Tachyzoite Invasion Research Articles

Toxoplasma gondii surface antigen 1 (SAG1) interacts in vitro with host cell receptor for activated C kinase 1 (RACK1)

Toxoplasma gondii (T. gondii) surface antigen 1 (SAG1) is crucial for tachyzoite invasion into host cells. However, the role of SAG1 in interaction with host cells remains unknown. The primary objective of this study was to analyze and validate the interaction between SAG1 and host cells. RACK1, an intracellular multifunctional protein, was identified as a SAG1 binding partner in host cells. Furthermore, the expression of RACK1 is manipulated by SAG1, and depletion of RACK1 negatively regulated host cell viability. These results imply that through interaction with RACK1, SAG1 preserves the viability of host cells to satisfy the survival needs of T. gondii. Our findings suggest a novel role for SAG1 in intracellular parasitism.

Extracellular H2O2, peroxiredoxin, and glutathione reductase alter Neospora caninum invasion and proliferation in Vero cells

Neospora caninum is a protozoan member of the Apicomplexa phylum and is closely connected with abortion in cattle. The development of the parasite in host cells is characterized by the active secretion of proteins, allied to the tight control of the redox status. In this sense, elucidating the mechanisms related to the role of the redox agents and enzymes during the invasion and proliferation of N. caninum may contribute to developing novel forms of neosporosis control. In this study we verified the effects of the recombinant forms of N. caninum glutathione reductase (rNcGR) and thioredoxin-dependent peroxide reductase (rNcPrx), as well as H2O2 in the tachyzoite invasion and proliferation. rNcPrx interfered in the N. caninum invasion in a redox state manner. Oxidized rNcPrx inhibited the N. caninum invasion and proliferation with no toxic effects observed in Vero cells. In contrast, lower concentrations of H2O2 (10 μM) stimulated the N. caninum invasion, which was reverted in higher doses (>100 μM). H2O2 inhibited the parasite proliferation in lower concentrations than cytotoxicity in host cells, resulting in a positive selectivity index (1.8). Besides, rNcPrx (reduced and non-reduced) and rNcGR inhibited the parasite proliferation without affecting the host cell. Our results indicate the connection between the N. caninum development and the redox state, contributing to the elucidation of parasite propagation and control mechanisms.

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.

Two old drugs, NVP-AEW541 and GSK-J4, repurposed against the Toxoplasma gondii RH strain

BackgroundToxoplasma gondii is a zoonotic pathogen that causes toxoplasmosis and leads to serious public health problems in developing countries. However, current clinical therapeutic drugs have some disadvantages, such as serious side effects, a long course of treatment and the emergence of drug-resistant strains. The urgent need to identify novel anti-Toxoplasma drugs has initiated the effective strategy of repurposing well-characterized drugs. As a principled screening for the identification of effective compounds against Toxoplasma gondii, in the current study, a collection of 666 compounds were screened for their ability to significantly inhibit Toxoplasma growth.MethodsThe inhibition of parasite growth was determined using a luminescence-based β-galactosidase activity assay. Meanwhile, the effect of compounds on the viability of host cells was measured using CCK8. To assess the inhibition of the selected compounds on discrete steps of the T. gondii lytic cycle, the invasion, intracellular proliferation and egress abilities were evaluated. Finally, a murine infection model of toxoplasmosis was used to monitor the protective efficacy of drugs against acute infection of a highly virulent RH strain.ResultsA total of 68 compounds demonstrated more than 70% parasite growth inhibition. After excluding compounds that impaired host cell viability, we further characterized two compounds, NVP-AEW541 and GSK-J4 HCl, which had IC50 values for parasite growth of 1.17 μM and 2.37 μM, respectively. In addition, both compounds showed low toxicity to the host cell. Furthermore, we demonstrated that NVP-AEW541 inhibits tachyzoite invasion, while GSK-J4 HCl inhibits intracellular tachyzoite proliferation by halting cell cycle progression from G1 to S phase. These findings prompted us to analyse the efficacy of the two compounds in vivo by using established mouse models of acute toxoplasmosis. In addition to prolonging the survival time of mice acutely infected with T. gondii, both compounds had a remarkable ability to reduce the parasite burden of tissues.ConclusionsOur findings suggest that both NVP-AEW541 and GSK-J4 could be potentially repurposed as candidate drugs against T. gondii infection.

