Tachyzoite Replication Research Articles

Toxoplasma gondii eIF-5A Modulates the Immune Response of Murine Macrophages In Vitro.

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan that can elicit a robust immune response during infection. Macrophage cells have been shown to play an important role in the immune response against T. gondii. In our previous study, the eukaryotic translation initiation factor 5A (eIF-5A) gene of T. gondii was found to influence the invasion and replication of tachyzoites. In this study, the recombinant protein of T. gondii eIF-5A (rTgeIF-5A) was incubated with murine macrophages, and the regulatory effect of TgeIF-5A on macrophages was characterized. Immunofluorescence assay showed that TgeIF-5A was able to bind to macrophages and partially be internalized. The Toll-like receptor 4 (TLR4) level and chemotaxis of macrophages stimulated with TgeIF-5A were reduced. However, the phagocytosis and apoptosis of macrophages were amplified by TgeIF-5A. Meanwhile, the cell viability experiment indicated that TgeIF-5A can promote the viability of macrophages, and in the secretion assays, TgeIF-5A can induce the secretion of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and nitric oxide (NO) from macrophages. These findings demonstrate that eIF-5A of T. gondii can modulate the immune response of murine macrophages in vitro, which may provide a reference for further research on developing T. gondii vaccines.

Besnoitia besnoiti tachyzoite replication in bovine primary endothelial cells relies on host Niemann-Pick type C protein 1 for cholesterol acquisition.

Besnoitia besnoiti is a cyst-forming apicomplexan parasite and the causal agent of bovine besnoitiosis. During early phase of infection, tachyzoites replicate within host endothelial cells in a host cell cholesterol-dependent process. By applying U18666A treatments, we here evaluated the role of Niemann-Pick type C protein 1 (NPC1) in both, intracellular B. besnoiti replication and host cellular cholesterol distribution. Additionally, B. besnoiti-driven changes in NPC1 gene transcription were studied by qPCR. Overall, U18666A treatments significantly reduced B. besnoiti proliferation and induced cholesterol accumulation in host cytoplasmic dense vesicles. However, NPC1 gene transcription was not affected by B. besnoiti infection.

Phytohormones regulate asexual Toxoplasma gondii replication.

The protozoan Toxoplasma gondii (T. gondii) is a zoonotic disease agent causing systemic infection in warm-blooded intermediate hosts including humans. Duringthe acute infection, the parasite infects host cells and multiplies intracellularly in the asexual tachyzoite stage. In this stage of the life cycle, invasion, multiplication, and egress are themost critical events in parasite replication. T. gondii features diverse cell organelles to support these processes, including the apicoplast, an endosymbiont-derived vestigial plastid originating from an alga ancestor. Previous studies have highlighted that phytohormones can modify the calcium-mediated secretion, e.g., of adhesins involved in parasite movement and cell invasion processes. The present study aimed to elucidate the influence of different plant hormones on the replication of asexual tachyzoites in a human foreskin fibroblast (HFF) host cell culture. T. gondii replication was measured by the determination of T. gondii DNA copies via qPCR. Three selected phytohormones, namely abscisic acid (ABA), gibberellic acid (GIBB), and kinetin (KIN) as representatives of different plant hormone groups were tested. Moreover, the influence of typical cell culture media components on the phytohormone effects was assessed. Our results indicate that ABA is able to induce a significant increase of T. gondii DNA copies in atypical supplemented cell culture medium when applied in concentrations of 20 ng/μl or 2 ng/μl, respectively. In contrast, depending on the culture medium composition, GIBB may potentially serve as T. gondii growth inhibitor and may be further investigated as a potential treatment for toxoplasmosis.

