Attachment of Toxoplasma gondii to a specific membrane fraction of CHO cells

We have observed previously that attachment of Toxoplasma gondii to synchronized host cells is considerably increased at the mid-S phase (4 h postrelease). Synchronized CHO host cells at the mid-S phase were fractionated by molecular weight, and the antigens were used to produce a panel of polyclonal mouse antisera. The polyclonal antisera raised against fraction 4 with molecular mass ranging approximately from 18 to 40 kDa significantly reduced attachment to mid-S-phase host cells. Immunofluorescence assays demonstrated strong reactivity to mid-S-phase host cells and identified a number of potential receptors on Western blots. These data indicate that there is a specific host membrane receptor for parasite attachment that is upregulated during the mid-S phase of the host cell cycle.     

Defining the cell cycle for the tachyzoite stage of Toxoplasma gondii.

Tachyzoite endodyogeny is characterized by a three phase cell cycle comprised of major G1 and S phases with mitosis following immediately upon the conclusion of DNA replication. Cytokinesis, which begins with the formation of daughter apical complexes, initiates in late S phase and overlaps mitosis. There is no evidence to support an extended G2 period in these parasites. In all strains, parasites with a 2 N DNA content are a relatively small subpopulation and when tachyzoites expressing a fluorescent nuclear marker (green-fluorescent-protein fused to proliferating-cell-nuclear-antigen) were observed by time-lapse microscopy, there appeared to be little delay between S phase and mitosis. Measurements of the DNA content of RH parasites by flow cytometry demonstrated that the G1 and S periods were approximately 60 and approximately 30% of a single division cycle, although these phases were longer in strains that display a slower growth rate. The overall length of S phase was determined by [3H]-thymidine autoradiography using transgenic parasites expressing herpes simplex thymidine kinase and validated by Northern analysis of S phase specific genes during synchronous growth. The fraction of S phase parasites by flow cytometry paralleled autoradiography, however, within S phase, the distribution of parasites was bimodal in all strains examined. Parasites containing a 1-1.7 N DNA complement were a small fraction when compared to the major S phase population which contained a near-diploid ( approximately 1.8 N) complement, suggesting parasites in late S phase have a slower rate of DNA replication. In lieu of a short or missing G2, where checkpoints are thought to operate in other eukaryotes, the bimodal replication of tachyzoite chromosomes may represent a distinct premitotic checkpoint associated with endodyogeny.     

Inhibition of Toxoplasma gondii growth by pyrrolidine dithiocarbamate is cell cycle specific and leads to population synchronization

Successful completion of the Toxoplasma cell cycle requires the coordination of a series of complex and ordered processes that results in the formation of two daughters by internal budding. Although we now understand the order and timing of intracellular events associated with the parasite cell cycle, the molecular details of the checkpoints that regulate each step in Toxoplasma gondii division is still uncertain. In other eukaryotic cells, the use of cytostatic inhibitors that are able to arrest replication at natural checkpoints have been exploited to induce synchronization of population growth. Herein, we describe a novel method to synchronize T. gondii tachyzoites based on the reversible growth inhibition by the drug and pyrrolidine dithiocarbamate. This method is an improvement over other strategies developed for this parasites as no prior genetic manipulation of the parasite was required. RH tachyzoites blocked by pyrrolidine dithiocarbamate exhibited a near uniform haploid DNA content and single centrosome indicating that this compound arrests parasites in the G1 phase of the tachyzoite cell cycle with a minor block in late cytokinesis. Thus, these studies support the existence of a natural checkpoint that regulates passage through the G1 period of the cell cycle. Populations released from pyrrolidine dithiocarbamate inhibition completed progression through G1 and entered S phase approximately 2 h post-drug release. The transit of drug-synchronized populations through S phase and mitosis followed a similar timeframe to previous studies of the tachyzoite cell cycle. Tachyzoites treated with pyrrolidine dithiocarbamate were fully viable and completed two identical division cycles post-drug release demonstrating that this is a robust method for synchronizing population growth in Toxoplasma.     

