T. gondii is a highly successful global pathogen that is remarkable in its ability to infect nearly any nucleated cell in any warm-blooded animal. Infection with T. gondii typically occurs through the ingestion of contaminated food or water, but the parasite then breaches the intestinal epithelial barrier and spreads from the lamina propria to a large variety of other organs in the body. A key feature of T. gondii pathogenesis is the parasite's ability to cross formidable biological barriers in the infected host and enter tissues such as the brain, eye, and placenta. The dissemination of T. gondii into these organs underlies the severe disease that accompanies human toxoplasmosis. In this review we will focus on seminal studies as well as exciting recent findings that have shaped our current understanding of the cellular and molecular mechanisms by which T. gondii journeys throughout the host and enters the vital organs to cause disease. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Toxoplasma gondii is a highly successful parasite that can manipulate host immune responses to optimise its persistence and spread. As a result, a highly complex relationship exists between T. gondii and the immune system of the host. Advances in imaging techniques, and in particular, the application of two-photon microscopy to mouse infection models, has made it possible to directly visualize interactions between parasites and the host immune system as they occur in living tissues. Here we will discuss how dynamic imaging techniques have provided unexpected new insight into (1) how immune responses are dynamically regulated by cells and structures in the local tissue environment, (2) how protective responses to T. gondii are generated, and (3) how the parasite exploits the immune system for its own benefit. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Toxoplasma encephalitis is caused by the opportunistic protozoan parasite Toxoplasma gondii. Primary infection with T. gondii in immunocompetent individuals remains largely asymptomatic. In contrast, in immunocompromised individuals, reactivation of the parasite results in severe complications and mortality. Molecular changes at the protein level in the host central nervous system and proteins associated with pathogenesis of toxoplasma encephalitis are largely unexplored. We used a global quantitative proteomic strategy to identify differentially regulated proteins and affected molecular networks in the human host during T. gondii infection with HIV co-infection.
We identified 3,496 proteins out of which 607 proteins were differentially expressed (≥1.5-fold) when frontal lobe of the brain from patients diagnosed with toxoplasma encephalitis was compared to control brain tissues. We validated differential expression of 3 proteins through immunohistochemistry, which was confirmed to be consistent with mass spectrometry analysis. Pathway analysis of differentially expressed proteins indicated deregulation of several pathways involved in antigen processing, immune response, neuronal growth, neurotransmitter transport and energy metabolism.
Global quantitative proteomic approach adopted in this study generated a comparative proteome profile of brain tissues from toxoplasma encephalitis patients co-infected with HIV. Differentially expressed proteins include previously reported and several new proteins in the context of T. gondii and HIV infection, which can be further investigated. Molecular pathways identified to be associated with the disease should enhance our understanding of pathogenesis in toxoplasma encephalitis.
Toxoplasma gondii parasites must actively invade host cells to propagate. Secretory microneme proteins have been shown to be important for both gliding motility and active invasion. MIC2-M2AP is a protein complex that is essential for productive motility and rapid invasion by binding to host cell surface receptors. To investigate the architecture of the MIC2 and M2AP complex, we identified the minimal domains sufficient for interaction and solved the NMR solution structure of the globular domain of M2AP. We found that M2AP adopts a modified galectin fold similar to the C-terminal domain of another microneme protein, MIC1. NMR and immunoprecipitation analyses implicated hydrophobic residues on one face of the M2AP galectin fold in binding to the membrane proximal 6th thrombospondin type I repeat (TSR) domain of MIC2. Our findings provide a second example of a galectin fold adapted for microneme protein-protein interactions and suggest a conserved strategy for the assembly and folding of diverse protein complexes.
Parasites change their transcriptional systems in different developmental stages and in response to environmental changes. To investigate the molecular mechanisms that underlie transcriptional regulation, it is essential to identify the exact positions of the transcriptional start sites (TSSs) and characterize the upstream promoter regions. However, it has been essentially impossible to obtain comprehensive information using conventional methods. Here, we introduce our TSS-seq method, which combines full-length technology, oligo-capping, and rapidly developing next-generation sequencing technology. TSS-seq has enabled identification of TSS positions and upstream promoter activities as digital TSS tag counts within a reasonable cost and time frame. In this chapter, we describe in detail the TSS-seq method for the identification and characterization of the promoters in Toxoplasma gondii.