Immune recognition of latent neuronal Toxoplasma gondiiinfection

Abstract Toxoplasma gondii is a neurotropic parasite with high mortality rates in immunocompromised individuals. Successful therapies have been developed against the acute tachyzoite stage, but no treatment options exist to target the latent form of the parasite – bradyzoites that form cysts within neurons. It is thought that residence of cysts in neurons shelters T. gondii from immune recognition and clearance. However, a decline in cyst numbers over time suggests the presence of anti-cyst immunity. Additionally, neuronal deletion of STAT1 results in increased cyst size and number, but no change in tachyzoite replication, providing evidence for IFN-γ-mediated cyst control. T cells are a major source of IFN-γ and are crucial for parasite control in the CNS, but it remains unclear if T cells directly target cyst-infected neurons. The use of transgenic parasites with bradyzoite-restricted OVA expression (BagOVA) reveals that cyst-derived antigen can induce OT-I CD8+ T cell responses in the brains of chronically infected mice. These cyst-specific OT-Is have distinct effector phenotypes and cytokine production when compared to T cells responding to antigen expressed by both tachyzoites and bradyzoites (ptubOVA). To determine whether neurons directly present cyst antigen to induce CD8+ T cell responses in BagOVA infection, mice with neuronal-specific loss of MHCI were generated. Infection of these mice showed no significant change in the magnitude of T cell responses or parasite burden in the CNS. Together these findings reveal that T cell responses can be mounted against cyst antigens, but another cell type likely mediates cross presentation of cyst derived antigens. Supported by grants from NIH (R01 580-5803-4-580697-XXXX-2000-3000)

Moringa oleifera Leaf Ethanol Extract Inhibits Toxoplasma gondii Tachyzoites Replication

The various infection routes of Toxoplasma gondii that are close to daily life strongly support the incidence of toxoplasmosis. The emergence of drug-resistant Toxoplasma gondii strains raises future concerns. Moringa leaf ethanol extract has been shown to have several anti-pathogen activities, which could have an anti-Toxoplasma effect. This research was conducted to analyze the anti-Toxoplasma effect of moringa leaf ethanol extract against tachyzoites replication in Toxoplasma gondii and the correlation between extract doses with the number of tachyzoites. Mice were divided into five groups. The negative control group (Group I) received CMC-Na solution. The positive control group (Group II) received spiramycin 100 mg/kg BW. The treatment groups received moringa leaf ethanol extract 250 mg/kg BW (group III), 500 mg/kg BW (group IV), and 1000 mg/kg BW (group V), respectively. Mice were injected with 1 x 105 tachyzoites/0.1 mL/mice intraperitoneally on the first day. Moringa leaf ethanol extract and spiramycin were given orally once daily for three days. The number of tachyzoites in the intraperitoneal fluid was calculated on the fifth day. The results have shown that there were significantly lower differences (P < 0.05) in group IV (P = 0.021) and group V (P = 0.022) compared to group I. There was also a significant negative correlation between the extract doses and the number of tachyzoites (P = 0.000; r = -0.781). Moringa oleifera leaf ethanol extract has an anti-Toxoplasma effect by inhibiting the tachyzoite replication at 500 mg/kg BW and 1000 mg/kg BW.

Not your Mother's MAPKs: Apicomplexan MAPK function in daughter cell budding.

Reversible phosphorylation by protein kinases is one of the core mechanisms by which biological signals are propagated and processed. Mitogen-activated protein kinases, or MAPKs, are conserved throughout eukaryotes where they regulate cell cycle, development, and stress response. Here, we review advances in our understanding of the function and biochemistry of MAPK signaling in apicomplexan parasites. As expected for well-conserved signaling modules, MAPKs have been found to have multiple essential roles regulating both Toxoplasma tachyzoite replication and sexual differentiation in Plasmodium. However, apicomplexan MAPK signaling is notable for the lack of the canonical kinase cascade that normally regulates the networks, and therefore must be regulated by a distinct mechanism. We highlight what few regulatory relationships have been established to date, and discuss the challenges to the field in elucidating the complete MAPK signaling networks in these parasites.

