Biology subjects

Sullivan, W. J.

Publications and source records attributed to Sullivan, W. J..

17 recordsLinked to original sources

bioRxiv2026

Histone H2A.X contributes to Gene Expression Regulation and Cell-Cycle Progression in Toxoplasma gondii Tachyzoites

Histone H2A.X is a conserved variant classically associated with the DNA damage response (DDR), where its phosphorylation ({gamma}H2A.X) marks double-strand breaks and recruits repair factors. H2A.X is absent in many parasitic protozoans such as Plasmodium, Trypanosoma and Leishmania. In Toxoplasma gondii, the obligate intracellular parasite, causative agent of toxoplasmosis, however, its presence raises the question of whether it contributes to genome stability. To tackle this, we generated a T. gondii knockout of H2A.X (RH{Delta}H2A.X) and evaluated its phenotype. Although H2A.X is phosphorylated in response to DNA damage in T. gondii as in other systems, RH{Delta}H2A.X parasites showed no increased sensitivity to genotoxic agents, indicating that H2A.X is dispensable for canonical DDR. Instead, loss of H2A.X caused reduced gliding motility, increased host cell invasion and clustering of extracellular parasites after egress. Despite no defects in replication rate, RH{Delta}H2A.X showed delayed mitotic progression with abnormal vacuole morphology. Transcriptomic profiling revealed a moderate yet coordinated reprogramming of protein-coding genes. Specifically, H2A.X loss altered the expression of key apical secretory and virulence factors, especially ROP18, while inducing the transcriptional activation of mitotic and DNA replication machinery. Differential enrichment analysis further revealed that upregulated genes were preferentially associated with S phase, whereas downregulated genes were enriched during mitosis and cytokinesis. This is consistent with the delayed mitotic progression and spindle abnormalities observed by microscopy and flow cytometry and suggests a cell cycle phase-specific mechanism of H2A.X-dependent transcriptional control. Rather than a driver of DDR, these findings suggest that T. gondii H2A.X operates as a chromatin organizer that coordinates cell division fidelity and virulence gene expression.

cell biology↗

bioRxiv2025

Toxoplasma gondii RAD51 recombinase is required to overcome DNA replication stress and its inactivation leads to bradyzoite differentiation

Toxoplasma gondii is an obligate intracellular parasite with a high replication rate that can lead to DNA replicative stress, in turn associated with the generation of DNA double-strand breaks (DSBs). Cells have two main pathways to repair DSBs: non-homologous end joining and homologous recombination repair (NHEJ and HRR respectively). RAD51 is the key recombinase in the HRR pathway. In this work, we achieved endogenous tagging of the RAD51 gene using the Auxin Inducible Degron (AID) system, to generate the clonal line RH RAD51HA-AID. Here we demonstrate that RAD51 is expressed in replicative tachyzoites and establishes damage foci. Auxin-induced knock-down (KD) affects the correct replication of tachyzoites which show loss of synchronization. The use of the RAD51 inhibitor B02 also affects parasite growth, with an IC50 of 4.8 {micro}M. B02 produced alterations in tachyzoite replication and arrest in the S phase of the cell cycle. Additionally, B02 induced tachyzoite to bradyzoite differentiation showing small cyst-like structures. In conclusion, the HRR pathway is necessary for maintaining proper tachyzoite replication under normal growth conditions, supporting that replicative stress occurs during the cell cycle. Our findings also suggest that DNA replication stress can induce bradyzoite differentiation.

molecular biology↗

bioRxiv2025

GCN5a is a telomeric lysine acetyltransferase whose loss primes Toxoplasma gondii for latency

