Cryogenic electron tomography reveals novel structures in the apical complex of Plasmodium falciparum.

Intracellular infectious agents, like the malaria parasite, Plasmodium falciparum, face the daunting challenge of how to invade a host cell. This problem may be even harder when the host cell in question is the enucleated red blood cell, which lacks the host machinery co-opted by many pathogens for internalization. Evolution has provided P. falciparum and related single-celled parasites within the phylum Apicomplexa with a collection of organelles at their apical end that mediate invasion. This apical complex includes at least two sets of secretory organelles, micronemes and rhoptries, and several structural features like apical rings and a putative pore through which proteins may be introduced into the host cell during invasion. We perform cryogenic electron tomography (cryo-ET) equipped with Volta Phase Plate on isolated and vitrified merozoites to visualize the apical machinery. Through tomographic reconstruction of cellular compartments, we see new details of known structures like the rhoptry tip interacting directly with a rosette resembling the recently described rhoptry secretory apparatus (RSA), or with an apical vesicle docked beneath the RSA. Subtomogram averaging reveals that the apical rings have a fixed number of repeating units, each of which is similar in overall size and shape to the units in the apical rings of tachyzoites of Toxoplasma gondii. Comparison of these polar rings in Plasmodium and Toxoplasma parasites also reveals them to have a structurally conserved assembly pattern. These results provide new insight into the essential and structurally conserved features of this remarkable machinery used by apicomplexan parasites to invade their respective host cells. IMPORTANCE: Malaria is an infectious disease caused by parasites of the genus Plasmodium and is a leading cause of morbidity and mortality globally. Upon infection, Plasmodium parasites invade and replicate in red blood cells, where they are largely protected from the immune system. To enter host cells, the parasites employ a specialized apparatus at their anterior end. In this study, advanced imaging techniques like cryogenic electron tomography (cryo-ET) and Volta Phase Plate enable unprecedented visualization of whole Plasmodium falciparum merozoites, revealing previously unknown structural details of their invasion machinery. Key findings include new insights into the structural conservation of apical rings shared between Plasmodium and its apicomplexan cousin, Toxoplasma. These discoveries shed light on the essential and conserved elements of the invasion machinery used by these pathogens. Moreover, the research provides a foundation for understanding the molecular mechanisms underlying parasite-host interactions, potentially informing strategies for combating diseases caused by apicomplexan parasites.

Host MOSPD2 enrichment at the parasitophorous vacuole membrane varies between Toxoplasma strains and involves complex interactions.

Toxoplasma gondii is an obligate, intracellular parasite. Infection of a cell produces a unique niche for the parasite named the parasitophorous vacuole (PV) initially composed of host plasma membrane invaginated during invasion. The PV and its membrane (parasitophorous vacuole membrane [PVM]) are subsequently decorated with a variety of parasite proteins allowing the parasite to optimally grow in addition to manipulate host processes. Recently, we reported a proximity-labeling screen at the PVM-host interface and identified host endoplasmic reticulum (ER)-resident motile sperm domain-containing protein 2 (MOSPD2) as being enriched at this location. Here we extend these findings in several important respects. First, we show that the extent and pattern of host MOSPD2 association with the PVM differ dramatically in cells infected with different strains of Toxoplasma. Second, in cells infected with Type I RH strain, the MOSPD2 staining is mutually exclusive with regions of the PVM that associate with mitochondria. Third, immunoprecipitation and liquid chromatography tandem mass spectrometry (LC-MS/MS) with epitope-tagged MOSPD2-expressing host cells reveal strong enrichment of several PVM-localized parasite proteins, although none appear to play an essential role in MOSPD2 association. Fourth, most MOSPD2 associating with the PVM is newly translated after infection of the cell and requires the major functional domains of MOSPD2, identified as the CRAL/TRIO domain and tail anchor, although these domains were not sufficient for PVM association. Lastly, ablation of MOSPD2 results in, at most, a modest impact on Toxoplasma growth in vitro. Collectively, these studies provide new insight into the molecular interactions involving MOSPD2 at the dynamic interface between the PVM and the host cytosol. IMPORTANCE Toxoplasma gondii is an intracellular pathogen that lives within a membranous vacuole inside of its host cell. This vacuole is decorated by a variety of parasite proteins that allow it to defend against host attack, acquire nutrients, and interact with the host cell. Recent work identified and validated host proteins enriched at this host-pathogen interface. Here, we follow up on one candidate named MOSPD2 shown to be enriched at the vacuolar membrane and describe it as having a dynamic interaction at this location depending on a variety of factors. Some of these include the presence of host mitochondria, intrinsic domains of the host protein, and whether translation is active. Importantly, we show that MOSPD2 enrichment at the vacuole membrane differs between strains indicating active involvement of the parasite with this phenotype. Altogether, these results shed light on the mechanism and role of protein associations in the host-pathogen interaction.