Transmission and scanning electron microscopy of host cell entry by Toxoplasma gondii.

Entry by tachyzoites of Toxoplasma gondii, the causative agent of toxoplasmosis, into peritoneal cells was investigated with transmission and scanning electron microscopy. The process of entry is initiated by the parasite contacting the host cell with its anterior end, creating a small depression in the plasmalemma of the host cell. Occasionally, a small portion of the host cell cytoplasm protrudes and contaccts the anterior end of the parasite. A cylindrical structure (35 nm in diameter) extends from the pellicle of the parasite to the host cell. Such structures appear to assist host cell entry by T. gondii. As the entry process progresses, pseudopods of the host cell surround theparasite and finally T gondii becomes intracellular, being located in a vacuole separated from the host cell cytoplasm by a unit membrane. (Am J. Pathol 87:285-296, 1977).     

Toxoplasma gondii homologue of plasmodium apical membrane antigen 1 is involved in invasion of host cells

Proteins with constitutive or transient localization on the surface of Apicomplexa parasites are of particular interest for their potential role in the invasion of host cells. We describe the identification and characterization of TgAMA1, the Toxoplasma gondii homolog of the Plasmodium apical membrane antigen 1 (AMA1), which has been shown to elicit a protective immune response against merozoites dependent on the correct pairing of its numerous disulfide bonds. TgAMA1 shows between 19% (Plasmodium berghei) and 26% (Plasmodium yoelii) overall identity to the different Plasmodium AMA1 homologs and has a conserved arrangement of 16 cysteine residues and a putative transmembrane domain, indicating a similar architecture. The single-copy TgAMA1 gene is interrupted by seven introns and is transcribed into an mRNA of approximately 3.3 kb. The TgAMA1 protein is produced during intracellular tachyzoite replication and initially localizes to the micronemes, as determined by immunofluorescence assay and immunoelectron microscopy. Upon release of mature tachyzoites, TgAMA1 is found distributed predominantly on the apical end of the parasite surface. A approximately 54-kDa cleavage product of the large ectodomain is continuously released into the medium by extracellular parasites. Mouse antiserum against recombinant TgAMA1 blocked invasion of new host cells by approximately 40%. This and our inability to produce a viable TgAMA1 knock-out mutant indicate that this phylogenetically conserved protein fulfills a key function in the invasion of host cells by extracellular T. gondii tachyzoites.     

Toxoplasma gondii uses sulfated proteoglycans for substrate and host cell attachment

Toxoplasma gondii is an obligate intracellular parasite that actively invades a wide variety of vertebrate cells, although the basis of this pervasive cell recognition is not understood. We demonstrate here that binding to the substratum and to host cells is partially mediated by interaction with sulfated glycosaminoglycans (GAGs). Addition of excess soluble GAGs blocked parasite attachment to serum-coated glass, thereby preventing gliding motility of extracellular parasites. Similarly, excess soluble GAGs decreased the attachment of parasites to human host cells from a variety of lineages, including monocytic, fibroblast, endothelial, epithelial, and macrophage cells. The inhibition of parasite attachment by GAGs was observed with heparin and heparan sulfate and also with chondroitin sulfates, indicating that the ligands for attachment are capable of recognizing a broad range of GAGs. The importance of sulfated proteoglycan recognition was further supported by the demonstration that GAG-deficient mutant host cells, and wild-type cells treated enzymatically to remove GAGs, were partially resistant to parasite invasion. Collectively, these studies reveal that sulfated proteoglycans are one determinant used for substrate and cell recognition by Toxoplasma. The widespread distribution of these receptors may contribute to the broad host and tissue ranges of this highly successful intracellular parasite.     

IP-10 is critical for effector T cell trafficking and host survival in Toxoplasma gondii infection

The generation of an adaptive immune response against intracellular pathogens requires the recruitment of effector T cells to sites of infection. Here we show that the chemokine IP-10, a specific chemoattractant for activated T cells, controls this process in mice naturally infected with Toxoplasma gondii. Neutralization of IP-10 in infected mice inhibited the massive influx of T cells into tissues and impaired antigen-specific T cell effector functions. This resulted in >1000-fold increase in tissue parasite burden and a marked increase in mortality compared to control antibody-treated mice. These observations suggest that IP-10 may play a broader role in the localization and function of effector T cells at sites of Th1 inflammation.     