Toxoplasma gondii is a protozoan parasite that causes persistent infection in warm-blooded vertebrates by undergoing differentiation from a replicative stage (tachyzoites) to a latent encysted stage (bradyzoites). Stage differentiation is critical for transmission and pathogenesis and relies on gene regulation driven by a network of transcription and epigenetic factors. We previously found in non-cystogenic type I RH strain parasites that the lysine acetyltransferase (KAT), GCN5a, is dispensable in tachyzoites but required to upregulate stress-response genes, suggesting a link with bradyzoite conversion. To address this possibility, we generated endogenously tagged GCN5a parasites and a genetic knockout in cystogenic type II Pru strain. We show that GCN5a protein, but not mRNA, increases during differentiation and complexes with unique protein partners, most of which contain AP2 domains. Pru strain tachyzoites lacking GCN5a augment bradyzoite-specific gene expression in the absence of stress. Loss of GCN5a slowed tachyzoite replication and heightened sensitivity to bradyzoite conversion but resulted in smaller cyst sizes compared to wildtype. Using CUT&Tag, we delineated the chromosomal occupancy of GCN5a relative to the essential KAT, GCN5b. While GCN5b localizes to coding regions, GCN5a surprisingly localizes exclusively to telomeres. These findings suggest that the loss of GCN5a leads to telomere dysfunction, which slows replication and promotes the transition to latency. IMPORTANCEToxoplasma gondii is a single-celled parasite that persists in warm-blooded hosts, including humans, because it converts into latent tissue cysts. Switching from its replicating form into dormant cysts is a tightly regulated process that involves epigenetic factors such as lysine acetyltransferases GCN5a and GCN5b. This study is the first to examine the role of GCN5a in a cyst-forming Toxoplasma strain. We found that GCN5a protein, but not mRNA, increases during cyst development. Additionally, parasites lacking GCN5a replicate more slowly and are quicker to form cysts when stressed. We show that GCN5a and GCN5b work in different multi-protein complexes and localize to different areas of the genome; while GCN5b targets promoters of gene coding regions, GCN5a is exclusively found at telomeric regions. Our findings suggest a novel role for GCN5a in telomere biology that, when depleted, produces a fitness defect that favors development of latent stages.

microbiology↗

bioRxiv2024

Cap-independent translation directs stress-induced differentiation of the protozoan parasite Toxoplasma gondii

Translational control mechanisms modulate microbial latency of eukaryotic pathogens, enabling them to evade immunity and drug treatments. The protozoan parasite Toxoplasma gondii persists in hosts by differentiating from proliferative tachyzoites to latent bradyzoites, which are housed inside tissue cysts. Transcriptional changes facilitating bradyzoite conversion are mediated by a Myb domain transcription factor called BFD1, whose mRNA is present in tachyzoites but not translated into protein until stress is applied to induce differentiation. We addressed the mechanisms by which translational control drives BFD1 synthesis in response to stress-induced parasite differentiation. Using biochemical and molecular approaches, we show that the 5-leader of BFD1 mRNA is sufficient for preferential translation upon stress. The translational control of BFD1 mRNA is maintained when ribosome assembly near its 5-cap is impaired by insertion of a 5-proximal stem-loop and upon knockdown of the Toxoplasma cap-binding protein, eIF4E1. Moreover, we show that a trans-acting RNA-binding protein called BFD2/ROCY1 is necessary for cap-independent translation of BFD1 through its binding to the 5-leader. Translation of BFD2 mRNA is also suggested to be preferentially induced under stress, but by a cap-dependent mechanism. These results show that translational control and differentiation in Toxoplasma proceed through cap-independent mechanisms in addition to canonical cap-dependent translation. Our identification of cap-independent translation in protozoa underscores the antiquity of this mode of gene regulation in cellular evolution and its central role in stress-induced life-cycle events.

microbiology↗

bioRxiv2023

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

AbstractsThe 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 ({Delta}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 {Delta}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 {Delta}eif1.2 parasites. Together, our findings suggest that eIF1.2 functions by regulating the translation of key differentiation factors necessary to establish chronic toxoplasmosis.

microbiology↗

bioRxiv2023

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 eIF2 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. SignificanceToxoplasma gondii is an opportunistic pathogen of importance 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 are required. Here we show that the mRNA cap-binding protein, eIF4E1, is involved in regulating 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.

