Host Cell Penetration of Toxoplasma gondii

The host cell penetration of Toxoplasma gondii was studied with reference to synthesis of a penetration-enhancing factor and the penetrative capacity of the parasites during the various phases of the infectious cycle. HeLa cells as suspension or monolayer cultures were infected with Toxoplasma. After a lag phase shorter than 1 hr, there was a synthesis of penetration-enhancing factor demonstrable in the parasites. Exposure of the factor to host cells resulted in disappearance of its penetration-enhancing activity. The findings were in accord with the hypothesis that Toxoplasma gondii is capable of synthesizing an enzyme-like factor, reacting with the host cell surface, and, thereby, facilitating the penetration of the parasites. This hypothesis was further supported by the observations that the penetrative capacity of extracellular parasites gradually was reduced in presence of host cells and that intracellular parasites released artificially from host cells shortly after penetration were found to have a reduced penetrative capacity. Parasites allowed to remain for 4 hr or more intracellularly could restore and markedly improve their penetrative capacity.     

Cell free synthesis of Toxoplasma gondii antigens

Functionally active poly(A)-containing mRNA was isolated from tachyzoites of the RH strain of Toxoplasma gondii. The T. gondii mRNA was capable of directing the synthesis of proteins in a wheat germ in vitro translation system, but not in a cell free system derived from rabbit reticulocyte lysate. Efficient translation in the wheat germ system required spermine and exogenous tRNA. Amino acid incorporation was maximal at 110 mM K+ and 1.8 mM Mg2+. Tachyzoite antigens synthesized in vitro were immunoprecipitated with T. gondii antibodies from rabbits, mice and humans. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitated polypeptides yielded patterns that differed according to antibody source, but all T. gondii antibody preparations reacted with a translation product with an apparent molecular weight of 24 000.     

Immunological Study on the Host Cell Penetration Factor of Toxoplasma gondii

The host cell penetration factor (PEF) of Toxoplasma gondii was studied by biochemical and immunological techniques. Sephadex gel filtration of an ammonium sulfate-precipitated PEF yielded two components with different molecular weight, but both having penetration-enhancing activity. The methods for purification removed at least 99.9% of extraneous protein. For demonstration of a significant enhancement of penetration, 0.001 μg of protein was sufficient. Biochemically, they appeared to be acid proteins with the same electrophoretic mobility. Both components showed maximal enhancement of penetration at pH 7.6 and 37 C. PEF antisera reduced the penetrative capacity of Toxoplasma parasites. The penetration-enhancing effect of the two components of PEF was inhibited by antiserum against any of them. Moreover, immunologically identical immunoprecipitates were obtained when antiserum reacted with the two components. The results thus indicated that the two components of PEF were immunogenically identical and that the difference in molecular weights might result from aggregation. Immunofluorescence indicated that PEF was related to cytoplasmic structures located in the anterior end of Toxoplasma. A possible relation between these structures and the paired organelle or the convoluted tubes was discussed. The number of parasites with immunofluorescence was low shortly after host cell penetration and increased during the intracellular life of the parasites after kinetics, previously observed for synthesis of PEF as well as for lysosomal activity of Toxoplasma.     

Cell surface heparan sulfate promotes replication of Toxoplasma gondii

Previous work suggests that cell surface heparan sulfate acts as a receptor for the Apicomplexan parasite Toxoplasma gondii. Using Chinese hamster ovary cell mutants defective in heparan sulfate biosynthesis, we show that heparan sulfate is necessary and sufficient for infectivity. Further, we demonstrate that the parasite requires N sulfation of heparan sulfate initiated by N-deacetylase/N-sulfotransferase-1, but 2-O sulfation and 6-O sulfation appear to be dispensable. In order to study the role of heparan sulfate in other cell types, we created a conditional allele for N-deacetylase/N-sulfotransferase-1 by using Cre-loxP technology. Mammary tumor cells lacking N-deacetylase/N-sulfotransferase-1 exhibited reduced toxoplasma infectivity like Chinese hamster ovary cell mutants. Surprisingly, heparin, chemically modified heparinoids, and monoclonal antibodies to heparan sulfate had no effect on toxoplasma infection. T. gondii attachment and invasion were unchanged in N-deacetylase/N-sulfotransferase-1-inactivated cells as well, but replication was reduced. Thus, heparan sulfate does not appear to function as a receptor for T. gondii but instead facilitates parasite replication postinvasion.     

