drug selection. We expect this advance to enable investigations, especially in Trypanosoma cruzi and Leishmania spp., that have eluded investigators for decades.

The human eukaryotic pathogen Trichomonas vaginalis causes trichomoniasis, a prevalent sexually transmitted infection. This extracellular protozoan is intimately associated with the human vaginal mucosa and microbiota, but key aspects of the complex interactions between the parasite and the vaginal bacteria remain elusive. We report that T. vaginalis has acquired, by lateral gene transfer from bacteria, genes encoding peptidoglycan hydrolases of the NlpC/P60 family. Two of the T. vaginalis enzymes were active against bacterial peptidoglycan, retaining the active-site fold and specificity as dl-endopeptidases. The endogenous NlpC/P60 genes are transcriptionally upregulated in T. vaginalis in the presence of bacteria. The overexpression of an exogenous copy enables the parasite to outcompete bacteria from mixed cultures, consistent with the biochemical activity of the enzyme. Our study results highlight the relevance of the interactions of this eukaryotic pathogen with bacteria, a poorly understood aspect of the biology of this important human parasite.IMPORTANCETrichomonas vaginalis is a parasitic protozoan of the human urogenital tract that causes trichomoniasis, a very common sexually transmitted disease. Despite residing extracellularly and in close association with the vaginal bacteria (i.e., the microbiota), very little is known about the nature of the parasite-bacterium interactions. Our study showed that this parasite had acquired genes from bacteria which retained their original function. They produce active enzymes capable of degrading peptidoglycan, a unique polymer of the bacterial cell envelope, helping the parasite to outcompete bacteria in mixed cultures. This study was the first to show that a laterally acquired group of genes enables a eukaryotic mucosal pathogen to control bacterial population. We highlight the importance of understanding the interactions between pathogens and microbiota, as the outcomes of these interactions are increasingly understood to have important implications on health and disease.

Tick-borne diseases (TBDs) considerably impair equine health and productivity. Moreover, TBDs, particularly equine piroplasmosis, impede international movement and trade of equids, which is a vital component of the global horse racing industry. In the Philippines, horse racing is a lucrative industry generating millions of USD annually. However, information on equine TBDs is scarce. This study intended to describe molecularly the equine tick-borne infections in a racehorse park in Cavite, Philippines and identify the risk factors associated with the infections. One hundred twenty-four (n = 124) thoroughbred racehorses were sampled and screened for selected tick-borne protozoan and bacterial pathogens using polymerase chain reaction (PCR) assays. Racehorses were positive for Babesia caballi (12.10%; 15/124), Theileria equi (0.81%; 1/124), Anaplasma phagocytophilum (10.48%; 13/124), Borrelia burgdorferi sensu lato (38.71%; 48/124), A. marginale (0.81%; 1/124), and Coxiella burnetii (0.81%; 1/124). Rickettsia was not detected in the samples. Gender was determined as a significant risk factor for B. caballi infection. Sequencing analysis revealed that seven partial 18S rRNA B. caballi isolates shared 98.63-100% identity with each other and were classified as genotype A. Meanwhile, the sequence obtained from the lone T. equi-positive sample was 99.77% identical to isolates from Spain, Switzerland, China, Saudi Arabia, and South Korea, and was confirmed as genotype E based on the 18S rRNA gene. Eight Anaplasma 16S rRNA partial sequences were highly identical to A. phagocytophilum and A. ovis. Partial sequences of Borrelia 5-23S rRNA were most closely related to B. japonica and other Borrelia sp. isolates from various countries. This study reports the first molecular detection of Borrelia and Anaplasma and the identification of B. caballi and T. equi genotypes in racehorses in the Philippines. Findings from this study shall be useful in crafting equine tick and TBD control and prevention programs in the country.

Perkinsus marinus (Perkinsozoa), a close relative of apicomplexans, is an osmotrophic facultative intracellular marine protozoan parasite responsible for "Dermo" disease in oysters and clams. Although there is no clinical evidence of this parasite infecting humans, HLA-DR40 transgenic mice studies strongly suggest the parasite as a natural adjuvant in oral vaccines. P. marinus is being developed as a heterologous gene expression platform for pathogens of medical and veterinary relevance and a novel platform for delivering vaccines. We previously reported the transient expression of two rodent malaria genes Plasmodium berghei HAP2 and MSP8. In this study, we optimized the original electroporation-based protocol to establish a stable heterologous expression method. Using 20 μg of pPmMOE[MOE1]:GFP and 25.0 × 106 P. marinus cells resulted in 98% GFP-positive cells. Furthermore, using the optimized protocol, we report for the first time the successful knock-in of GFP at the C-terminus of the PmMOE1 using ribonucleoprotein (RNP)-based CRISPR/Cas9 gene editing methodology. The GFP was expressed 18 h post-transfection, and expression was observed for 8 months post-transfection, making it a robust and stable knock-in system.