Cryo-electron tomography with mixed-scale dense neural networks reveals key steps in deployment of Toxoplasma invasion machinery.

Host cell invasion by intracellular, eukaryotic parasites within the phylum Apicomplexa is a remarkable and active process involving the coordinated action of apical organelles and other structures. To date, capturing how these structures interact during invasion has been difficult to observe in detail. Here, we used cryogenic electron tomography to image the apical complex of Toxoplasma gondii tachyzoites under conditions that mimic resting parasites and those primed to invade through stimulation with calcium ionophore. Through the application of mixed-scale dense networks for image processing, we developed a highly efficient pipeline for annotation of tomograms, enabling us to identify and extract densities of relevant subcellular organelles and accurately analyze features in 3-D. The results reveal a dramatic change in the shape of the anteriorly located apical vesicle upon its apparent fusion with a rhoptry that occurs only in the stimulated parasites. We also present information indicating that this vesicle originates from the vesicles that parallel the intraconoidal microtubules and that the latter two structures are linked by a novel tether. We show that a rosette structure previously proposed to be involved in rhoptry secretion is associated with apical vesicles beyond just the most anterior one. This result, suggesting multiple vesicles are primed to enable rhoptry secretion, may shed light on the mechanisms Toxoplasma employs to enable repeated invasion attempts. Using the same approach, we examine Plasmodium falciparum merozoites and show that they too possess an apical vesicle just beneath a rosette, demonstrating evolutionary conservation of this overall subcellular organization.

Transcriptional signatures of clonally derived Toxoplasma tachyzoites reveal novel insights into the expression of a family of surface proteins.

Toxoplasma gondii has numerous, large, paralogous gene families that are likely critical for supporting its unparalleled host range: nearly any nucleated cell in almost any warm-blooded animal. The SRS (SAG1-related sequence) gene family encodes over 100 proteins, the most abundant of which are thought to be involved in parasite attachment and, based on their stage-specific expression, evading the host immune response. For most SRS proteins, however, little is understood about their function and expression profile. Single-parasite RNA-sequencing previously demonstrated that across an entire population of lab-grown tachyzoites, transcripts for over 70 SRS genes were detected in at least one parasite. In any one parasite, however, transcripts for an average of only 7 SRS genes were detected, two of which, SAG1 and SAG2A, were extremely abundant and detected in virtually all. These data do not address whether this pattern of sporadic SRS gene expression is consistently inherited among the progeny of a given parasite or arises independently of lineage. We hypothesized that if SRS expression signatures are stably inherited by progeny, subclones isolated from a cloned parent would be more alike in their expression signatures than they are to the offspring of another clone. In this report, we compare transcriptomes of clonally derived parasites to determine the degree to which expression of the SRS family is stably inherited in individual parasites. Our data indicate that in RH tachyzoites, SRS genes are variably expressed even between parasite samples subcloned from the same parent within approximately 10 parasite divisions (72 hours). This suggests that the pattern of sporadically expressed SRS genes is highly variable and not driven by inheritance mechanisms, at least under our conditions.

Cryo-ET of Toxoplasma parasites gives subnanometer insight into tubulin-based structures.