Autophagy activated by Toxoplasma gondii infection in turn facilitates Toxoplasma gondii proliferation

Autophagy was found to play an antimicrobial or antiparasitic role in the activation of host cells to defend against intracellular pathogens, at the same time, pathogens could compete with host cell and take advantage of autophagy to provide access for its proliferation, but there are few articles for studying the outcome of this competition between host cell and pathogens. Therefore, the aim of our study was to investigate the relationship between autophagy activated by Toxoplasma gondii (T. gondii) and proliferation of T. gondii affected by autophagy in vitro. Firstly, human embryonic fibroblasts (HEF) cells were infected with T. gondii for different times. The monodansylcadaverine (MDC) staining, acridine orange (AO) staining, punctuate GFP-LC3 distribution, and transmission electron microscopy (TEM) assays were conducted, and the results were consistent in showing that gondii infection could induce autophagy. Secondly, HEF cells were infected with T. gondii and treated with autophagy inhibitor bafilomycin A1 or inducer lithium chloride for different times. Giemsa staining was conducted, and the results exhibited that T. gondii infection-induced autophagy could in turn promote T. gondii proliferation. Simultaneously, the results of Giemsa staining also revealed that autophagy inhibitor could reduce the number of each cell infected with T. gondii and inhibit T. gondii proliferation. In contrast, autophagy inducer could increase the number of each cell infected with T. gondii and encourage T. gondii proliferation. Therefore, our study suggests that T. gondii infection could activate autophagy, and this autophagy could in turn facilitate T. gondii proliferation in HEF cells for limiting nutrients.     

Immunology of Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite. Following oral infection the parasite crosses the intestinal epithelial barrier to disseminate throughout the body and establish latent infection in central nervous tissues. The clinical presentation ranges from asymptomatic to severe neurological disorders in immunocompromised individuals. Since the clinical presentation is diverse and depends, among other factors, on the immune status of the host, in the present review, we introduce parasitological, epidemiological, clinical, and molecular biological aspects of infection with T. gondii to set the stage for an in-depth discussion of host immune responses. Since immune responses in humans have not been investigated in detail the present review is exclusively referring to immune responses in experimental models of infection. Systemic and local immune responses in different models of infection are discussed, and a separate chapter introduces commonly used animal models of infection.     

Endopolygenie bei Toxoplasma gondii

Zusammenfassung Die ungeschlechtliche Vermehrung von T. gondii in den Epithelzellen des Katzendarms setzt mit einer zweifachen Teilung des Schizonten-Zellkerns ein, ohne die Ausbildung der bei der Endodyogenie bekannten Organellen. Erst im vierkernigen Stadium beginnt, unter Erhaltung des Mutterkonoids, die Entwicklung von Merozoiten. Dieser Vorgang verläuft nach dem Muster der Endodyogenie, führt jedoch nicht zur Ausbildung von nur 2, sondern von vermutlich 32 Tochterindividuen.Daher wird dieser Vermehrungsmodus, der eine Abwandlung der Endodyogenie darstellt, von uns Endopolygenie genannt.

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Endopolygeny on Toxoplasma gondii

Summary The asexual multiplication of T. gondii in the epithelial cells of cat intestine starts with a division of the cell nucleus of the schizont without the formation of the organelles known in endodyogeny. The development of merozoites begins at the four-nucleus-stage; in this case the mother conoids are maintained. This development follows the pattern of endodyogeny but produces 32 daughter cells instead of 2.Therefore this form of multiplication, which is related to endodyogeny, is called by us endopolygeny.

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Vorgetragen auf dem 2. Internationalen Kongreß für Parasitologie, September 1970 in Washington D.C.     

Complex Immune Cell Interplay in the Gamma Interferon Response during Toxoplasma gondii Infection

Toxoplasma gondii is an obligate intracellular parasite of clinical importance, especially in immunocompromised patients. Investigations into the immune response to the parasite found that T cells are the primary effector cells regulating gamma interferon (IFN-γ)-mediated host resistance. However, recent studies have revealed a critical role for the innate immune system in mediating host defense independently of the T cell responses to the parasite. This body of knowledge is put into perspective by the unifying theme that immunity to the protozoan parasite requires a strong IFN-γ host response. In the following review, we discuss the role of IFN-γ-producing cells and the signals that regulate IFN-γ production during T. gondii infection.     

Genomic drift of Toxoplasma gondii

 We review herein studies concerning the genomic polymorphism of Toxoplasma gondii including 3 clones (1 linked with mouse pathogenicity), 5 zymodemes, and 13 schizodemes. Because mutations occur with some frequency and several allelic configurations are present in isolates grown in the same environment, we conclude that many of the mutations may not be affected by selection pressure. However, the gametocyte-forming ability is under selection pressure from the host and depends on the development of bradyzoites in tissue cysts. After prolonged multiplication exclusively in the tachyzoite stage in mice and, possibly, in patients the gametocyte-forming ability may be lost. To avoid this genomic change, isolates should be passed in the laboratory, permitting bradyzoite and tissue-cyst formation. Mouse pathogenicity is selected for during mouse passage. We find no major genomic instability justifying species or subspecies distinctions. Received: 13 July 1996 / Accepted: 2 August 1996     