microbiology↗

bioRxiv2023

Histone variant H2B.Z acetylation is necessary for maintenance of Toxoplasma gondii biological fitness

Through regulation of DNA packaging, histone proteins are fundamental to a wide array of biological processes. A variety of post-translational modifications (PTMs), including acetylation, constitute a proposed histone code that is interpreted by "reader" proteins to modulate chromatin structure. Canonical histones can be replaced with variant versions that add an additional layer of regulatory complexity. The protozoan parasite Toxoplasma gondii is unique among eukaryotes in possessing a novel variant of H2B designated H2B.Z. The combination of PTMs and the use of histone variants is important for gene regulation in T. gondii, offering new targets for drug development. In this work, T. gondii parasites were generated in which the 5 N-terminal acetylatable lysines in H2B.Z were mutated to either alanine (c-Myc-A) or arginine (c-Myc-R). c-Myc-A mutant only displayed a mild effect in its ability to kill mice. c-Myc-R mutant presented an impaired ability to grow and an increase in differentiation to latent bradyzoites. This mutant line was also more sensitive to DNA damage, displayed no virulence in mice, and provided protective immunity against future infection. While nucleosome composition was unaltered, key genes were abnormally expressed during in vitro bradyzoite differentiation. Our results show that the N-terminal positive charge patch of H2B.Z is important for these procceses. Pull down assays with acetylated N-terminal H2B.Z peptide and unacetylated one retrieved common and differential interactors. Acetylated peptide pulled down proteins associated with chromosome maintenance/segregation and cell cycle, opening the question of a possible link between H2B.Z acetylation status and mitosis.

cell biology↗

bioRxiv2022

AP2XII-2 coordinates transcriptional repression for Toxoplasma gondii sexual commitment

Toxoplasma gondii is a widespread protozoan parasite that has 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 spurious gene expression of sexually committed parasites, the specific factors that recruit this generalized machinery to the appropriate genes remains 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 the epigenetic regulators of transcriptional silencing, HDAC3 and MORC. Despite widespread association with gene promoters, AP2XII-2 is required for silencing of relatively few genes. Using CUT&Tag 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. IMPORTANCEToxoplasma 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 parasites life cycle stage transitions is needed for the development of new therapies aimed to treat toxoplasmosis and 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 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 by infected cats.

microbiology↗

bioRxiv2022

Eukaryotic initiation factor 2α kinases regulate virulence functions, stage conversion, and the stress response in Entamoeba invadens

Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. This pathogen possesses a two-stage life cycle consisting of an environmentally stable cyst and a pathogenic amoeboid trophozoite. Since infection is acquired by ingestion of cysts from contaminated food and water, this parasite is prevalent in underdeveloped countries. A reptilian pathogen, Entamoeba invadens, which can encyst in culture, has long-served as a surrogate to study stage conversion. In the host, the Entamoebae must manage stress including nutrient deprivation and host immune pressure. In many systems, the stress response is characterized by down-regulation of translation, which is initiated by the phosphorylation of eukaryotic initiation factor-2 alpha (eIF2). In mammalian cells, this phosphorylation is carried out by a family of eIF2 kinases. A canonical eIF2 translational control system exists in the Entamoebae; however, no eIF2 kinases have been characterized. In this study, we identified two eIF2 kinases in E. invadens, EiIF2K-A and EiIF2K-B. Their identity as eIF2 kinases was validated using a heterologous yeast system. We used an RNAi Trigger-mediated silencing system to reduce expression of EiIF2K-A, which also reduced expression of EiIF2K-B. Parasites with decreased kinase expression exhibited decreased phosphorylation of eIF2 and increased sensitivity to oxidative stress. Diminished kinase expression also correlates with an increased rate of encystation, a decreased the rate of excystation, and an increase in several virulence functions, erythrophagocytosis and adhesion to host cells. Taken together, these data suggest that EiIF2K-A and EiIF2K-B are authentic eIF2 kinases that may regulate the Entamoeba stress response. ImportanceEntamoeba histolytica is a human pathogen that causes dysentery and affects millions of people worldwide. This parasite possesses a two-stage life cycle: an environmentally stable cyst and the pathogenic trophozoite. Cysts are ingested from contaminated food and water; thus, this parasite in prevalent in underdeveloped countries. Current therapies commonly cause adverse side effects; therefore, new treatments are needed. In the host, Entamoeba experiences stress brought on, in part, by the host immune system. Understanding stage conversion and the stress response of this pathogen may lead to new drug therapies. Using the model organism, E. invadens, we identified two kinases, similar to those involved in stress and stage conversion in other systems. We determined that these kinases may regulate the oxidative stress response, stage conversion, and virulence. This work is significant as it will inform future studies on the life cycle and pathogenicity of the Entamoeba species.