Tubulin is a conserved protein that polymerizes into different forms of filamentous structures in Toxoplasma gondii, an obligate intracellular parasite in the phylum Apicomplexa. Two key tubulin-containing cytoskeletal components are subpellicular microtubules (SPMTs) and conoid fibrils (CFs). The SPMTs help maintain shape and gliding motility, while the CFs are implicated in invasion. Here, we use cryogenic electron tomography to determine the molecular structures of the SPMTs and CFs in vitrified intact and detergent-extracted parasites. Subvolume densities from detergent-extracted parasites yielded averaged density maps at subnanometer resolutions, and these were related back to their architecture in situ. An intralumenal spiral lines the interior of the 13-protofilament SPMTs, revealing a preferred orientation of these microtubules relative to the parasite's long axis. Each CF is composed of nine tubulin protofilaments that display a comma-shaped cross-section, plus additional associated components. Conoid protrusion, a crucial step in invasion, is associated with an altered pitch of each CF. The use of basic building blocks of protofilaments and different accessory proteins in one organism illustrates the versatility of tubulin to form two distinct types of assemblies, SPMTs and CFs.

Proximity-Labeling Reveals Novel Host and Parasite Proteins at the Toxoplasma Parasitophorous Vacuole Membrane.

Toxoplasma gondii is a ubiquitous, intracellular parasite that envelops its parasitophorous vacuole with a protein-laden membrane (PVM). The PVM is critical for interactions with the infected host cell, such as nutrient transport and immune defense. Only a few parasite and host proteins have so far been identified on the host-cytosolic side of the Toxoplasma PVM. We report here the use of human foreskin fibroblasts expressing the proximity-labeling enzyme miniTurbo, fused to a domain that targets it to this face of the PVM, in combination with quantitative proteomics to specifically identify proteins present at this interface. Out of numerous human and parasite proteins with candidate PVM localization, we validate three parasite proteins (TGGT1_269950 [GRA61], TGGT1_215360 [GRA62], and TGGT1_217530 [GRA63]) and four new host proteins (PDCD6IP/ALIX, PDCD6, CC2D1A, and MOSPD2) as localized to the PVM in infected human cells through immunofluorescence microscopy. These results significantly expand our knowledge of proteins present at the Toxoplasma PVM and, given that three of the validated host proteins are components of the ESCRT (endosomal sorting complexes required for transport) machinery, they further suggest that novel biology is operating at this crucial host-pathogen interface. IMPORTANCEToxoplasma is an intracellular pathogen which resides and replicates inside a membrane-bound vacuole in infected cells. This vacuole is modified by both parasite and host proteins which participate in a variety of host-parasite interactions at this interface, including nutrient exchange, effector transport, and immune modulation. Only a small number of parasite and host proteins present at the vacuolar membrane and exposed to the host cytosol have thus far been identified. Here, we report the identification of several novel parasite and host proteins present at the vacuolar membrane using enzyme-catalyzed proximity-labeling, significantly increasing our knowledge of the molecular players present and novel biology occurring at this crucial interface.

Seizing control: How dense granule effector proteins enable Toxoplasma to take charge.

Control of the host cell is crucial to the Apicomplexan parasite, Toxoplasma gondii, while it grows intracellularly. To achieve this goal, these single-celled eukaryotes export a series of effector proteins from organelles known as "dense granules" that interfere with normal cellular processes and responses to invasion. While some effectors are found attached to the outer surface of the parasitophorous vacuole (PV) in which Toxoplasma tachyzoites reside, others are found in the host cell's cytoplasm and yet others make their way into the host nucleus, where they alter host transcription. Among the processes that are severely altered are innate immune responses, host cell cycle, and association with host organelles. The ways in which these crucial processes are altered through the coordinated action of a large collection of effectors is as elegant as it is complex, and is the central focus of the following review; we also discuss the recent advances in our understanding of how dense granule effector proteins are trafficked out of the PV.

Toxoplasma Uses GRA16 To Upregulate Host c-Myc.

