P-glycoprotein inhibitors modulate accumulation and efflux of xenobiotics in extra and intracellular Toxoplasma gondii

We examined xenobiotic transport and the effects of P-glycoprotein (Pgp) inhibitors on efflux function in Toxoplasma gondii tachyzoites. The fluorescence emission of JC-1 and daunorubicin (Pgp substrates) was determined in both extracellular tachyzoites and T. gondii-infected human KB cells. Dye accumulation and efflux were modulated by verapamil (Vp) and cyclosporin A (CsA), both of which are Pgp inhibitors. Red JC-1 emission was measured from 10(6) extracellular tachyzoites, using spectrofluorometry. The increase in red emission was significant from 1 microM concentration of both drugs and was higher with CsA than with Vp. Compared with untreated tachyzoites, JC-1 efflux was inhibited by 3 microM CsA and 3 microM Vp. With intracellular tachyzoites, the fluorescence distribution of daunorubicin (DNR) between the parasitophorous vacuole and the host cell was modulated by Vp and CsA. In media free of CsA and Vp, DNR emission inside intracellular tachyzoites was very weak, as observed by confocal microscopy. In the presence of CsA or Vp, DNR emission was markedly enhanced in tachyzoites but not in the whole vacuole. The modulation of DNR uptake seems to involve the tachyzoite membrane rather than the parasitophorous vacuole or host cell membranes. It suggests that Vp would inhibit the DNR efflux from intracellular tachyzoites through a transitory effect. In conclusion, these two Pgp inhibitors increase both extracellular and intracellular dye accumulation in living T. gondii, pointing to the existence of a transmembrane transport mediated by a Pgp homologue located on the parasite membrane complex.     

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.

---

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.

---

---

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

In vitro activity of cotrimoxazole on the intracellular multiplication of Toxoplasma gondii.

Cotrimoxazole, from 60 mug/ml, inhibited the replication of Toxoplasma gondii within Hela cells and mouse peritoneal macrophages. The percentage of inhibition reached practically 100% after 18 hours of treatment at 37 degrees C. More prolonged treatment resulted in an eradication of the organisms from the cell monolayers. In contrast, similar doses of spiramycin were quite ineffective against intracellular toxoplasma. The active doses of cotrimoxazole used were not harmful for cell cultures. Cotrimoxazole also destroyed clones (rosaces) of toxoplasma which were formed during the past 18 hours of intracellular replication in the absence of the drug. Trimethoprim was the only effective compound of cotrimoxazole on intracellular parasites; the adjunction of sulfamethoxazole produced a marked synergistic effect. The present findings confirm the great efficiency of cotrimoxazole in the treatment of experimental toxoplasmosis in mice performed previously in this laboratory.     

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.     

Toxoplasma gondii: redistribution of tachyzoite surface protein during host cell invasion and intracellular development

Immunoperoxidase localisation of antigen at the electron microscope level confirms that parasite surface proteins, in association with membrane, are shed from the surface of the zoite on invasion, while varying amounts are also internalised. SAG 1 is stable on intracellular zoites for up to 48h, although new protein is also synthesised. SAG1 is present on the surface of daughter zoites and is found throughout the infected cell in distinct vacuoles; these vacuoles represent either direct extensions of the parasitophorous vacuole or true export of parasite surface material.     

Interferon-gamma-activated primary enterocytes inhibit Toxoplasma gondii replication: a role for intracellular iron.

Toxoplasma gondii is an obligate intracellular parasite that infects a wide variety of nucleated cells in its numerous intermediate hosts including man. The oral route is the natural portal of entry of T. gondii. Ingested organisms are released from cysts or oocysts within the gastrointestinal tract and initially invade the intestinal epithelium. We show that T. gondii invades and proliferates in cultured primary rat enterocytes, obtained with an original procedure. Activation of the enterocytes with rat recombinant interferon-gamma (IFN-gamma) inhibits T. gondii replication, the inhibition being dose dependent. Neither nitrogen and oxygen derivatives nor tryptophan starvation appear to be involved in the inhibition of parasite replication by IFN-gamma. Experiments using Fe2+ salt, carrier and chelator indicate that intracellular T. gondii replication is iron dependent, suggesting that IFN-gamma-treated enterocytes inhibit T. gondii replication by limiting the availability of intracellular iron to the parasite. Our data show that enterocytes probably play a major role on mucosal surfaces as a first line of defence against this coccidia, and possibly other pathogens, through an immune mechanism. The results suggest that limiting the availability of iron could represent a broad antimicrobial mechanism through which the activated enterocytes exert control over intracellular pathogens.     