The Putative TCP-1 Chaperonin Is an Important Player Involved in Sialic Acid-Dependent Host Cell Invasion by Toxoplasma gondii.

Host cell invasion by Toxoplasma gondii is crucial for the survival and proliferation of parasite. The process of T. gondii tachyzoite invasion requires interaction between parasite proteins and receptors on the surface of host cells. Sialic acid is one of the important receptors for host cell invasion by T. gondii. However, the parasite-derived proteins interacting with sialic acid have not been well characterized. In this study, a novel protein named putative TCP-1 chaperonin (TGME49_318410) in T. gondii (TgTCP-1) was targeted and characterized. TgTCP-1 protein colocalized with MIC3 protein, which could be secreted from T. gondii tachyzoites, and this protein showed a specific binding activity to sialic acid, and DC and Vero cells in vitro. The binding of TgTCP-1 protein to DC and Vero cells were inhibited by either pre-incubation with free sialic acid or neuraminidase treatment of the cells. Moreover, a significant reduction of T. gondii invasion in Vero cells was observed after pre-incubation of the cells with recombinant TgTCP-1 protein. These results illustrated that TgTCP-1 is an important molecule involved in sialic acid-dependent host cell invasion by T. gondii.

Protein O-fucosyltransferase 2–mediated O-glycosylation of the adhesin MIC2 is dispensable for Toxoplasma gondii tachyzoite infection

Toxoplasma gondii is a ubiquitous, obligate intracellular eukaryotic parasite that causes congenital birth defects, disease in immunocompromised individuals, and blindness. Protein glycosylation plays an important role in the infectivity and evasion of immune responses of many eukaryotic parasites and is also of great relevance to vaccine design. Here we demonstrate that micronemal protein 2 (MIC2), a motility-associated adhesin of T. gondii, has highly glycosylated thrombospondin repeat (TSR) domains. Using affinity-purified MIC2 and MS/MS analysis along with enzymatic digestion assays, we observed that at least seven C-linked and three O-linked glycosylation sites exist within MIC2, with >95% occupancy at these O-glycosylation sites. We found that addition of O-glycans to MIC2 is mediated by a protein O-fucosyltransferase 2 homolog (TgPOFUT2) encoded by the TGGT1_273550 gene. Even though POFUT2 homologs are important for stabilizing motility-associated adhesins and for host infection in other apicomplexan parasites, loss of TgPOFUT2 in T. gondii had only a modest impact on MIC2 levels and the wider parasite proteome. Consistent with this, both plaque formation and tachyzoite invasion were broadly similar in the presence or absence of TgPOFUT2. These findings indicate that TgPOFUT2 O-glycosylates MIC2 and that this glycan, in contrast to previous findings in another study, is dispensable in T. gondii tachyzoites and for T. gondii infectivity.

Extracellular Trap Formation by Donkey Polymorphonuclear Neutrophils Against Toxoplasma gondii

Toxoplasma (T.) gondii is an obligatory intracellular apicomplexan parasite. The donkey is one of the intermediate hosts of T. gondii. There are almost no data about T. gondii infection in donkeys, apart from a few seroprevalence reports. The aim of the study was to detect the formation of extracellular traps by donkey polymorphonuclear neutrophils (PMNs) when exposed to T. gondii tachyzoites in vitro. Tachyzoites were observed to be entrapped within extracellular traps (NET) structures. Myeloperoxidase, neutrophil elastase (NE), and histone (H3) were observed in NET structures. NE and reactive oxygen species activity increased with time and was higher in the PMN-tachyzoite cocultures. Some tachyzoites were seen close localization to the nucleus of neutrophils. Degranulation and extracellular traps were observed simultaneously in some donkey neutrophils after incubation for 1 hour. The tachyzoite invasion rate decreased in PMN-tachyzoite cocultures in comparison to the controls. NETs can negatively affect the viability of entrapped tachyzoites in addition to their action of physical entrapment. NE may play a key role in the viability of T. gondii in donkeys. The amount of extracellular DNA increased with incubation time in the PMN-tachyzoite cocultures (P < .001). This is a first report regarding the formation of donkeys NETs after exposure to T. gondii tachyzoites in vitro. Unlike sheep and cattle PMN, degranulation and extracellular traps developed in the same neutrophil. Further studies focusing on signaling pathways may shed light on what determines the behavior of donkey neutrophils after exposure to T. gondii.