TNFα rs1799964 TT genotype may be a susceptibility factor for vertical transmission of Toxoplasma gondii and clinical signs in newborns from pregnant women with acute toxoplasmosis.

One of the main impacts of Toxoplasma gondii infection occurs during pregnancy and is related to the vertical transmission of the parasite (congenital toxoplasmosis), which can cause severe clinical outcomes and fetal death. During acute infection, in order to control the rapid replication of tachyzoites, different host immune response genes are activated, and these include cytokine-encoding genes. Considering that polymorphisms in cytokine genes may increase susceptibility to vertical transmission of T. gondii by determining the immune status of the pregnant woman, this study evaluated the influence of polymorphisms of tumor necrosis factor alpha (TNFα) rs1799964 (- 1031) and interleukin 1 beta (IL1β) rs16944 (- 511) genes on gestational toxoplasmosis and on the vertical transmission of the parasite and verified the allele and genotype frequency of these polymorphisms in pregnant patients whose respective newborn did or did not present clinical abnormalities suggestive of congenital toxoplasmosis. A total of 204 pregnant patients with (n = 114) or without (n = 90) infection by T. gondii were enrolled. No associations were found involving the polymorphisms rs1799964 (- 1031) of the TNFα gene and rs16944 (- 511) of the IL1β gene with the increased chance of T. gondii infection during pregnancy. However, it was observed that the maternal TT genotype referring to the polymorphism of the TNFα gene seems to influence the vertical transmission of the parasite (P = 0.01; χ2 = 6.05) and the presence of clinical manifestation in newborns from pregnancies with acute toxoplasmosis (P = 0.007; χ2 = 9.68). The TNFα rs1799964 TT genotype may act as a susceptibility factor for the vertical transmission of parasite and for the presence of clinical signs in newborns from pregnant women with acute toxoplasmosis.

Investigation of Antiparasitic Activity of Two Marine Natural Products, Estradiol Benzoate, and Octyl Gallate, on Toxoplasma gondii In Vitro.

Toxoplasmosis, caused by Toxoplasma gondii, is a common disease worldwide and could be severe and even fatal in immunocompromised individuals and fetuses. Limitation in current available treatment options drives the need to develop novel therapeutics. This study assessed the anti-T. gondii potential of 103 marine natural products. A luminescence-based β-galactosidase activity assay was used to screen the marine natural products library. Afterward, those compounds that displayed over 70% parasite inhibition ratio were further chosen to assess their cytotoxicity. Compounds exhibiting low cytotoxicity (≥80% cell viability) were applied to evaluate the inhibition efficacy on discrete steps of the T. gondii lytic cycle, including invasion, intracellular growth, and egress abilities as well as the cell cycle. We found that both estradiol benzoate and octyl gallate caused >70% inhibition of tachyzoite growth with IC50 values of 4.41 ± 0.94 and 5.66 ± 0.35 μM, respectively, and displayed low cytotoxicity with TD50 values of 34.11 ± 2.86 and 26.4 ± 0.98 μM, respectively. Despite their defects in inhibition of invasion and egress of tachyzoite, the two compounds markedly inhibited the tachyzoite intracellular replication. Flow cytometric analyses further suggested that the anti-T. gondii activity of estradiol benzoate, rather than octyl gallate, may be linked to halting cell cycle progression of tachyzoite from G1 to S phase. Taken together, these findings suggest that both estradiol benzoate and octyl gallate are potential inhibitors for anti-T. gondii infection and support the further exploration of marine natural products as a thinkable source of alternative and active agents against T. gondii.