microbiology↗

bioRxiv2021

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 within the consensus sequence, YGCAUGCR. Knockdown of the m6A writer enzyme METTL3 resulted in diminished m6A marks, loss of a target transcript, 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. Together, these findings establish that the m6A epitranscriptome is essential for parasite viability by contributing to the processing of mRNA 3-ends. Author SummaryToxoplasma gondii is a parasite of medical importance that causes disease upon immuno-suppression. Uncovering essential pathways that the parasite uses for its basic biological processes may reveal opportunities for new anti-parasitic drug therapies. Here, we describe the machinery that Toxoplasma uses to modify specific adenosine residues within its messenger RNAs (mRNA) by N6-adenosine methylation (m6A). We discovered that m6A mRNA methylation is prevalent in multiple stages of the parasite life cycle and is required for parasite replication. We also establish that m6A plays a major role in the proper maturation of mRNA. Two proteins that bind m6A modifications on mRNA associate with factors responsible for the cleavage and final processing steps of mRNA maturation. Since all of the machinery is conserved from plants to Toxoplasma and other related parasites, we propose that this system operates similarly in these organisms.

microbiology↗

bioRxiv2020

TgIF2K-B is an eIF2α kinase in Toxoplasma gondii that responds to oxidative stress and optimizes pathogenicity

Toxoplasma gondii is an obligate intracellular parasite that persists in its vertebrate hosts in the form of dormant tissue cysts, which facilitate transmission through predation. The parasite must strike a balance that allows it to disseminate throughout its host without killing it, which requires the ability to properly counter host cell defenses. For example, oxidative stress encountered by Toxoplasma is suggested to impair parasite replication and dissemination. However, the strategies by which Toxoplasma mitigates oxidative stress are not completely clear. Among eukaryotes, environmental stresses induce the integrated stress response via phosphorylation of a translation initiation factor, eIF2. Here, we show that the Toxoplasma eIF2 kinase, TgIF2K-B, is activated in response to oxidative stress and affords protection. Knockout of TgIF2K-B, {Delta}tgif2k-b, disrupted parasite responses to oxidative stresses and enhanced replication, diminishing the ability of the parasite to differentiate into tissue cysts. In addition, parasites lacking TgIF2K-B exhibited resistance to activated macrophages and showed greater virulence in an in vivo model of infection. Our results establish that TgIF2K-B is essential for Toxoplasma responses to oxidative stress, which are important for the parasites ability to establish persistent infection in its host. IMPORTANCEToxoplasma gondii is a single-celled parasite that infects nucleated cells of warm-blooded vertebrates, including one-third of the human population. The parasites are not cleared by the immune response and persist in the host by converting into a latent tissue cyst form. Development of tissue cysts can be triggered by cellular stresses, which activate a family of TgIF2 kinases to phosphorylate the eukaryotic translation initiation factor, TgIF2. Here, we establish that the TgIF2 kinase TgIF2K-B is activated by oxidative stress and is critical for maintaining oxidative balance in the parasite. Depletion of TgIF2K-B alters gene expression, leading to accelerated growth and a diminished ability to convert into tissue cysts. This study establishes that TgIF2K-B is essential for the parasites oxidative stress response and its ability to persist in the host as a latent infection.