Toxoplasma gondii is a prevalent zoonotic pathogen infecting livestock as well as humans. The exceptional ability of this parasite to reproduce in several types of nucleated host cells necessitates a coordinated usage of endogenous and host-derived nutritional resources for membrane biogenesis. Phosphatidylethanolamine is the second most common glycerophospholipid in T. gondii, but how its requirement in the acutely-infectious fast-dividing tachyzoite stage is satisfied remains enigmatic. This work reveals that the parasite deploys de novo synthesis and salvage pathways to meet its demand for ester- and ether-linked PtdEtn. Auxin-mediated depletion of the phosphoethanolamine cytidylyltransferase (ECT) caused a lethal phenotype in tachyzoites due to impaired invasion and cell division, disclosing a vital role of the CDP-ethanolamine pathway during the lytic cycle. In accord, the inner membrane complex appeared disrupted concurrent with a decline in its length, parasite width and major phospholipids. Integrated lipidomics and isotope analyses of the TgECT mutant unveiled the endogenous synthesis of ester-PtdEtn, and salvage of ether-linked lipids from host cells. In brief, this study demonstrates how T. gondii operates various means to produce distinct forms of PtdEtn while featuring the therapeutic relevance of its de novo synthesis.

Background. Plasmodium yoelii 17XNL is a nonlethal malaria strain in mice of different genetic backgrounds including the C57BL/6 mice (I-Ab/I-Enull) used in this study as a control strain. We have compared the trends of blood stage infection with the nonlethal murine strain of P. yoelii 17XNL malaria protozoan in immunocompetent Nonobese Diabetic (NOD) mice prone to type 1 diabetes (T1D) and C57BL/6 mice (control mice) that are not prone to T1D and self-cure the P. yoelii 17XNL infection. Prediabetic NOD mice could not mount a protective antibody response to the P. yoelii 17XNL-infected red blood cells (iRBCs), and they all succumbed shortly after infection. Our data suggest that the lack of anti-P. yoelii 17XNL-iRBCs protective antibodies in NOD mice is a result of parasite-induced, Foxp3+ T regulatory (Treg) cells able to suppress the parasite-specific antibody secretion. Conclusions. The NOD mouse model may help in identifying new mechanisms of B-cell evasion by malaria parasites. It may also serve as a more accurate tool for testing antimalaria therapeutics due to the lack of interference with a preexistent self-curing mechanism present in other mouse strains.

The obligate intracellular parasite Toxoplasma gondii reprograms host gene expression through multiple mechanisms that promote infection, including the up-regulation of mTOR-dependent host mRNA translation. In addition to the mTOR-4E-BP1/2 axis, MAPK-interacting kinases 1 and 2 (MNK1/2) control the activity of the mRNA cap-binding protein eIF4E. Herein, we show that T. gondii inhibits the phosphorylation of MNK1/2 and their downstream target eIF4E in murine and human macrophages. Exposure to soluble T. gondii antigens (STAg) failed to fully recapitulate this phenotype indicating the requirement of live infection. Treatment with okadaic acid, a potent phosphatase inhibitor, restored phosphorylation of MNK1/2 and eIF4E regardless of infection. T. gondii replication was higher in macrophages isolated from mice mutated at the residue where eIF4E is phosphorylated (eIF4E S209A knock-in) than in wild-type (WT) control cells despite no differences in infection rates. Similarly, parasitemia in the mesenteric lymph nodes and spleen, as well as brain cyst burden were significantly augmented in infected eIF4E S209A knock-in mice compared to their WT counterparts. Of note, mutant mice were more susceptible to acute toxoplasmosis and displayed exacerbated levels of IFNγ. In all, these data suggest that the MNK1/2-eIF4E axis is required to control T. gondii infection and that its inactivation represents a strategy exploited by the parasite to promote its survival.