In silico restriction site analysis for characterization of Toxoplasma gondii isolate

Toxoplasma gondii infection is a serious major public health concern. Toxoplasmosis is a disease that affects humans and warm-blooded animals all over the world. The virulence and severity of the sickness may be influenced by parasite load. The three clonal lineages for biological research are T. gondii genotypes I, II, and III. Using primer genes for gra1, rop1, and mic3, which have been identified as essential proteins for tachyzoite invasion and replication in host cells. PCR was used to determine the genotype of a T. gondii isolate from the Indonesian research center for veterinary science. In silico restriction site analysis was performed on T. gondii isolates using CLC sequence viewer 8.0 software to assess sequence data for the existence of restriction enzyme patterns. Predictable restriction fragment length polymorphism using In silico analysis. T. gondii sequencing genes are In silico digested with different restriction enzymes. The findings show that they can easily distinguish archetypal parasites from biotypes and estimate the genetic diversity of the parasites. According to the interpretation of the data, isolate T. gondii belongs to strain RH genotype I.

Tityus serrulatus (Scorpion): From the Crude Venom to the Construction of Synthetic Peptides and Their Possible Therapeutic Application Against Toxoplasma gondii Infection.

Toxoplasmosis, caused by Toxoplasma gondii, is a major public concern owing to its neurotropic nature and high morbidity and mortality rates in immunocompromised patients and newborns. Current treatment for this disease is inefficient and produces side effects. Inflammatory mediators produced during T. gondii infection (e.g., cytokines and nitric oxide) are crucial in controlling parasite replication. In this context, Tityus serrulatus venom (TsV) induces the production of inflammatory mediators by immune cells. Thus, this study aimed to isolate and identify the components of TsV with potential anti-T. gondii activity. TsV was extracted from scorpions and lyophilized or loaded onto a column to obtain its fractions. TsV subfractions were obtained using chromatography, and its amino acid sequence was identified and applied to peptide design using bioinformatics tools. The C57BL/6 mice and their harvested macrophages were used to test the anti-Toxoplasma activity of TsV components and peptides. TsV and its fraction F6 attenuated the replication of tachyzoites in macrophages and induced nitric oxide and cytokine (IL-12, TNF, and IL-6) production by infected cells, without host cell toxicity. Moreover, Su6-B toxin, a subfraction of F6, demonstrated anti-T. gondii activity. The partially elucidated and characterized amino acid sequence of Sub6-B demonstrated 93% similarity with T. serrulatus 2 toxin (Ts2). Ts2 mimetic peptides (“Pep1,” “Pep2a,” and “Pep2b”) were designed and synthesized. Pep1 and Pep2a, but not Pep2b, reduced the replication of tachyzoites in macrophages. In vivo, treatment of T. gondii-infected mice with Pep1, Pep2a, or Pep2b decreased the number of cerebral cysts and did not induce hepatotoxicity in the animals. Taken together, our data show promising immunomodulatory and antiparasitic activity of TsV that could be explored and applied in future therapies for treating infectious parasitic diseases such as toxoplasmosis.

Ezetimibe blocks Toxoplasma gondii-, Neospora caninum- and Besnoitia besnoiti-tachyzoite infectivity and replication in primary bovine endothelial host cells.

Coccidia are obligate apicomplexan parasites that affect humans and animals. In fast replicating species, in vitro merogony takes only 24–48 h. In this context, successful parasite proliferation requires nutrients and other building blocks. Coccidian parasites are auxotrophic for cholesterol, so they need to obtain this molecule from host cells. In humans, ezetimibe has been applied successfully as hypolipidaemic compound, since it reduces intestinal cholesterol absorption via blockage of Niemann−Pick C-1 like-1 protein (NPC1L1), a transmembrane protein expressed in enterocytes. To date, few data are available on its potential anti-parasitic effects in primary host cells infected with apicomplexan parasites of human and veterinary importance, such as Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti. Current inhibition experiments show that ezetimibe effectively blocks T. gondii, B. besnoiti and N. caninum tachyzoite infectivity and replication in primary bovine endothelial host cells. Thus, 20 μm ezetimibe blocked parasite proliferation by 73.1−99.2%, via marked reduction of the number of tachyzoites per meront, confirmed by 3D-holotomographic analyses. The effects were parasitostatic since withdrawal of the compound led to parasite recovery with resumed proliferation. Ezetimibe-glucuronide, the in vivo most effective metabolite, failed to affect parasite proliferation in vitro, thereby suggesting that ezetimibe effects might be NPC1L1-independent.