Manipulation of the host cell is a crucial part of life for many intracellular organisms. We have recently come to appreciate the extent to which the intracellular pathogen Toxoplasma gondii reprograms its host cell, and this is illustrated by the marked upregulation of the central regulator c-Myc, an oncogene that coordinates myriad cellular functions. In an effort to identify an effector protein capable of regulating c-Myc, our laboratory constructed a screen for mutant parasites unable to accomplish this upregulation. Interestingly, this screen identified numerous components of a complex located in/on the parasitophorous vacuole membrane necessary to translocate Toxoplasma proteins out into the host cytosol, but it never identified a specific effector protein. Thus, how the parasite upregulates c-Myc has largely been a mystery. Previously, the Toxoplasma dense granule protein GRA16 has been described to bind to one isoform of PP2A-B, a regulatory subunit that coordinates the activity of the catalytic protein phosphatase PP2A. As other PP2A subunits have been reported to target PP2A protein phosphatase activity to c-Myc, subsequently leading to c-Myc destabilization, we examined whether GRA16 has an impact on host c-Myc accumulation. Expression of Toxoplasma's GRA16 protein in Neospora caninum, a close relative of Toxoplasma that does not naturally upregulate host c-Myc, conferred the ability on Neospora to do this now. Further support was obtained by deleting the GRA16 gene from Toxoplasma and observing a severely diminished ability of Toxoplasma tachyzoites to upregulate host c-Myc. Thus, GRA16 is an effector protein central to Toxoplasma's ability to upregulate host c-Myc.IMPORTANCE The proto-oncogene c-Myc plays a crucial role in the growth and division of many animal cells. Previous studies have identified an active upregulation of c-Myc by Toxoplasma tachyzoites, suggesting the existence of one or more exported "effector" proteins. The identity of such an effector, however, has not previously been known. Here, we show that a previously known secreted protein, GRA16, plays a crucial role in c-Myc upregulation. This finding will enable further dissection of the precise mechanism and role of c-Myc upregulation in Toxoplasma-infected cells.

Differential Impacts on Host Transcription by ROP and GRA Effectors from the Intracellular Parasite Toxoplasma gondii.

The intracellular parasite Toxoplasma gondii employs a vast array of effector proteins from the rhoptry and dense granule organelles to modulate host cell biology; these effectors are known as ROPs and GRAs, respectively. To examine the individual impacts of ROPs and GRAs on host gene expression, we developed a robust, novel protocol to enrich for ultrapure populations of a naturally occurring and reproducible population of host cells called uninfected-injected (U-I) cells, which Toxoplasma injects with ROPs but subsequently fails to invade. We then performed single-cell transcriptomic analysis at 1 to 3 h postinfection on U-I cells (as well as on uninfected and infected controls) arising from infection with either wild-type parasites or parasites lacking the MYR1 protein, which is required for soluble GRAs to cross the parasitophorous vacuole membrane (PVM) and reach the host cell cytosol. Based on comparisons of infected and U-I cells, the host's earliest response to infection appears to be driven primarily by the injected ROPs, which appear to induce immune and cellular stress pathways. These ROP-dependent proinflammatory signatures appear to be counteracted by at least some of the MYR1-dependent GRAs and may be enhanced by the MYR-independent GRAs (which are found embedded within the PVM). Finally, signatures detected in uninfected bystander cells from the infected monolayers suggest that MYR1-dependent paracrine effects also counteract inflammatory ROP-dependent processes.IMPORTANCE This work performs transcriptomic analysis of U-I cells, captures the earliest stage of a host cell's interaction with Toxoplasma gondii, and dissects the effects of individual classes of parasite effectors on host cell biology.

A single-parasite transcriptional atlas of Toxoplasma Gondii reveals novel control of antigen expression.

Toxoplasma gondii, a protozoan parasite, undergoes a complex and poorly understood developmental process that is critical for establishing a chronic infection in its intermediate hosts. Here, we applied single-cell RNA-sequencing (scRNA-seq) on >5,400 Toxoplasma in both tachyzoite and bradyzoite stages using three widely studied strains to construct a comprehensive atlas of cell-cycle and asexual development, revealing hidden states and transcriptional factors associated with each developmental stage. Analysis of SAG1-related sequence (SRS) antigenic repertoire reveals a highly heterogeneous, sporadic expression pattern unexplained by measurement noise, cell cycle, or asexual development. Furthermore, we identified AP2IX-1 as a transcription factor that controls the switching from the ubiquitous SAG1 to rare surface antigens not previously observed in tachyzoites. In addition, comparative analysis between Toxoplasma and Plasmodium scRNA-seq results reveals concerted expression of gene sets, despite fundamental differences in cell division. Lastly, we built an interactive data-browser for visualization of our atlas resource.