The glideosome is an actomyosin-based machinery that powers motility in Apicomplexa and participates in host cell invasion and egress from infected cells. The central component of the glideosome, myosin A (MyoA), is a motor recruited at the pellicle by the acylated gliding-associated protein GAP45. In Toxoplasma gondii, GAP45 also contributes to the cohesion of the pellicle, composed of the inner membrane complex (IMC) and the plasma membrane, during motor traction. GAP70 was previously identified as a paralog of GAP45 that is tailored to recruit MyoA at the apical cap in the coccidian subgroup of the Apicomplexa. A third member of this family, GAP80, is demonstrated here to assemble a new glideosome, which recruits the class XIV myosin C (MyoC) at the basal polar ring. MyoC shares the same myosin light chains as MyoA and also interacts with the integral IMC proteins GAP50 and GAP40. Moreover, a central component of this complex, the IMC-associated protein 1 (IAP1), acts as the key determinant for the restricted localization of MyoC to the posterior pole. Deletion of specific components of the MyoC-glideosome underscores the installation of compensatory mechanisms with components of the MyoA-glideosome. Conversely, removal of MyoA leads to the relocalization of MyoC along the pellicle and at the apical cap that accounts for residual invasion. The two glideosomes exhibit a considerable level of plasticity to ensure parasite survival.
Using high through-put RNA sequencing, we assayed the transcriptomes of three different strains of Toxoplasma gondii representing three common genotypes under both in vitro tachyzoite and in vitro bradyzoite-inducing alkaline stress culture conditions. Strikingly, the differences in transcriptional profiles between the strains, RH, PLK, and CTG, is much greater than differences between tachyzoites and alkaline stressed in vitro bradyzoites. With an FDR of 10%, we identified 241 genes differentially expressed between CTG tachyzoites and in vitro bradyzoites, including 5 putative AP2 transcription factors. We also observed a close association between cell cycle regulated genes and differentiation. By Gene Set Enrichment Analysis (GSEA), there are a number of KEGG pathways associated with the in vitro bradyzoite transcriptomes of PLK and CTG, including pyrimidine metabolism and DNA replication. These functions are likely associated with cell-cycle arrest. When comparing mRNA levels between strains, we identified 1,526 genes that were differentially expressed regardless of culture-condition as well as 846 differentially expressed only in bradyzoites and 542 differentially expressed only in tachyzoites between at least two strains. Using GSEA, we identified that ribosomal proteins were expressed at significantly higher levels in the CTG strain than in either the RH or PLK strains. This association holds true regardless of life cycle stage.
Autophagy plays a crucial role in intracellular defense against various pathogens. Xenophagy is a form of selective autophagy that targets intracellular pathogens for degradation. In addition, several related, yet distinct, intracellular defense responses depend on autophagy-related genes. This review gives an overview of these processes, pathogen strategies to subvert them, and their crosstalk with various cell death programs.
The recruitment of autophagy-related proteins plays a key role in multiple intracellular defense programs, specifically xenophagy, microtubule-associated protein 1 light chain 3 alpha (LC3)-associated phagocytosis, and the interferon gamma-mediated elimination of pathogens, such as Toxoplasma gondii and murine norovirus. Recent progress has revealed methods employed by pathogens to resist these intracellular defense mechanisms and/or persist in spite of them. The intracellular pathogen load can tip the balance between cell survival and cell death. Further, it was recently observed that LC3-associated phagocytosis is indispensable for the efficient clearance of dying cells.
Autophagy-dependent and autophagy-related gene-dependent pathways are essential in intracellular defense against a broad range of pathogens.
Toxoplasma gondii is an obligate intracellular parasite that causes serious opportunistic infections, birth defects and blindness in humans. Microtubules are critically important components of diverse structures that are used throughout the Toxoplasma life cycle. As in other eukaryotes, spindle microtubules are required for chromosome segregation during replication. Additionally, a set of membrane-associated microtubules is essential for the elongated shape of invasive zoites and motility follows a spiral trajectory that reflects the path of these microtubules. Toxoplasma zoites also construct an intricate, tubulin-based apical structure termed the conoid which is important for host cell invasion and associates with proteins typically found in the flagellar apparatus. Lastly, microgametes specifically construct a microtubule-containing flagellar axoneme in order to fertilize macrogametes, permitting genetic recombination. The specialized roles of these microtubule populations are mediated by distinct sets of associated proteins. This review summarizes our current understanding of the role of tubulin, microtubule populations, and associated proteins in Toxoplasma; these components are used for both novel and broadly conserved processes that are essential for parasite survival.