A Family of Toxoplasma gondii Genes Related to GRA12 Regulate Cyst Burdens and Cyst Reactivation.

ABSTRACTToxoplasma gondii causes a chronic infection that renders the immunocompromised human host susceptible to toxoplasmic encephalitis triggered by cyst reactivation in the central nervous system. The dense granule protein GRA12 is a major parasite virulence factor required for parasite survival during acute infection. Here, we characterized the role of four GRA12-related genes in acute and chronic stages of infection. While GRA12A, GRA12B, and GRA12D were highly expressed in asexual stage tachyzoites and bradyzoites, expression of GRA12C appeared to be restricted to the sexual stages. In contrast to deletion of GRA12 (Δgra12), no major defects in acute virulence were observed in Δgra12A, Δgra12B, or Δgra12D parasites, though Δgra12B parasites exhibited an increased tachyzoite replication rate. Bradyzoites secreted GRA12A, GRA12B, and GRA12D and incorporated these molecules into the developing cyst wall, as well as the cyst matrix in distinct patterns. Similar to GRA12, GRA12A, GRA12B, and GRA12D colocalized with the dense granules in extracellular tachyzoites, with GRA2 and the intravacuolar network in the tachyzoite stage parasitophorous vacuole and with GRA2 in the cyst matrix and cyst wall. Chronic stage cyst burdens were decreased in mice infected with Δgra12A parasites and were increased in mice infected with Δgra12B parasites. However, Δgra12B cysts were not efficiently maintained in vivo. Δgra12A, Δgra12B, and Δgra12D in vitro cysts displayed a reduced reactivation efficiency, and reactivation of Δgra12A cysts was delayed. Collectively, our results suggest that a family of genes related to GRA12 play significant roles in the formation, maintenance, and reactivation of chronic stage cysts.IMPORTANCE If host immunity weakens, Toxoplasma gondii cysts recrudesce in the central nervous system and cause a severe toxoplasmic encephalitis. Current therapies target acute stage infection but do not eliminate chronic cysts. Parasite molecules that mediate the development and persistence of chronic infection are poorly characterized. Dense granule (GRA) proteins such as GRA12 are key virulence factors during acute infection. Here, we investigated four GRA12-related genes. GRA12-related genes were not major virulence factors during acute infection. Instead, GRA12-related proteins localized at the cyst wall and cyst matrix and played significant roles in cyst development, persistence, and reactivation during chronic infection. Similar to GRA12, the GRA12-related proteins selectively associated with the intravacuolar network of membranes inside the vacuole. Collectively, our results support the hypothesis that GRA12 proteins associated with the intravacuolar membrane system support parasite virulence during acute infection and cyst development, persistence, and reactivation during chronic infection.

Evaluation of ATM Kinase Inhibitor KU-55933 as Potential Anti-Toxoplasma gondii Agent.

Toxoplasma gondii is an apicomplexan protozoan parasite with a complex life cycle composed of multiple stages that infect mammals and birds. Tachyzoites rapidly replicate within host cells to produce acute infection during which the parasite disseminates to tissues and organs. Highly replicative cells are subject to Double Strand Breaks (DSBs) by replication fork collapse and ATM, a member of the phosphatidylinositol 3-kinase (PI3K) family, is a key factor that initiates DNA repair and activates cell cycle checkpoints. Here we demonstrate that the treatment of intracellular tachyzoites with the PI3K inhibitor caffeine or ATM kinase-inhibitor KU-55933 affects parasite replication rate in a dose-dependent manner. KU-55933 affects intracellular tachyzoite growth and induces G1-phase arrest. Addition of KU-55933 to extracellular tachyzoites also leads to a significant reduction of tachyzoite replication upon infection of host cells. ATM kinase phosphorylates H2A.X (γH2AX) to promote DSB damage repair. The level of γH2AX increases in tachyzoites treated with camptothecin (CPT), a drug that generates fork collapse, but this increase was not observed when co-administered with KU-55933. These findings support that KU-55933 is affecting the Toxoplasma ATM-like kinase (TgATM). The combination of KU-55933 and other DNA damaging agents such as methyl methane sulfonate (MMS) and CPT produce a synergic effect, suggesting that TgATM kinase inhibition sensitizes the parasite to damaged DNA. By contrast, hydroxyurea (HU) did not further inhibit tachyzoite replication when combined with KU-55933.