Toxoplasma gondii is a single-celled parasite that can infect most warm-blooded animals, but only reproduces sexually in domestic and wild cats. Distantly related to the malaria agent, it currently infects over a quarter of the world's human population. Although it is benign in most cases, the condition can still be dangerous for foetuses and people whose immune system is compromised. In the human body, Toxoplasma cells infiltrate muscle and nerve cells; there it undergoes a complex transformation that helps the parasites to stop dividing quickly and instead hide from the immune system in a dormant state. It is still unclear how this transition unfolds, and in particular which genes are switched on and off at any given time. To understand this transformation, scientists often measure which genes are active across a group of parasites. However, this approach gives only an 'average' picture and does not allow each parasite to be profiled, missing out on the diversity that may exist between individuals. One area of particular interest, for example, is a set of genes called SAG1-related sequences. They code for the 'molecular overcoat' of the parasite, an array of proteins that sit on the surface of Toxoplasma cells. More than 120 SAG1-related genes exist in the genome of each Toxoplasma parasite, creating a whole wardrobe of proteins that potentially hide the parasites from the immune system. Here, Xue et al. harnessed a technique called single-cell RNA sequencing, which allowed them to screen which genes were active in 5,400 individual Toxoplasma parasites from different strains. The analysis included both the rapidly dividing form of the parasite (present in the initial stage of an infection), and the slowly dividing form found in people who carry Toxoplasma without any symptoms. The resulting 'atlas' contains previously hidden information about the genes used at each stage of parasite development: this included unexpected similarities between Toxoplasma and the malaria agent, as well as subtle differences between two of the Toxoplasma strains. The atlas also sheds light on how individual parasites turns on SAG1-related sequences. It reveals a surprising diversity in the composition of the protein coats sported by Toxoplasma cells at the same developmental stage, a strategy that may help to thwart the immune system. One individual parasite in particular had an unusual combination of coat and other proteins found in both the fast and slow-dividing human forms. This parasite had been grown in human cells, yet a closer analysis revealed that it had activated several genes (including ones encoding the protein coat) that are normally only 'on' in the parasites going through sexual reproduction in domestic and wild cats. This new data atlas helps to understand how Toxoplasma are transmitted to and grow within humans, which could aid the development of treatments. Ultimately, a better knowledge of these parasites could also bring new information about the agent that causes malaria.

Coimmunoprecipitation with MYR1 Identifies Three Additional Proteins within the Toxoplasma gondii Parasitophorous Vacuole Required for Translocation of Dense Granule Effectors into Host Cells.

Toxoplasma gondii is a ubiquitous, intracellular protozoan that extensively modifies infected host cells through secreted effector proteins. Many such effectors must be translocated across the parasitophorous vacuole (PV), in which the parasites replicate, ultimately ending up in the host cytosol or nucleus. This translocation has previously been shown to be dependent on five parasite proteins: MYR1, MYR2, MYR3, ROP17, and ASP5. We report here the identification of several MYR1-interacting and novel PV-localized proteins via affinity purification of MYR1, including TGGT1_211460 (dubbed MYR4), TGGT1_204340 (dubbed GRA54), and TGGT1_270320 (PPM3C). Further, we show that three of the MYR1-interacting proteins, GRA44, GRA45, and MYR4, are essential for the translocation of the Toxoplasma effector protein GRA16 and for the upregulation of human c-Myc and cyclin E1 in infected cells. GRA44 and GRA45 contain ASP5 processing motifs, but like MYR1, processing at these sites appears to be nonessential for their role in protein translocation. These results expand our understanding of the mechanism of effector translocation in Toxoplasma and indicate that the process is highly complex and dependent on at least eight discrete proteins.IMPORTANCEToxoplasma is an extremely successful intracellular parasite and important human pathogen. Upon infection of a new cell, Toxoplasma establishes a replicative vacuole and translocates parasite effectors across this vacuole to function from the host cytosol and nucleus. These effectors play a key role in parasite virulence. The work reported here newly identifies three parasite proteins that are necessary for protein translocation into the host cell. These results significantly increase our knowledge of the molecular players involved in protein translocation in Toxoplasma-infected cells and provide additional potential drug targets.