About 30% of people on Earth have latent toxoplasmosis. Infected subjects do not express any clinical symptoms, however, they carry dormant stages of parasite Toxoplasma for the rest of their life. This form of toxoplasmosis is mostly considered harmless, however, recent studies showed its specific effects on physiology, behaviour and its associations with various diseases, including psychiatric disorders such as schizophrenia. Individuals who suffer from schizophrenia have about 2.7 times higher prevalence of Toxoplasma-seropositivity than controls, which suggests that some traits characteristic of schizophrenic patients, including the sex difference in schizophrenia onset, decrease of grey matter density in specific brain areas and modification of prepulse inhibition of startle reaction could in fact be caused by toxoplasmosis for those patients who are Toxoplasma-seropositive.
We measured the effect of prepulse inhibition/facilitation of the startle reaction on reaction times. The students, 170 women and 66 men, were asked to react as quickly as possible to a startling acoustic signal by pressing a computer mouse button. Some of the startling signals were without the prepulse, some were 20 msec. preceded by a short (20 msec.) prepulse signal of lower intensity. Toxoplasma-seropositive subjects had longer reaction times than the controls. Acoustic prepulse shorted the reaction times in all subjects. This effect of prepulse on reaction times was stronger in male subjects and increased with the duration of infection, suggesting that it represented a cumulative effect of latent toxoplasmosis, rather than a fading out after effect of past acute toxoplasmosis.
Different sensitivity of Toxoplasma-seropositive and Toxoplasma-seronegative subjects on effect of prepulses on reaction times (the toxoplasmosis-prepulse interaction) suggested, but of course did not prove, that the alternations of prepulse inhibition of startle reaction observed in schizophrenia patients probably joined the list of schizophrenia symptoms that are in fact caused by latent toxoplasmosis.
Lysine succinylation is a new posttranslational modification (PTM) identified in histone proteins of Toxoplasma gondii, an obligate intracellular parasite of the phylum Apicomplexa. However, very little is known about their scope and cellular distribution. Here, using LC-MS/MS to identify parasite peptides enriched by immunopurification with succinyl-lysine antibody, we produced the first lysine succinylome in this parasite. Overall, a total of 425 lysine succinylation sites occurred on 147 succinylated proteins were identified in extracellular Toxoplasma tachyzoites, which is a proliferative stage that results in acute toxoplasmosis. With the bioinformatics analysis, it is shown that these succinylated proteins are evolutionarily conserved and involved in a wide variety of cellular functions such as metabolism, epigenetic gene regulation, and exhibit diverse subcellular localizations. Moreover, we defined five types of definitively conserved succinylation site motifs and the results imply that lysine residue of a polypeptide with lysine on the +3 position and without lysine at the -1 to +2 position is a preferred to be substrate of lysine succinyltransferase. In conclusion, our findings suggest that lysine succinylation in Toxoplasma involves in a diverse array of cellular functions although the succinylation occurs at a low level.