T91. DEVELOPMENT OF NOVEL BIS-AMIDINES FOR THE TREATMENT OF TOXOPLASMOSIS

BackgroundToxoplasma infections constitute a worldwide public health problem responsible for significant morbidity. Symptoms of acute infection by the agent, the apicomplexan parasite Toxoplasma gondii, can range from mild to life-threatening depending upon the immune status of the host. Toxoplasma infections are often unrecognized in immune competent hosts but infection can lead to the long-term establishment of cysts in the brain and thus a persistent infection. Such tissue cysts are associated with altered behavior and cognition in mouse models. Furthermore, serological evidence of Toxoplasma infection has been associated with an increased risk of recent onset psychosis, schizophrenia, and other psychiatric disorders in humans. Adjunct treatment of Toxoplasma-seropositive individuals afflicted with schizophrenia with effective anti-parasitic medications could be beneficial by eliminating the parasite cysts, thereby possibly alleviating psychiatric symptoms. However, currently available medications are ineffective against the cyst form of the organism responsible for persistent infection. Others have published the anti-Toxoplasma potential of derivatives of the bis-amidine pentamidine. We designed and synthesized a panel of novel bis-amidines and explored their anti-Toxoplasma efficacy.MethodsWe designed and synthesized an array of twelve novel bis-amidines. These compounds were examined for in vitro activity against T. gondii tachyzoites first using a colorimetric assay employing the β-gal producing strain RH-2F to examine the effect of the compounds over 5 days of parasite growth in human fibroblast host cells. This was followed by several immunofluorescence-based assays to determine if compounds can directly act on tachyzoites (red/green invasion assay), on an established infection of host cells (replication assay), and whether the compounds are parasiticidal, ie. can rid the host cells of tachyzoite infection after one dose (recovery assay). A rodent model of acute toxoplasmosis was established for examining in vivo efficacy of lead compounds.ResultsFour of these compounds proved highly efficacious in vitro with 50% inhibitory (IC50) values ranging from 200 nM to 1.3 µM. Therapeutic indices based on the ratio between the median cell cytotoxic dose and the IC50 ranged from 4 to 84. These four compounds all inhibited tachyzoite invasion and significantly inhibited in vitro replication over a 24-hour period at nanomolar concentrations. Additionally, two of the compounds evaluated thus far appear to be parasiticidal in vitro at 1 - 2 µM.DiscussionOur results suggest that bis-amidines can be designed to be effective against experimental Toxoplasma infections. The parasiticidal activity of some of the compounds make them serious candidates for further drug development. Further experiments to determine 1) in vivo efficacy in mouse models of persistent infection, and 2) synergy with antipsychotics and mood stabilizers will investigate the possibility that these compounds could be used as adjunct treatment in Toxoplasma-positive individuals suffering with schizophrenia and other psychiatric disorders.

Differential susceptibility of bovine caruncular and trophoblast cell lines to infection with high and low virulence isolates of Neospora caninum