A Toxoplasma gondii rhoptry protein associated with host cell penetration has unusual charge asymmetry.

The monoclonal antibody Tg49 both recognizes a Toxoplasma gondii rhoptry protein (ROP1) and inhibits penetration enhancing factor. The latter is a proteinaceous factor found in Toxoplasma lysates or conditioned media that increases the efficiency with which parasites invade host cells. Tg49 was used to screen a lambda gt11 cDNA library and the clone obtained was identified as the cognate gene for ROP1 by several criteria: (1) recombinant protein reacted with the monoclonal; (2) antiserum against the recombinant reacted with the same bands on Western blots as did Tg49; and (3) antiserum against the recombinant recognized a protein in the rhoptries. The ROP1 gene is a single copy gene with a message of approximately 2.1 kb. The predicted polypeptide sequence of ROP1 shows an unusual charge and amino acid asymmetry. There is a highly acidic, proline-rich domain in the amino-terminal portion of the predicted protein, followed by a strongly basic carboxy-terminal domain. An octapeptide repeat is found almost midway through the peptide sequence toward the end of the acidic domain. The ROP1 gene was expressed in a bacterial system, and the resulting polypeptide exhibited anomalous migration on polyacrylamide gel electrophoresis. Given that Tg49 inhibits penetration enhancing factor, it seems likely that the ROP1 protein is a component of that factor, and that the unusual sequence of this protein plays some role in host cell penetration by T. gondii.     

Protein phosphorylation during the process of interaction of Toxoplasma gondii with host cells

Previous studies have shown that tachyzoites of Toxoplasma gondii were able to penetrate into macrophages using two mechanisms: phagocytosis and active penetration. We show here that previous incubation of the macrophages or the parasites with staurosporine, a wide range inhibitor of protein kinases, tyrfostin and genistein, specific inhibitors of tyrosine kinases, significantly interfered with the process of parasite-macrophage interaction. Staurosporine treatment induced the formation of many filopodium-like surface projections of the macrophages and markedly increased the attachment of the tachyzoites to the cell surface. Genistein inhibited about 50% penetration of T. gondii into macrophages. Previous incubation of tachyzoites with genistein also significantly inhibited their attachment to and penetration into the macrophages. Confocal laser scanning microscopy was used to locate phosphoproteins in macrophages interacting with tachyzoites. Antiphosphotyrosine antibodies labeled the surface of macrophages, but not L929 cells, incubated in presence of T. gondii, even those cells did not show associated parasites. Anti phosphotyrosine, phosphothreonine and phosphoserine antibodies labeled the region surrounding the parasitophorous vacuoles. These observations suggest that protein phosphorylation is a key event in the process of T. gondii-host cell interaction.     

Toll-like receptors and their role in host resistance to Toxoplasma gondii

Toxoplasma gondii and other apicomplexan parasites are widely distributed obligate intracellular protozoa. A critical host mediator produced in response to T. gondii infection is IL-12. This cytokine is synthesized by dendritic cells, macrophages and neutrophils and plays a pivotal role in the production of IFN-gamma, which in turn activates anti-microbial effector cells. In the past several years, many of the receptors and signaling pathways that link pathogen detection to induction of IL-12 have been identified and characterized. Among these receptors the Toll-like receptor (TLR) family can recognize all classes of pathogens and induce different types of immune responses. In the following review, I summarize the evidence for specific TLR function in host resistance to T. gondii.     