What a Difference 30 Years Makes! A Perspective on Changes in Research Methodologies Used to Study Toxoplasma gondii.

Toxoplasma gondii is a remarkable species with a rich cell, developmental, and population biology. It is also sometimes responsible for serious disease in animals and humans and the stages responsible for such disease are relatively easy to study in vitro or in laboratory animal models. As a result of all this, Toxoplasma has become the subject of intense investigation over the last several decades, becoming a model organism for the study of the phylum of which it is a member, Apicomplexa. This has led to an ever-growing number of investigators applying an ever-expanding set of techniques to dissecting how Toxoplasma "ticks" and how it interacts with its many hosts. In this perspective piece I first wind back the clock 30 years and then trace the extraordinary pace of methodologies that have propelled the field forward to where we are today. In keeping with the theme of this collection, I focus almost exclusively on the parasite, rather than host side of the equation. I finish with a few thoughts about where the field might be headed-though if we have learned anything, the only sure prediction is that the pace of technological advance will surely continue to accelerate and the future will give us still undreamed of methods for taking apart (and then putting back together) this amazing organism with all its intricate biology. We have so far surely just scratched the surface.

Translocation of Dense Granule Effectors across the Parasitophorous Vacuole Membrane in Toxoplasma-Infected Cells Requires the Activity of ROP17, a Rhoptry Protein Kinase.

Toxoplasma gondii tachyzoites co-opt host cell functions through introduction of a large set of rhoptry- and dense granule-derived effector proteins. These effectors reach the host cytosol through different means: direct injection for rhoptry effectors and translocation across the parasitophorous vacuolar membrane (PVM) for dense granule (GRA) effectors. The machinery that translocates these GRA effectors has recently been partially elucidated, revealing three components, MYR1, MYR2, and MYR3. To determine whether other proteins might be involved, we returned to a library of mutants defective in GRA translocation and selected one with a partial defect, suggesting it might be in a gene encoding a new component of the machinery. Surprisingly, whole-genome sequencing revealed a missense mutation in a gene encoding a known rhoptry protein, a serine/threonine protein kinase known as ROP17. ROP17 resides on the host cytosol side of the PVM in infected cells and has previously been known for its activity in phosphorylating and thereby inactivating host immunity-related GTPases. Here, we show that null or catalytically dead mutants of ROP17 are defective in GRA translocation across the PVM but that translocation can be rescued "in trans" by ROP17 delivered by other tachyzoites infecting the same host cell. This strongly argues that ROP17's role in regulating GRA translocation is carried out on the host cytosolic side of the PVM, not within the parasites or lumen of the parasitophorous vacuole. This represents an entirely new way in which the different secretory compartments of Toxoplasma tachyzoites collaborate to modulate the host-parasite interaction.IMPORTANCE When Toxoplasma infects a cell, it establishes a protective parasitophorous vacuole surrounding it. While this vacuole provides protection, it also serves as a barrier to the export of parasite effector proteins that impact and take control of the host cell. Our discovery here that the parasite rhoptry protein ROP17 is necessary for export of these effector proteins provides a distinct, novel function for ROP17 apart from its known role in protecting the vacuole. This will enable future research into ways in which we can prevent the export of effector proteins, thereby preventing Toxoplasma from productively infecting its animal and human hosts.

Translocation of effector proteins into host cells by Toxoplasma gondii.

The Apicomplexan parasite, Toxoplasma gondii, is an obligate intracellular organism that must co-opt its host cell to survive. To this end, Toxoplasma parasites introduce a suite of effector proteins from two secretory compartments called rhoptries and dense granules into the host cells. Once inside, these effectors extensively modify the host cell to facilitate parasite penetration, replication and persistence. In this review, we summarize the most recent advances in current understanding of effector translocation from Toxoplasma's rhoptry and dense granule organelles into the host cell, with comparisons to Plasmodium spp. for broader context.