BackgroundNeospora caninum, one of the main causes of abortion in cattle, is very effective at crossing the placental barrier and placental damage is crucial in the pathogenesis of abortion. Bovine trophoblast and caruncular cell layers are key cellular components in the maternal-foetal interface in placentomes, playing a fundamental role in placental functionality.MethodsWe studied tachyzoite adhesion, invasion, proliferation and egress of high- (Nc-Spain7) and low- (Nc-Spain1H) virulence N. caninum isolates in established cultures of bovine caruncular epithelial (BCEC-1) and trophoblast (F3) cells. The parasite invasion rate (pInvR) and the cell infection rate (cInfR) were determined by immunostaining plaque assay at different time points and multiplicities of infection (MOIs), respectively. In addition, tachyzoite growth kinetics were investigated using real-time PCR (qPCR) analysis and immunostaining plaque assay at different times.ResultsNeospora caninum invaded and proliferated in both cell lines. The pInvR was higher in F3 compared to BCEC-1 cells for the Nc-Spain7 isolate (P < 0.05), and higher for the Nc-Spain7 than the Nc-Spain1H in F3 cells (P < 0.01). The cInfR was also higher in F3 cells than in BCEC-1 cells for both isolates (P < 0.0001), and the cInfR for the Nc-Spain7 isolate was higher than for the Nc-Spain1H isolate in both cell lines (P < 0.05). Tachyzoite growth kinetics showed tachyzoite exponential growth until egress at 58 hpi for both isolates in F3, whereas Nc-Spain1H showed a non-exponential growth pattern in BCEC-1. Asynchronous egress of both isolates was observed from 22 h post-infection onwards in BCEC-1. In addition, the tachyzoite yield (TY58h) was higher in F3 than in BCEC-1 infected by both isolates (P < 0.0001), highlighting better replication abilities of both parasites in F3. Nc-Spain7 showed shorter doubling times and higher TY58h compared to Nc-Spain1H in F3 cells; adhesion, invasion and proliferation mechanisms were very similar for both isolates in BCEC-1.ConclusionsOur results indicate a highly similar behavior of high- and low-virulence isolates in their interactions with maternal caruncular cells and suggest an important role of foetal trophoblasts in the pathogenesis of N. caninum infection.

Characterization of a Toxoplasma gondii calcium calmodulin-dependent protein kinase homolog

BackgroundToxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa and a major pathogen of animals and immunocompromised humans, in whom it causes encephalitis. Understanding the mechanism of tachyzoite invasion is important for the discovery of new drug targets and may serve as a model for the study of other apicomplexan parasites. We previously showed that Plasmodium falciparum expresses a homolog of human calcium calmodulin-dependent protein kinase (CaMK) that is important for host cell invasion. In this study, to identify novel targets for the treatment of Toxoplasma gondii infection (another apicomplexan parasite), we sought to identify a CaMK-like protein in the T. gondii genome and to characterize its role in the life-cycle of this parasite.MethodsAn in vitro kinase assay was performed to assess the phosphorylation activities of a novel CaMK-like protein in T. gondii by using purified proteins with various concentrations of calcium, calmodulin antagonists, or T. gondii glideosome proteins. Indirect immunofluorescence microscopy was performed to detect the localization of this protein kinase by using the antibodies against this protein and organellar maker proteins of T. gondii.ResultsWe identified a novel CaMK homolog in T. gondii, T. gondii CaMK-related kinase (TgCaMKrk), which exhibits calmodulin-independent autophosphorylation and substrate phosphorylation activity. However, calmodulin antagonists had no effect on its kinase activity. In T. gondii-infected cells, TgCaMKrk localized to the apical ends of extracellular and intracellular tachyzoites. TgCaMKrk phosphorylated TgGAP45 for phosphorylation in vitro.ConclusionsOur data improve our understanding of T. gondii motility and infection, the interaction between parasite protein kinases and glideosomes, and drug targets for protozoan diseases.

Characterization of the Neospora caninum NcROP40 and NcROP2Fam-1 rhoptry proteins during the tachyzoite lytic cycle.

Virulence factors from the ROP2-family have been extensively studied in Toxoplasma gondii, but in the closely related Neospora caninum only NcROP2Fam-1 has been partially characterized to date. NcROP40 is a member of this family and was found to be more abundantly expressed in virulent isolates. Both NcROP2Fam-1 and NcROP40 were evaluated as vaccine candidates and exerted a synergistic effect in terms of protection against vertical transmission in mouse models, which suggests that they may be relevant for parasite pathogenicity. NcROP40 is localized in the rhoptry bulbs of tachyzoites and bradyzoites, but in contrast to NcROP2Fam-1, the protein does not associate with the parasitophorous vacuole membrane due to the lack of arginine-rich amphipathic helix in its sequence. Similarly to NcROP2Fam-1, NcROP40 mRNA levels are highly increased during tachyzoite egress and invasion. However, NcROP40 up-regulation does not appear to be linked to the mechanisms triggering egress. In contrast to NcROP2Fam-1, phosphorylation of NcROP40 was not observed during egress. Besides, NcROP40 secretion into the host cell was not successfully detected by immunofluorescence techniques. These findings indicate that NcROP40 and NcROP2Fam-1 carry out different functions, and highlight the need to elucidate the role of NcROP40 within the lytic cycle and to explain its relative abundance in tachyzoites.