The zoonotic pathogen Toxoplasma gondii infects over 1/3 of the human population. The intracellular parasite can persist lifelong in the CNS within neurons modifying their function and structure, thus leading to specific behavioral changes of the host. In recent years, several in vitro studies and murine models have focused on the elucidation of these modifications. Furthermore, investigations of the human population have correlated Toxoplasma seropositivity with changes in neurological functions; however, the complex underlying mechanisms of the subtle behavioral alteration are still not fully understood. The parasites are able to induce direct modifications in the infected cells for example by altering dopamine metabolism, by functionally silencing neurons as well as by hindering apoptosis. Moreover, indirect effects of the peripheral immune system and alterations of the immune status of the CNS, observed during chronic infection, might also contribute to changes in neuronal connectivity and synaptic plasticity. In this review we will provide an overview and highlight recent advances, which describe changes in the neuronal function and morphology upon T. gondii infection. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Pathogenic microbes rely on environmental cues to initiate key events during infection such as differentiation, motility, egress and invasion of cells or tissues. Earlier investigations showed that an acidic environment activates motility of the protozoan parasite T. gondii. Conversely, potassium ions, which are abundant in the intracellular milieu that bathes immotile replicating parasites, suppress motility. Since motility is required for efficient parasite cell invasion and egress we sought to better understand its regulation by environmental cues. We found that low pH stimulates motility by triggering Ca2+-dependent secretion of apical micronemes, and that this cue is sufficient to overcome suppression by potassium ions and drive parasite motility, cell invasion and egress. We also discovered that acidification promotes membrane binding and cytolytic activity of perforin-like protein 1 (PLP1), a pore-forming protein required for efficient egress. Agents that neutralize pH reduce the efficiency of PLP1-dependent perforation of host membranes and compromise egress. Finally, although low pH stimulation of microneme secretion promotes cell invasion, it also causes PLP1-dependent damage to host cells, suggesting a mechanism by which neutral extracellular pH subdues PLP1 activity to allow cell invasion without overt damage to the target cell. These findings implicate acidification as a signal to activate microneme secretion and confine cytolytic activity to egress without compromising the viability of the next cell infected.
The protozoan Toxoplasma gondii expresses large amounts of a 37 kDa Type 2C serine-threonine phosphatase, the so-called TgPP2C which has been suggested to contribute to parasite growth regulation. Ectopic expression in mammalian cells also indicated that the enzyme could regulate growth and survival. In this study, we aimed to investigate the interaction of TgPP2C with human SSRP1 (structure-specific recognition protein 1) and the effects of TgPP2C on cell viability.
The yeast two hybrid system, His-tag pull-down and co-immunoprecipitation assays were used to confirm the interaction of TgPP2C with SSRP1 and determine the binding domain on SSRP1. The evaluation of cell apoptosis was performed using cleaved caspase-3 antibody and Annexin-V/PI kit combined with flow cytometry.
We identified human SSRP1 as an interacting partner of TgPP2C. The C-terminal region of SSRP1 including the amino acids 471 to 538 was specifically mapped as the region responsible for interaction with TgPP2C. The overexpression of TgPP2C down-regulated cell apoptosis and negatively regulated apoptosis induced by DRB, casein kinase II (CKII) inhibitor, through enhanced interaction with SSRP1.
Toxoplasma gondii and malaria parasites contain a unique and essential relict plastid called the apicoplast. Most apicoplast proteins are encoded in the nucleus and are transported to the organelle via the endoplasmic reticulum (ER). Three trafficking routes have been proposed for apicoplast membrane proteins: (i) vesicular trafficking from the ER to the Golgi and then to the apicoplast, (ii) contiguity between the ER membrane and the apicoplast allowing direct flow of proteins, and (iii) vesicular transport directly from the ER to the apicoplast. Previously, we identified a set of membrane proteins of the T. gondii apicoplast which were also detected in large vesicles near the organelle. Data presented here show that the large vesicles bearing apicoplast membrane proteins are not the major carriers of luminal proteins. The vesicles continue to appear in parasites which have lost their plastid due to mis-segregation, indicating that the vesicles are not derived from the apicoplast. To test for a role of the Golgi body in vesicle formation, parasites were treated with brefeldin A or transiently transfected with a dominant-negative mutant of Sar1, a GTPase required for ER to Golgi trafficking. The immunofluorescence patterns showed little change. These findings were confirmed using stable transfectants, which expressed the toxic dominant-negative sar1 following Cre-loxP mediated promoter juxtaposition. Our data support the hypothesis that the large vesicles do not mediate the trafficking of luminal proteins to the apicoplast. The results further show that the large vesicles bearing apicoplast membrane proteins continue to be observed in the absence of Golgi and plastid function. These data raise the possibility that the apicoplast proteome is generated by two novel ER to plastid trafficking pathways, plus the small set of proteins encoded by the apicoplast genome.