A change in the premitotic period of the cell cycle is associated with bradyzoite differentiation in Toxoplasma gondii

We have demonstrated that bradyzoites return to the tissue-cyst stage by a developmental pathway that is indistinguishable from that initiated by sporozoites. Mature bradyzoites, like sporozoites from oocysts, were non-proliferative as they contained uniform 1N DNA contents, and replication occurred only in parasites that de-differentiated back into tachyzoites. Moreover, tachyzoites emergent from the bradyzoite-initiated pathway underwent a spontaneous growth shift prior to the onset of tissue cyst formation in a timeframe that was identical to cultures infected with sporozoites. In sporozoite-infected cultures, a novel premitotic, near-diploid subpopulation was detected during bradyzoite differentiation that co-expressed tachyzoite and bradyzoite markers. These observations suggest that activation of a G2-related cell cycle mechanism is required during bradyzoite development, and indicates that equivalent cell cycle mechanisms may govern development in the intermediate life cycle regardless of the origin of infection.     

Induction and regulation of IL-12-dependent host resistance to Toxoplasma gondii

Resistance to the intracellular protozoan parasite Toxoplasma gondii is initiated by the induction of interleukin-12 (IL-12), which stimulates interferon (IFN)-gamma synthesis by natural killer (NK) cells and T lymphocytes. This review summarizes the work of our laboratory on the mechanisms by which the parasite triggers IL-12 synthesis, and how this response is regulated to avoid the lethal effects of excessive tissue inflammation. In addition, we present an overview of our studies investigating the mechanisms by which the IFN-gamma produced as a consequence of IL-12 stimulation controls intracellular replication of the parasite in host cells.     

Calomys callosus (Rodentia: Cricetidae) trophoblast cells as host cells to Toxoplasma gondii in early pregnancy

The potential of the RH strain of Toxoplasma gondii to invade trophoblast cells of the cricetid rodent Calomys callosus in a congenital infection in the initial third of pregnancy was investigated in this study using morphological and immunocytochemical approaches. The animals were intraperitoneally inoculated on the 1st day of pregnancy and the infection was observed on day 7. Various numbers of parasites could be observed inside the parasitophorous vacuoles in trophoblastic cells under light and electron microscopy. The trophoblast cells showed characteristics of healthy cells, and no alteration other than parasite vacuoles in their cytoplasm could be detected. Polyclonal or monoclonal anti-T. gondii antibodies (respectively, anti-T. gondii components and the major surface parasite antigen p30) labeled both the parasite surface and parasitophorous vacuole membranes, regardless of the number of parasites inside the compartment. In addition, p30-containing trails were detected in the extracellular matrix surrounding trophoblastic cells similar to those found with other parasites during locomotion and the invasion process. Our results show the ability of T. gondii to infect trophoblast cells during the early blastocyst-endometrial relationship and open new possibilities for more accurate study of the invasion process of this parasite and the role of the trophoblast as an embryo defense barrier. Received: 30 December 1998 / Accepted: 7 February 1999     

Rosette-forming cells during immune response to Toxoplasma gondii in mice.

The rosette-forming cell (RFC) response of mice immunized with varying doses of Toxoplasma gondii was studied by immunocytoadherence (ICA). The specificity of ICA in the present system was tested by passive sensitization with hyperimmune serum in vivo and in vitro. A slight increase in RFC was observed with the latter. Prior treatment of spleen cells from immunized animals with rabbit anti-mouse immunoglobulin resulted in total inhibition of ICA. During the primary and the secondary response after 10 days, the number of RFC rose rapidly to reach the peak on the 3rd day. With secondary immunization 30 days later, the peak shifted to the 2nd day. Mice infected with a lower dose of Toxoplasma had a greater number of RFC during the secondary response after 10 days than with a larger dose.     

Induction and regulation of IL-12-dependent host resistance to Toxoplasma gondii

Resistance to the intracellular protozoan parasite Toxoplasma gondii is initiated by the induction of interleukin-12 (IL-12), which stimulates interferon (IFN)-γ synthesis by natural killer (NK) cells and T lymphocytes. This review summarizes the work of our laboratory on the mechanisms by which the parasite triggers IL-12 synthesis, and how this response is regulated to avoid the lethal effects of excessive tissue inflammation. In addition, we present an overview of our studies investigating the mechanisms by which the IFN-γ produced as a consequence of IL-12 stimulation controls intracellular replication of the parasite in host cells.