The distribution pattern of α2,3- and α2,6-linked sialic acids affects host cell preference in Toxoplasma gondii

Tachyzoites of Toxoplasma gondii, an obligate intracellular parasite, actively invade almost all types of nucleated cells. However, T. gondii tachyzoites preferentially infect particular types of animal tissue cells. The mechanism underlying the host cell preference of T. gondii is not yet known. In this study, we found that enzymatic removal of α2,3- but not α2,6-linked sialic acids on the surface of Vero cells decreased T. gondii tachyzoite adhesion or invasion to the treated cells. Although Chinese hamster ovary (CHO) cells express only α2,3-linked sialic acid, a genetically modified CHO cell line constructed by transfection with the α2,6-sialiltransferase gene contains subpopulations with a variety of expression patterns of α2,3- and α2,6-linked sialic acids. When T. gondii tachyzoites were added to the modified CHO cells, the tachyzoites preferentially attached to cells belonging to a subpopulation of cells that highly expressed α2,3-linked sialic acids. Additionally, multiple regression analysis performed to analyse the relationship between the amount of α2,3-linked/α2,6-linked sialic acids and parasite-expressed fluorescence intensity suggested that more tachyzoites adhered to individual α2,3-linked sialic acid rich-cells than to α2,3-linked sialic acid-poor/null cells. The results of confocal laser microscopy confirmed this finding. These results indicate that the host cell preference of T. gondii was, at least partially, affected by the distribution pattern of α2,3-, but almost never α2,6-linked sialic acids on host cells.

Host cell invasion by apicomplexan parasites: the junction conundrum.

Host cell invasion by apicomplexan parasites: the junction conundrum.

Intestinal epithelial barrier dysfunction protects against pathogen translocation during initial infection (1111.2)

Myosin light chain kinase (MLCK) is essential for pathophysiological epithelial tight junction regulation. Transgenic mice with intestinal epithelial‐specific expression of constitutively‐active MLCK (CA‐MLCK) demonstrate increased permeability and susceptibility to immune‐mediated colitis. However, the effect of barrier dysfunction on enteric infection remains unknown. Within 30 min after mucosal application of DsRed‐labeled Salmonella typhimurium (SL3201 108 CFU), CA‐MLCK mice exhibited a 5‐fold reduction in bacterial translocation compared to wildtype (WT). This protection also occurred 1 h after oral infection with Toxoplasma gondii, in which tachyzoite invasion was reduced in CA‐MLCK mice relative to WT, 1.5±0.6 vs 4.0±1.0 tachyzoite/mm2 villus, respectively. Intraepithelial lymphocytes expressing the γδ T‐cell receptor (γδ IEL) protect against pathogen invasion. Time‐lapse confocal microscopy revealed enhanced γδ IEL surveillance of the villous epithelium in CA‐MLCK mice compared to WT with more rapid migration (4.2±0.1 vs 3.8±0.1μm/min) and reduced localization to the lateral intercellular space (26±1.7 vs 32±3.3%). However, loss of γδ T cells in CA‐MLCK mice did not alter Salmonella transmigration compared to WT. These data indicate that increased γδ IEL epithelial surveillance is not sufficient for the reduced pathogen invasion observed during early infection in CA‐MLCK mice.Grant Funding Source: Supported by NIH DK61931 and DK68271, UChicago DDRCC, Gastro‐intestinal Research Foundation

Rapid cytoskeleton remodelling in dendritic cells following invasion byToxoplasma gondiicoincides with the onset of a hypermigratory phenotype