microbiology↗

bioRxiv2020

Toxoplasma gondii AP2XII-2 contributes to proper progression through S-phase of the cell cycle

Toxoplasma gondii is a protozoan parasite that causes lifelong chronic infection that can reactivate in immunocompromised individuals. Upon infection, the replicative stage (tachyzoite) converts into a latent tissue cyst stage (bradyzoite). Like other apicomplexans, T. gondii possesses an extensive linage of proteins called ApiAP2s that contain plant-like DNA-binding domains. The function of most ApiAP2s is unknown. We previously found that AP2IX-4 is a cell cycle-regulated ApiAP2 expressed only in dividing parasites as a putative transcriptional repressor. In this study, we purified proteins interacting with AP2IX-4, finding it to be a component of the recently characterized microrchidia (MORC) transcriptional repressor complex. We further analyzed AP2XII-2, another cell cycle-regulated factor that associates with AP2IX-4. We monitored parallel expression of AP2IX-4 and AP2XII-2 proteins in tachyzoites, detecting peak expression during S/M phase. Unlike AP2IX-4, which is dispensable in tachyzoites, loss of AP2XII-2 resulted in a slowed tachyzoite growth due to a delay in S-phase progression. We also found that AP2XII-2 depletion increased the frequency of bradyzoite differentiation in vitro. These results suggest that multiple AP2 factors collaborate to ensure proper cell cycle progression and tissue cyst formation in T. gondii. ImportanceToxoplasma gondii is a single-celled parasite that causes opportunistic infection due to its ability to convert into a latent cyst stage. This work describes a new transcriptional factor called AP2XII-2 that plays a role in properly maintaining the growth rate of replicating parasites, which contributes to signals required for development into its dormant stage. Without AP2XII-2, Toxoplasma parasites experience a delay in their cell cycle that increases the frequency of latent cyst formation. In addition, we found that AP2XII-2 operates in a multi-subunit complex with other AP2 factors and chromatin remodeling machinery that represses gene expression. These findings add to our understanding of how Toxoplasma parasites balance replication and dormancy, revealing novel points of potential therapeutic intervention to disrupt this clinically relevant process.

microbiology↗

bioRxiv2020

Toxoplasma gondii co-opts the unfolded protein response to enhance migration and dissemination of infected host cells

Toxoplasma gondii is an intracellular parasite that reconfigures its host cell to promote pathogenesis. One consequence of Toxoplasma parasitism is increased migratory activity of host cells, which facilitates dissemination. Here we show that Toxoplasma triggers the unfolded protein response (UPR) in host cells through calcium release from the endoplasmic reticulum (ER). We further found that host IRE1, an ER stress sensor protein activated during Toxoplasma infection, also plays a noncanonical role in actin remodeling by binding filamin A in infected cells. By inducing cytoskeletal remodeling via IRE1 oligomerization in host cells, Toxoplasma enhances host cell migration in vitro and dissemination of the parasite to host organs in vivo. Our study identifies novel mechanisms used by Toxoplasma to induce dissemination of infected cells, providing new insights into strategies for treatment of toxoplasmosis. ImportanceCells that are infected with the parasite Toxoplasma gondii exhibit heightened migratory activity, which facilitates dissemination of the infection throughout the body. In this study, we identify a new mechanism used by Toxoplasma to hijack its host cell and increase its mobility. We further show that the ability of Toxoplasma to increase host cell migration does not involve the enzymatic activity of IRE1, but rather IRE1 engagement with actin cytoskeletal remodeling. Depletion of IRE1 from infected host cells reduces their migration in vitro and significantly hinders dissemination of Toxoplasma in vivo. Our findings reveal a new mechanism underlying host-pathogen interactions, demonstrating how host cells are co-opted to spread a persistent infection around the body.