High mobility group box 1 (HMGB1) is a nuclear factor that usually binds DNA and modulates gene expression in multicellular organisms. Three HMGB1 orthologs were predicted in the genome of Toxoplasma gondii, an obligate intracellular protozoan pathogen, termed TgHMGB1a, b and c. Phylogenetic and bioinformatic analyses indicated that these proteins all contain a single HMG box and which shared in three genotypes. We cloned TgHMGB1a, a 33.9 kDa protein that can stimulates macrophages to release TNF-α, and, we demonstrated that the TgHMGB1a binds distorted DNA structures such as cruciform DNA in electrophoretic mobility shift assays (EMSA). Immunofluorescence assay indicated TgHMGB1a concentrated in the nucleus of intracellular tachyzoites but translocated into the cytoplasm while the parasites release to extracellular. There were no significant phenotypic changes when the TgHMGB1a B box was deleted, while transgenic parasites that overexpressed TgHMGB1a showed slower intracellular growth and caused delayed death in mouse, further quantitative RT-PCR analyses showed that the expression levels of many important genes, including virulence factors, increased when TgHMGB1a was overexpressed, but no significant changes were observed in TgHMGB1a B box-deficient parasites. Our findings demonstrated that TgHMGB1a is indeed a nuclear protein that maintains HMG box architectural functions and is a potential proinflammatory factor during the T.gondii infection. Further studies that clarify the functions of TgHMGB1s will increase our knowledge of transcriptional regulation and parasite virulence, and might provide new insight into host-parasite interactions for T. gondii infection.
MicroRNA-132 (miR-132) has been demonstrated to affect multiple neuronal functions and its dysregulation is linked to several neurological disorders. We previously showed that acute Toxoplasma gondii infection induces miR-132 expression both in vitro and in vivo. To investigate the impact of chronic infection on miR-132, we infected mice with T. gondii PRU strain and performed assessment 5 months later in six brain regions (cortex, hypothalamus, striatum, cerebellum, olfactory bulb and hippocampus) by qPCR. We found that while acute infection of T. gondii increases the expression of miR-132, chronic infection has the opposite effect. The effect varied amongst different regions of the brain and presented in a sex-dependent manner, with females exhibiting more susceptibility than males. MiR-132 and brain-derived neurotrophic factor (BDNF, an inducer of miR-132) were not co-varies in the brain areas of infected mice. T. gondii DNA/RNA was found in all tested brain regions and a selective tropism towards the hippocampus, based on bradyzoite density, was observed in both males and females. However, the expressions of miR-132 or BDNF were poorly reflected by the density of T. gondii in brain areas. Our findings highlight the importance of investigating the miR-132-mediated neuronal function in mice infected with T. gondii.
This study analyzed the synthesis of Interferon gamma (IFN-γ), Tumor Necrosis Factor alpha (TNF-α), and Interleukin 10 (IL-10) in chronically infected patients which developed the symptomatic disease as cerebral or ocular toxoplasmosis. Blood from 61 individuals were divided into four groups: Cerebral toxoplasmosis/AIDS patients (CT/AIDS group) (n = 15), ocular toxoplasmosis patients (OT group) (n = 23), chronic toxoplasmosis individuals (CHR group) (n = 13) and healthy individuals (HI group) (n = 10). OT, CHR, and HI groups were human immunodeficiency virus (HIV) seronegative. The diagnosis was made by laboratorial (PCR and ELISA) and clinical subjects. For cytokine determination, peripheral blood mononuclear cells (PBMC) of each patient were isolated and stimulated in vitro with T. gondii antigen. IFN-γ, TNF-α, and IL-10 activities were determined by ELISA. Patients from CT/AIDS and OT groups had low levels of IFN-γ when were compared with those from CHR group. These data suggest the low resistance to develop ocular lesions by the low ability to produce IFN-γ against the parasite. The same patients, which developed ocular or cerebral toxoplasmosis had higher TNF-α levels than CHR individuals. High TNF-α synthesis contribute to the inflammatory response and damage of the choroid and retina in OT patients and in AIDS patients caused a high inflammatory response as the TNF-α synthesis is not affected since monocytes are the major source this cytokine in response to soluble T. gondii antigens. IL-10 levels were almost similar in CT/AIDS and OT patients but low when compared with CHR individuals. The deviation to Th2 immune response including the production of anti-inflammatory cytokines, such as IL-10 may promote the parasite's survival causing the tissue immune destruction. IL-10 production in T. gondii-infected brains may support the persistence of parasites as down-regulating the intracerebral immune response. All these indicate that OT and CT/AIDS patients produced low levels of IL-10 (Th2 response) and IFN-γ (Th1 response). They produced high TNF-α suggesting a high inflammatory response triggered by the parasite.