Host cell manipulation is an important feature of the obligate intracellular parasite Toxoplasma gondii. Recent reports have shown that the tachyzoite stages subvert dendritic cells (DC) as a conduit for dissemination (Trojan horse) during acute infection. To examine the cellular basis of these processes, we performed a detailed analysis of the early events following tachyzoite invasion of human monocyte-derived DC. We demonstrate that within minutes after tachyzoite penetration, profound morphological changes take place in DC that coincide with a migratory activation. Active parasite invasion of DC led to cytoskeletal actin redistribution with loss of adhesive podosome structures and redistribution of integrins (CD18 and CD11c), that concurred with the onset of DC hypermotility in vitro. Inhibition of parasite rhoptry secretion and invasion, but not inhibition of parasite or host cell protein synthesis, abrogated the onset of morphological changes and hypermotility in DC dose-dependently. Also, infected DC, but not by-stander DC, exhibited upregulation of C-C chemokine receptor 7 (CCR7). Yet, the onset of parasite-induced DC hypermotility preceded chemotactic migratory responsesin vitro. Collectively, present data reveal that invasion of DC by T. gondii initiates a series of regulated events, including rapid cytoskeleton rearrangements, hypermotility and chemotaxis, that promote the migratory activation of DC.

Toxoplasma gondii inhibits mast cell degranulation by suppressing phospholipase Cγ-mediated Ca2+ mobilization

Toxoplasma gondii is well-known to subvert normal immune responses, however, mechanisms are incompletely understood. In particular, its capacity to alter receptor-activated Ca2+-mediated signaling processes has not been well-characterized. In initial experiments, we found evidence that T. gondii infection inhibits Ca2+ responses to fMetLeuPhe in murine macrophages. To further characterize the mechanism of inhibition of Ca2+ mobilization by T. gondii, we used the well-studied RBL mast cell model to probe the capacity of T. gondii to modulate IgE receptor-activated signaling within the first hour of infection. Ca2+ mobilization that occurs via IgE/FcεRI signaling leads to granule exocytosis in mast cells. We found that T. gondii inhibits antigen-stimulated degranulation in infected cells in a strain-independent manner. Under these conditions, we found that cytoplasmic Ca2+ mobilization, particularly antigen-mediated Ca2+ release from intracellular stores, is significantly reduced. Furthermore, stimulation-dependent activation of Syk kinase leading to tyrosine phosphorylation and activation of phospholipase Cγ is inhibited by infection. Therefore, we conclude that inhibitory effects of infection are likely due to parasite-mediated inhibition of the tyrosine kinase signaling cascade that results in reduced hydrolysis of phosphatidylinositol 4,5-bisphosphate. Interestingly, inhibition of IgE/FcεRI signaling persists when tachyzoite invasion is arrested via cytochalasin D treatment, suggesting inhibition is mediated by a parasite-derived factor secreted into the cells during the invasion process. Our study provides direct evidence that immune subversion by T. gondii is initiated concurrently with invasion.

Toxoplasma Gondii Targets the Host Actin Cytoskeleton during Invasion, GO Figure

T. gondii belongs to the phylum apicomplexa, which also includes Plasmodium, the agent of malaria. T. gondii quickly establishes infection as a fast-replicating form, the tachyzoite, which invades numerous cell types throughout the body. The parasite can cause encephalitis and neurologic diseases. Unlike viruses and intracellular bacteria, Toxoplasma actively penetrates the host cell without using typical host-uptake pathways that involve coated pits or actin based engulfment. During invasion, the extracellular parasite triggers the assembly of a unique zoite-cell junction and in less than 20 sec progressively propels its ∼ 2um long body through the junction into the growing vacuole. How the growing PV and its parasite passes through the barrier of the host cell cortical actin remains unknown. Here we provide evidence that Toxofilin, an actin-binding protein secreted by the invading parasites into the host cell cytoplasm at the onset of invasion, targets the host cortical actin cytoskeleton to facilitate vacuole folding and tachytozoites invasion. Indeed, Toxoplasma tachyzoites lacking toxofilin exhibit delayed invasion kinetics. Correlative light and electron tomography allowed tracking toxofilin in action within the host cell and showed that integrity of the host cortical actin cytoskeleton was disrupted at the site of parasite entry, suggesting that toxofilin induces actin depolymerization in close proximity to the zoite apex at the onset of invasion. Quantitative fluorescent speckle microscopy indicates that toxofilin facilitate tachyzoite invasion by upregulating host cortical actin filament turnover, consistent with toxofilin monomer sequestration and barbed end filament-capping capabilities in vitro. The ability of toxofilin to expedite invasion by regulating the turnover of cortical actin filaments in the host cell is likely to confer a selective advantage to the parasite. Delorme-Walker et al, J Cell Sci. 2012 Jul 5. [Epub ahead of print] PubMed