Toxoplasma Controls Host Cyclin E Expression through the Use of a Novel MYR1-Dependent Effector Protein, HCE1.

Toxoplasma gondii is an obligate intracellular parasite that establishes a favorable environment in the host cells in which it replicates. We have previously reported that it uses MYR-dependent translocation of dense granule proteins to elicit a key set of host responses related to the cell cycle, specifically, E2F transcription factor targets, including cyclin E. We report here the identification of a novel Toxoplasma effector protein that is exported from the parasitophorous vacuole in a MYR1-dependent manner and localizes to the host's nucleus. Parasites lacking this inducer of host cyclin E (HCE1) are unable to modulate E2F transcription factor target genes and exhibit a substantial growth defect. Immunoprecipitation of HCE1 from infected host cells showed that HCE1 efficiently binds elements of the cyclin E regulatory complex, namely, DP1 and its partners E2F3 and E2F4. Expression of HCE1 in Neospora caninum, or in uninfected human foreskin fibroblasts (HFFs), showed localization of the expressed protein to the host nuclei and strong cyclin E upregulation. Thus, HCE1 is a novel effector protein that is necessary and sufficient to impact the E2F axis of transcription, resulting in co-opting of host functions to the advantage of ToxoplasmaIMPORTANCE Like most Apicomplexan parasites, Toxoplasma gondii has the remarkable ability to invade and establish a replicative niche within another eukaryotic cell, in this case, any of a large number of cell types in almost any warm-blooded animals. Part of the process of establishing this niche is the export of effector proteins to co-opt host cell functions in favor of the parasite. Here we identify a novel effector protein, HCE1, that the parasites export into the nucleus of human cells, where it modulates the expression of multiple genes, including the gene encoding cyclin E, one of the most crucial proteins involved in controlling when and whether a human cell divides. We show that HCE1 works through binding to specific transcription factors, namely, E2F3, E2F4, and DP1, that normally carefully regulate these all-important pathways. This represents a new way in which these consummately efficient infectious agents co-opt the human cells that they so efficiently grow within.

Toxoplasma gondii infection triggers chronic cachexia and sustained commensal dysbiosis in mice.

Toxoplasma gondii is a protozoan parasite with a predation-mediated transmission cycle between rodents and felines. Intermediate hosts acquire Toxoplasma by eating parasite cysts which invade the small intestine, disseminate systemically and finally establish host life-long chronic infection in brain and muscles. Here we show that Toxoplasma infection can trigger a severe form of sustained cachexia: a disease of progressive lean weight loss that is a causal predictor of mortality in cancer, chronic disease and many infections. Toxoplasma cachexia is characterized by acute anorexia, systemic inflammation and loss of 20% body mass. Although mice recover from symptoms of peak sickness, they fail to regain muscle mass or visceral adipose depots. We asked whether the damage to the intestinal microenvironment observed at acute time points was sustained in chronic infection and could thereby play a role in sustaining cachexia. We found that parasites replicate in the same region of the distal jejunum/proximal ileum throughout acute infection, inducing the development of secondary lymphoid structures and severe, regional inflammation. Small intestine pathology was resolved by 5 weeks post-infection. However, changes in the commensal populations, notably an outgrowth of Clostridia spp., were sustained in chronic infection. Importantly, uninfected animals co-housed with infected mice display similar changes in commensal microflora but never display symptoms of cachexia, indicating that altered commensals are not sufficient to explain the cachexia phenotype alone. These studies indicate that Toxoplasma infection is a novel and robust model to study the immune-metabolic interactions that contribute to chronic cachexia development, pathology and potential reversal.

Mitochondria Restrict Growth of the Intracellular Parasite Toxoplasma gondii by Limiting Its Uptake of Fatty Acids.