microbiology↗

bioRxiv2020

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 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 + ELQ-334 in vivo. While we were unable to completely eliminate brain cyst burden, we found that a combination treatment of ELQ-334 + pyrimethamine impressively reduced brain cysts to 95% in C57BL/6 mice, which approaches the limit of detection. These analyses highlight the importance of evaluating anti-infective drugs in multiple mouse strains and will help inform further preclinical cocktail therapy studies designed to treat chronic toxoplasmosis.

microbiology↗

bioRxiv2019

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 consistent with massive transcriptional rewiring while quiescent cells were best characterized by re-entry into the cell cycle. We also identified a translational control regimen consistent with 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 parasite and host.\n\nImportanceToxoplasma gondii is a single-celled parasite that has infected up to one-third of the worlds 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.

microbiology↗

bioRxiv2017

Toxoplasma gondii AP2IX-4 regulates gene expression during bradyzoite development

Toxoplasma gondii is a protozoan parasite of great importance to human and animal health. In the host, this obligate intracellular parasite persists as a tissue cyst form that is invisible to the immune response and unaffected by current therapies. The tissue cysts facilitate transmission through predation and give rise to chronic cycles of toxoplasmosis in immune compromised patients. Transcriptional changes accompany conversion of the rapidly replicating tachyzoites into the encysted bradyzoites, yet the mechanisms underlying these alterations in gene expression are not well-defined. Here we show that AP2IX-4 is a nuclear protein exclusively expressed in tachyzoites and bradyzoites undergoing division. Knockout of AP2IX-4 had no discernible effect on tachyzoite replication, but resulted in a reduced frequency of tissue cyst formation following alkaline stress induction - a defect that is reversible by complementation. AP2IX-4 has a complex role in regulating bradyzoite gene expression, as many bradyzoite mRNAs dramatically increased beyond normal stress-induction in AP2IX-4 knockout parasites exposed to alkaline media. The loss of AP2IX-4 also resulted in a modest virulence defect and reduced cyst burden in chronically infected mice, which was also reversed by complementation. These findings illustrate that the transcriptional mechanisms responsible for tissue cyst development operate across the intermediate life cycle from the dividing tachyzoite to the dormant bradyzoite.

microbiology↗

bioRxiv2017

Transcriptional repression by ApiAP2 factors is central to chronic toxoplasmosis

Bradyzoite differentiation 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 Toxoplasma transcriptional repressor mechanism controlling bradyzoite differentiation that operates exclusively 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 increased expression of bradyzoite mRNAs in replicating tachyzoites, and in two different genetic lineages we confirmed the misexpression of tissue cyst wall components (e.g. BPK1, MCP4, CST1) and the bradyzoite surface antigen SRS9 in the tachyzoite stage. In the murine animal model, the loss of AP2IV-4 had profound biological consequences. Type II prugniaud strain parasites lacking AP2IV-4 were unable to form tissue cysts in brain tissue and the absence of this factor also recruited a potent immune response characterized by increases inflammatory monocytes, IFN-{gamma} and higher numbers of both CD8+ and CD4+ T-cells. Altogether, these results indicate that suppression of bradyzoite antigens by AP2IV-4 during acute infection is required for Toxoplasma to establish a chronic infection in the immune-competent host.\n\nAuthor SummaryThe Toxoplasma biology that underlies the establishment of a chronic infection is developmental conversion of the acute tachyzoite stage into the latent bradyzoite-tissue cyst stage. Despite the important clinical consequences of this developmental pathway, the molecular basis of the switch mechanisms that control formation of the tissue cyst is still poorly understood. A fundamental feature of tissue cyst formation is the expression of bradyzoite-specific genes. Here we show the transcription factor AP2IV-4 directly silences bradyzoite mRNA and protein expression in the acute tachyzoite stage demonstrating that developmental control of tissue cyst formation is as much about when not to express bradyzoite genes as it is about when to activate them. Loosing the suppression of bradyzoite gene expression in the acute tachyzoite stage caused by deleting AP2IV-4 blocked the establishment of chronic disease in healthy animals through the pre-arming of the immune system suggesting a possible strategy for preventing chronic Toxoplasma infections.

microbiology↗