Interaction between Toxoplasma gondii and enterocyte

Enterocyte is the first cell to be invaded by Toxoplasma gondii when ingested parasites are released from cysts or oocysts within the gastrointestinal tract. Our data showed that the transcytotic pathway of IgA could interfere with intracellular replication of T. gondii. On another hand, IFN-gamma could activate enterocyte and inhibit the parasite replication through an iron-dependent mechanism.     

Macrophage Migration Inhibitory Factor contributes to drive phenotypic and functional macrophages activation in response to Toxoplasma gondii infection

Cytokines are small molecules secreted by numerous cells. Macrophage Migration Inhibitory Factor (MIF) is a cytokine initially described due to its function of inhibiting random macrophage migration. Currently, new functions have been described for MIF, such as stimulating inflammatory functions in response to infections by microorganisms including, Toxoplasma gondii. However, the primordial MIF function related to macrophages has been little addressed. The main purpose of the study was to recapitulate MIF function on macrophages in response to T. gondii infection. To achieve this goal, peritoneal macrophages were collected from C57BL/6WT and Mif1^-/- mice after recruitment with thioglycolate. Macrophages were cultured, treated with 4-Iodo-6-phenylpyrimidine (4-IPP), and infected or not by T. gondii for 24 h. Following this, the culture supernatant was collected for cytokine, urea and nitrite analysis. In addition, macrophages were evaluated for phagocytic activity and T. gondii proliferation rates. Results demonstrated that T. gondii infection triggered an increase in MIF production in the WT group as well as an increase in the secretion of IL-10, TNF, IFN-γ, IL-6 and IL-17 in the WT and Mif1^-/- macrophages. Regarding the comparison between groups, it was detected that Mif1^-/- macrophages secreted more IL-10 compared to WT. On the other hand, the WT macrophages produced greater amounts of TNF, IFN-γ, IL-6 and IL-17. Urea production was more pronounced in Mif1^-/- macrophages while nitrite production was higher in WT macrophages. T. gondii showed a greater ability to proliferate in Mif1^-/- macrophages and these cells also presented enhanced phagocytic activity. In conclusion, T. gondii infection induces macrophage activation inciting cytokine production. In presence of MIF, T. gondii infected macrophages produce pro-inflammatory cytokines compatible with the M1 activation profile. MIF absence caused a dramatic reduction in pro-inflammatory cytokines that are balanced by increased levels of urea and anti-inflammatory cytokines. These macrophages presented increased phagocytic capacity and shared features activation with the M2 profile.     

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.

---

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.     

Roles of IFN-gamma on stage conversion of an obligate intracellular protozoan parasite,Toxoplasma gondii

IFN-gamma downregulates the stage conversion of Toxoplasma gondii (T. gondii), from bradyzoites to tachyzoites, and the expression of heat shock protein 70 (HSP70) of T. gondii (T.g.HSP70) by tachyzoites. T.g.HSP70 has been shown to downregulate NO release from macrophages and also to induce auto-HSP70 antibody formation and IL-10 secretion by VH11-JH1 B-1 cells, resulting in the suppression of host defense responses to T. gondii infection. A novel category of virulent tachyzoite stage of T. gondii expressing T.g.HSP70 (virulent tachyzoite), which indirectly manifests its pathogenicity by downregulating host defense responses, has been proposed.     

Superoxide dismutase and catalase in Toxoplasma gondii

Catalase and superoxide dismutase detected in both RH and C strain Toxoplasma gondii tachyzoites were distinctly different in electrophoretic mobility from host cell enzymes. Catalase and superoxide dismutase activity levels were similar in both Toxoplasma strains and showed narrow pH optima around 8.0. Toxoplasma superoxide dismutase was resistant to cyanide but inhibited by azide or peroxide, consistent with an iron-containing enzyme typical of protozoan parasites. These enzymes may play a role in intracellular survival; however, they do not appear to be the basis for differences in virulence to mice.