How intracellular pathogens acquire essential non-diffusible host metabolites and whether the host cell counteracts the siphoning of these nutrients by its invaders are open questions. Here we show that host mitochondria fuse during infection by the intracellular parasite Toxoplasma gondii to limit its uptake of fatty acids (FAs). A combination of genetics and imaging of FA trafficking indicates that Toxoplasma infection triggers lipophagy, the autophagy of host lipid droplets (LDs), to secure cellular FAs essential for its proliferation. Indeed, Toxoplasma FA siphoning and growth are reduced in host cells genetically deficient for autophagy or triglyceride depots. Conversely, Toxoplasma FA uptake and proliferation are increased in host cells lacking mitochondrial fusion, required for efficient mitochondrial FA oxidation, or where mitochondrial FA oxidation is pharmacologically inhibited. Thus, mitochondrial fusion can be regarded as a cellular defense mechanism against intracellular parasites, by limiting Toxoplasma access to host nutrients liberated by lipophagy.

MYR1-Dependent Effectors Are the Major Drivers of a Host Cell's Early Response to Toxoplasma, Including Counteracting MYR1-Independent Effects.

The obligate intracellular parasite Toxoplasma gondii controls its host cell from within the parasitophorous vacuole (PV) by using a number of diverse effector proteins, a subset of which require the aspartyl protease 5 enzyme (ASP5) and/or the recently discovered MYR1 protein to cross the PV membrane. To examine the impact these effectors have in the context of the entirety of the host response to Toxoplasma, we used RNA-Seq to analyze the transcriptome expression profiles of human foreskin fibroblasts infected with wild-type RH (RH-WT), RHΔmyr1, and RHΔasp5 tachyzoites. Interestingly, the majority of the differentially regulated genes responding to Toxoplasma infection are MYR1 dependent. A subset of MYR1 responses were ASP5 independent, and MYR1 function did not require ASP5 cleavage, suggesting the export of some effectors requires only MYR1. Gene set enrichment analysis of MYR1-dependent host responses suggests an upregulation of E2F transcription factors and the cell cycle and a downregulation related to interferon signaling, among numerous others. Most surprisingly, "hidden" responses arising in RHΔmyr1- but not RH-WT-infected host cells indicate counterbalancing actions of MYR1-dependent and -independent activities. The host genes and gene sets revealed here to be MYR1 dependent provide new insight into the parasite's ability to co-opt host cell functions.IMPORTANCEToxoplasma gondii is unique in its ability to successfully invade and replicate in a broad range of host species and cells within those hosts. The complex interplay of effector proteins exported by Toxoplasma is key to its success in co-opting the host cell to create a favorable replicative niche. Here we show that a majority of the transcriptomic effects in tachyzoite-infected cells depend on the activity of a novel translocation system involving MYR1 and that the effectors delivered by this system are part of an intricate interplay of activators and suppressors. Removal of all MYR1-dependent effectors reveals previously unknown activities that are masked or hidden by the action of these proteins.

A Toxoplasma gondii locus required for the direct manipulation of host mitochondria has maintained multiple ancestral functions.

The Toxoplasma gondii locus mitochondrial association factor 1 (MAF1) encodes multiple paralogs, some of which mediate host mitochondrial association (HMA). Previous work showed that HMA was a trait that arose in T. gondii through neofunctionalization of an ancestral MAF1 ortholog. Structural analysis of HMA-competent and incompetent MAF1 paralogs (MAF1b and MAF1a, respectively) revealed that both paralogs harbor an ADP ribose binding macro-domain, with comparatively low (micromolar) affinity for ADP ribose. Replacing the 16 C-terminal residues of MAF1b with those of MAF1a abrogated HMA, and we also show that only three residues in the C-terminal helix are required for MAF1-mediated HMA. Importantly these same three residues are also required for the in vivo growth advantage conferred by MAF1b, providing a definitive link between in vivo proliferation and manipulation of host mitochondria. Co-immunoprecipitation assays reveal that the ability to interact with the mitochondrial MICOS complex is shared by HMA-competent and incompetent MAF1 paralogs and mutants. The weak ADPr coordination and ability to interact with the MICOS complex shared between divergent paralogs may represent modular ancestral functions for this tandemly expanded and diversified T. gondii locus.