Antiparasitic Efficacy of Curcumin Against Besnoitia besnoiti Tachyzoites in vitro.

Besnoitia besnoiti is the causative agent of bovine besnoitiosis. B. besnoiti infections lead to reduced fertility and productivity in cattle causing high economic losses, not only in Europe, but also in Asia and Africa. Mild to severe clinical signs, such as anasarca, oedema, orchitis, hyperkeratosis, and characteristic skin and mucosal cysts, are due to B. besnoiti tachyzoite and bradyzoite replication in intermediate host tissues. So far, there are no commercially available effective drugs against this parasite. Curcumin, a polyphenolic compound from Curcuma longa rhizome is well-known for its antioxidant, anti-inflammatory, immunomodulatory and also anti-protozoan effects. Hence, the objective of this study was to evaluate the effects of curcumin on viability, motility, invasive capacity, and proliferation of B. besnoiti tachyzoites replicating in primary bovine umbilical vein endothelial cells (BUVEC) in vitro. Functional inhibition assays revealed that curcumin treatments reduce tachyzoite viability and induce lethal effects in up to 57% of tachyzoites (IC50 in 5.93 μM). Referring to general motility, significant dose-dependent effects of curcumin treatments were observed. Interestingly, curcumin treatments only dampened helical gliding and twirling activities whilst longitudinal gliding motility was not significantly affected. In addition, curcumin pretreatments of tachyzoites resulted in a dose-dependent reduction of host cell invasion as detected by infections rates at 1 day p. i. These findings demonstrate feeding cattle with Curcuma longa rhizomes may represent a new strategy for besnoitiosis treatment.

Risk Assessment of Etanercept in Mice Chronically Infected With Toxoplasma gondii.

Toxoplasma gondii (T. gondii) is a zoonotic parasite that severely harms the health of the host. The cysts of T. gondii can reactivate from bradyzoites to tachyzoites, if the individual develops low or defective immunity, causing lethal toxoplasmosis. The host resists T. gondii infection by mediating Th1-type cellular immunity to generate pro-inflammatory cytokines. Tumor necrosis factor (TNF) is an important pro-inflammatory cytokine, which can induce lysosomal fusion of parasitophorous vacuole (PV) to kill parasites. Etanercept is a soluble TNF receptor fusion protein, which is widely used clinically to cure autoimmune diseases. The effects and specific molecular mechanisms of etanercept treatment on patients co-infected with autoimmune diseases and chronic toxoplasmosis are rarely reported. In our study, a mouse model of chronic infection with T. gondii and murine macrophages RAW264.7 cells infected with T. gondii were employed to investigate the impact of etanercept on the status of chronic infection. The cytokines levels and a series of phenotypic experiments in vivo and in vitro were measured. In the present study, the expression levels of TNF, IL-1β, and IL-6 were decreased and the brain cysts number was increased in mice chronically infected with T. gondii after being treated with etanercept. In vivo experiments confirmed that etanercept caused a decrease in the immune levels of the mice and activated the brain cysts, which would lead to conversion from chronic infection to acute infection, causing severe clinical and pathological symptoms. Murine macrophages RAW264.7 cells were pretreated with etanercept, and then infected with T. gondii. In vitro experiments, the expression levels of cytokines were decreased, indicating that etanercept could also reduce the cells’ immunity and promote the transformation of bradyzoites to tachyzoites, but did not affect the intracellular replication of tachyzoites. In summary, etanercept treatment could activate the conversion of bradyzoites to tachyzoites through reducing host immunity in vivo and in vitro. The results obtained from this study suggest that the use of etanercept in patients co-infected with autoimmune diseases and chronic toxoplasmosis may lead to the risk of activation of chronic infection, resulting in severe acute toxoplasmosis.