Transcriptomic Analysis of Toxoplasma Development Reveals Many Novel Functions and Structures Specific to Sporozoites and Oocysts

Sexual reproduction of Toxoplasma gondii occurs exclusively within enterocytes of the definitive felid host. The resulting immature oocysts are excreted into the environment during defecation, where in the days following, they undergo a complex developmental process. Within each oocyst, this culminates in the generation of two sporocysts, each containing 4 sporozoites. A single felid host is capable of shedding millions of oocysts, which can survive for years in the environment, are resistant to most methods of microbial inactivation during water-treatment and are capable of producing infection in warm-blooded hosts at doses as low as 1–10 ingested oocysts. Despite its extremely interesting developmental biology and crucial role in initiating an infection, almost nothing is known about the oocyst stage beyond morphological descriptions. Here, we present a complete transcriptomic analysis of the oocyst from beginning to end of its development. In addition, and to identify genes whose expression is unique to this developmental form, we compared the transcriptomes of developing oocysts with those of in vitro-derived tachyzoites and in vivo-derived bradyzoites. Our results reveal many genes whose expression is specifically up- or down-regulated in different developmental stages, including many genes that are likely critical to oocyst development, wall formation, resistance to environmental destruction and sporozoite infectivity. Of special note is the up-regulation of genes that appear “off” in tachyzoites and bradyzoites but that encode homologues of proteins known to serve key functions in those asexual stages, including a novel pairing of sporozoite-specific paralogues of AMA1 and RON2, two proteins that have recently been shown to form a crucial bridge during tachyzoite invasion of host cells. This work provides the first in-depth insight into the development and functioning of one of the most important but least studied stages in the Toxoplasma life cycle.

A4D12 monoclonal antibody recognizes a new linear epitope from SAG2A Toxoplasma gondii tachyzoites, identified by phage display bioselection

Toxoplasma gondii surface is coated by closely related antigens that belong to SRS (SAG-1 related sequences) superfamily. Two tachyzoite-specific SRS antigens, SAG1 and SAG2, are immunodominant proteins that apparently modulate the virulence of infection by inducing the host immune response against tachyzoites during the acute phase. In this study, we described a conformationally insensitive monoclonal antibody (A4D12mAb) that recognizes a linear epitope shared by two isoforms of p22 that is expressed in the surface of T. gondii tachyzoites. By using phage display approach and production of recombinant proteins, we clearly demonstrated that the A4D12mAb recognizes an epitope within C-terminal region of SAG2A. This mAb reacts with both T. gondii genotypes (I and II) but not with a closely related parasite, Neospora caninum. Also, the pretreatment of tachyzoites with A4D12 mAb did not inhibit T. gondii infection, suggesting that the epitope herein mapped is not crucial for tachyzoite invasion. However, a panel of human T. gondii positive sera showed significant degree of inhibition of A4D12 mAb reactivity against T. gondii native antigens, indicating that both A4D12 mAb and human sera recognize an overlapping immunodominant epitope within C-terminal region of SAG2A. To our knowledge, this is the first evidence using bioselection by phage display that identifies a T. gondii linear epitope recognized by a mAb specific to SAG2A. In conclusion, the results here presented add a new piece of information concerning T. gondii SAG2A molecule, emphasizing two dissimilar biological roles of this molecule, particularly for A4D12 epitope, suggesting that these characteristics may be important for parasite survival, since it is part of parasite components able to induce a strong immune response enough to allow host survival and establish long-term chronic infection.

Invasion of human retinal vascular endothelial cells by Toxoplasma gondii tachyzoites

Background:Toxoplasma gondii infection is a leading cause of posterior uveitis. Human retinal endothelial cells (HREC) are more susceptible to infection with T gondii tachyzoites than other subpopulations of endothelial cells....