Die Endodyogenie bei Toxoplasma gondii

Zusammenfassung Auf Grund systematischer elektronenmikroskopischer Untersuchungen an Serienschnitten durch die Vermehrungsformen der Toxoplasmen innerhalb des sog. Proliferations-Stadiums und des sog. Cysten-Stadiums konnte die Art der Vermehrung in den einzelnen Phasen beschrieben und belegt werden. Als einzige Vermehrungsart von Toxoplasma gondii fand sich die Endodyogenie. Beweisende Bilder für eine Vermehrung durch Zweiteilung oder Schizogonie ergaben sich bisher nicht.Eine besondere Rolle bei der Endodyogenie dürfte ein elektronendichter Kernkörper spielen, der durch histochemische Untersuchungen indirekt als DNS-Substanz angesprochen wurde und die Bezeichnung E-Körper (= Endodyogenie-) erhielt. Dieser E-Körper übernimmt die führende Rolle am Anfang der Endodyogenie. Der E-Körper wird zu Beginn der Endodyogenie aus dem mütterlichen Kern vorgeschoben, teilt sich und bildet in der weiteren Entwicklung den Grundstock für die Tochterzellkerne. Der mütterliche Zellkern wird anscheinend nicht vollständig zwischen den Tochterzellen aufgeteilt.

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Endodyogeny of Toxoplasma gondiiA morphologic analysis

Summary With electronmicroscopic studies of series sections a systematic investigation has been made of the method of reproduction of Toxoplasma gondii. Both, the so-termed proliferation and cyst stages could be closely followed and the various phases of the reproduction process plotted and described. Without a doubt, Toxoplasma gondii reproduce by means of endodyogeny. No electron micrographs have lent support to the concept of binary fission or schizogony.A special role in the process of endodyogeny seems to be played by an electrondense body of the nucleus, which upon histochemical examination has been identified as a DNA substance and been given the term E-body (E = Endodyogeny). The E-body is the instigating factor of endodyogeny. At the start of the process it protrudes itself from the maternal nucleus, divides and forms in the course of further development the matrix for the daughter-cell nuclei. The maternal nucleus, apparently, does not become fully distributed between the daughter cells.

     

Die Vermehrung von Toxoplasma gondii

Zusammenfassung Durch Anwendung des Mitosegiftes Colchicin konnte festgestellt werden, daß Toxoplasma gondii sich mitotisch teilt, 6 Chromosomen besitzt und sich einwandfrei durch Längsteilung, die ausschließlich intracellulär erfolgt, vermehrt. Ähnliche Verhältnisse finden sich bei Entwicklungsstadien gewisser Sporozoen.Mit Unterstützung der Deutschen Forschungsgemeinschaft.     

A novel rhoptry protein in Toxoplasma gondii bradyzoites and merozoites

The secretory organelles of Toxoplasma gondii orchestrate invasion of the host cell and establish the parasitophorous vacuole. Although much has been learned about the roles played by these organelles in invasion by the tachyzoite stage, little is known about the contents or functions of these organelles during bradyzoite development or pathogenesis. We identified a novel protein that localizes to the rhoptries of the bradyzoite stage, but is absent from the tachyzoite stage. This protein, BRP1, first appears in the nascent rhoptries during the first division of bradyzoite stage development. We observed secretion of BRP1 and other rhoptry proteins into the parasitophorous vacuole during bradyzoite development in vitro, but there was no evidence that this occurs in vivo. Brp1 knockout parasites did not appear to have any developmental or growth defects in vitro, and were able to establish infections in mice both as tachyzoites (via intraperitoneal injection of in vitro-derived tachyzoites) or bradyzoites (via oral gavage using cysts harvested from mouse brain). Mice infected using brain cysts from the brp1 knockout or the control strain developed similar numbers and sizes of brain cysts. Thus BRP1 does not appear to play an essential role in development of the bradyzoite stage, development of brain cysts, or oral infection of new hosts, at least in the mouse model used here. Since we also observed that BRP1 is expressed in the merozoite stages in the gut of infected cats, the coccidian phase of the life cycle may be where BRP1 plays its most important role.     

Strain-specific antigens of Toxoplasma gondii.

A detailed immunological assessment of strain-specific antigens of Toxoplasma gondii has not been reported. We developed rabbit antisera against three strains of toxoplasma obtained from divergent sources. These strains included the frequently studied laboratory strain RH, strain C, obtained from a naturally infected kitten, and strain P, which is maintained by passage in mice. The rabbit antisera were used to identify unique strain-specific and commonly shared tachyzoite antigens by radioiodination followed by immunoprecipitation and Western blot analysis. Both qualitative and quantitative differences of a number of the major tachyzoite antigens were found in these assays. A parasite plaque reduction assay using parasiticidal monoclonal antibody showed marked differences in the ability to kill these three different tachyzoite subtypes, further supporting antigenic variation among T. gondii strains.