One-pot, multicomponent synthesis of 2,3-disubstituted quinazolin-ones with potent and selective activity against Toxoplasma gondii

The discovery of two quinazolinones with selective, single-digit micromolar activity (IC50 = 6–7 µM) against the tachyzoites of the apicomplexan parasite Toxoplasma gondii is reported. These potent and selective third generation derivatives contain a benzyloxybenzyl substituent at C2 and a bulky aliphatic moiety at N3. Here we show that these quinazolinones inhibit T. gondii tachyzoite replication in an established infection, but do not significantly affect host cell invasion by the tachyzoites.

Characterisation of susceptibility of chicken macrophages to infection with Toxoplasma gondii of type II and III strains

Toxoplasma gondii is known to be able to infect any nucleated cell including immune cells like macrophages. In addition, it is assumed that macrophages serve as trojan horse during distribution in hosts. The underlying causes of parasite host interaction remain yet not fully understood. The aim of the present study was to investigate susceptibility of chicken macrophages to infection with T. gondii and the process of infection in avian cells in comparison to cells of mammalian origin. Primary avian blood monocyte-derived macrophages were infected with tachyzoites of type II (ME49) and III (NED) strains. Long term observations of parasite replication in primary macrophages were compared to data obtained in an avian macrophage cell line (HD11) and a standard cultivation mammalian cell line (VERO). Furthermore, we assessed the immune response of the primary macrophages by long-term investigation of gene expression of IL-1 beta, IL-12p40, Lipopolysaccharide induced TNF-alpha factor (LITAF) and inducible nitric oxide synthase (iNOS) comparing viable and heat-inactivated tachyzoites of the ME49 strain. Albeit, we found no differences between both strains, replication of tachyzoites in avian primary macrophages was significantly different from immortalized cell lines HD11 and VERO. The crucial period of parasite replication was between 8 and 24 h post-infection coinciding with the upregulation of gene expression of cytokines and iNOS revealing an active macrophage response at this period. Gene expression in macrophages was higher after infection with viable tachyzoites than by exposure of cells to heat-inactivated tachyzoites. Hence, we conclude that the process of penetration is pivotal for host cell response to the parasite both in avian as in mammalian cells.

Toxoplasma gondii reorganizes the host cell architecture during spontaneous cyst formation in vitro.

Toxoplasma gondii is an intracellular protozoan parasite that causes toxoplasmosis, a prevalent infection related to abortion, ocular diseases and encephalitis in immuno-compromised individuals. In the untreatable (and life-long) chronic stage of toxoplasmosis, parasitophorous vacuoles (PVs, containing T. gondii tachyzoites) transform into tissue cysts, containing slow-dividing bradyzoite forms. While acute-stage infection with tachyzoites involves global rearrangement of the host cell cytoplasm, focused on favouring tachyzoite replication, the cytoplasmic architecture of cells infected with cysts had not been described. Here, we characterized (by fluorescence and electron microscopy) the redistribution of host cell structures around T. gondii cysts, using a T. gondii strain (EGS) with high rates of spontaneous cystogenesis in vitro. Microtubules and intermediate filaments (but not actin microfilaments) formed a 'cage' around the cyst, and treatment with taxol (to inhibit microtubule dynamics) favoured cystogenesis. Mitochondria, which appeared adhered to the PV membrane, were less closely associated with the cyst wall. Endoplasmic reticulum (ER) profiles were intimately associated with folds in the cyst wall membrane. However, the Golgi complex was not preferentially localized relative to the cyst, and treatment with tunicamycin or brefeldin A (to disrupt Golgi or ER function, respectively) had no significant effect on cystogenesis. Lysosomes accumulated around cysts, while early and late endosomes were more evenly distributed in the cytoplasm. The endocytosis tracer HRP (but not BSA or transferrin) reached bradyzoites after uptake by infected host cells. These results suggest that T. gondii cysts reorganize the host cell cytoplasm, which may fulfil specific requirements of the chronic stage of infection.

ANK1 and DnaK-TPR, Two Tetratricopeptide Repeat-Containing Proteins Primarily Expressed in Toxoplasma Bradyzoites, Do Not Contribute to Bradyzoite Differentiation.

Toxoplasma gondii is an important zoonotic pathogen infecting one third of the world population and numerous animals. A key factor to its wide distribution is the ability to interconvert between fast replicating tachyzoites and slowly growing bradyzoites, and to establish lifelong chronic infection in intermediate hosts. Although it is well accepted that stage conversion plays key roles in the pathogenesis and transmission of the parasite, little is known about the molecular mechanisms behind it. Using existing gene expression data from TOXODB and published work, we looked for proteins with novel functional domains and whose expression is up-regulated in the bradyzoite stage, hoping to find molecules that have critical roles in regulating stage conversion and bradyzoite formation. In this study we characterized two such proteins ANK1 and DnaK-TPR, both of which are primarily expressed in bradyzoites and contain novel motifs to mediate protein-protein interactions. Through CRISPR/CAS9 directed gene editing technology, both genes were individually knocked out in type 1 strain TgHB2 and type 2 strain ME49. Disruption of neither of these two genes affected the growth or replication of tachyzoites in vitro, consistent with their minimal expression at this stage. However, mutants lacking ANK1 or DnaK-TPR displayed modest virulence attenuation during mice infection. Surprisingly, inactivation of neither ANK1 nor DnaK-TPR seemed to have a significant impact on bradyzoite differentiation in vitro or cyst formation in vivo. These results suggest that ANK1 and DnaK-TPR probably do not directly contribute to bradyzoite differentiation, but likely affect other aspects of bradyzoite biology.

The Toxoplasma Centrocone Houses Cell Cycle Regulatory Factors.

ABSTRACTOur knowledge of cell cycle regulatory mechanisms in apicomplexan parasites is very limited. In this study, we describe a novel Toxoplasma gondii factor that has a vital role in chromosome replication and the regulation of cytoplasmic and nuclear mitotic structures, and we named this factor ECR1 for essential for chromosome replication 1. ECR1 was discovered by complementation of a temperature-sensitive (ts) mutant that suffers lethal, uncontrolled chromosome replication at 40°C similar to a ts mutant carrying a defect in topoisomerase. ECR1 is a 52-kDa protein containing divergent RING and TRAF-Sina-like zinc binding domains that are dynamically expressed in the tachyzoite cell cycle. ECR1 first appears in the unique spindle compartment of the Apicomplexa (centrocone) of the nuclear envelope in early S phase and then in the nucleus in late S phase where it reaches maximum expression. Following nuclear division, but before daughter parasites separate from the mother parasite, ECR1 is downregulated and is absent in new daughter parasites. The proteomics of ECR1 identified interactions with the ubiquitin-mediated protein degradation machinery and the minichromosome maintenance complex, and the loss of ECR1 led to increased stability of a key member of this complex, MCM2. ECR1 also forms a stable complex with the cyclin-dependent kinase (CDK)-related kinase, T. gondii Crk5 (TgCrk5), which displays a similar cell cycle expression and localization during tachyzoite replication. Importantly, the localization of ECR1/TgCrk5 in the centrocone indicates that this Apicomplexa-specific spindle compartment houses important regulatory factors that control the parasite cell cycle.