Toxoplasma gondii-Vertebrate Cell Interactions. II. The Intracellular Reproductive Phase

SYNOPSIS.
The reproduction of Toxoplasma gondii RH-strain in vertebrate cells was studied in a controlled-environment culture system. The lag period before reproduction and the doubling time of individual parasites were determined using a least-squares linear regression method of analysis which does not artificially constrain the data. In the majority of cases, the time intercept of the linear regression line was either zero, implying the lack of a lag phase before reproduction, or negative, implying the parasite had completed part of its reproductive cycle before entering the host cell. The mean doubling time of T. gondii is 10.9 h in bovine embryo skeletal muscle cells and 8.3 h in HeLa cells. This difference is not significant at the 5% level. The population doubling times of mouse-derived parasites is best described by a gamma distribution.     

Trypanosoma cruzi: interaction with vertebrate cells in vitro. IV. Environmental temperature effects

The effects of 4 environmental temperatures (29, 32, 35, and 38 C) on the interaction between Trypanosoma cruzi and bovine embryo skeletal muscle cells were quantified. Three aspects of the interaction (penetration of host cells by trypomastigotes, the lag period prior to the reproductive phase, and the reproductive phase) were markedly affected by temperature. There was a linear increase in the number of trypomastigotes penetrating cells in the 29–35 C range. Temperatures above 35 C can be considered supraoptimal as no further increase in the rate of penetration occurred. The lag period decreased linearly as temperature increased in the 29–35 C range; at 38 C, the lag period was markedly shortened. The doubling time of amastigotes increased linearly as temperature increased in the 32–38 C range; at 29 C, the doubling time was markedly lengthened. At all temperatures, parasites reproduced for 9 generations before cell rupture. The changes in lag period and doubling time complemented each other in the 32–38 C range. Thus, there was essentially no change in the overall length of the intracellular cycle which lasted 6.1 to 6.5 days. At 29 C, however, the cycle was lengthened to 8.9 days. Thermodynamic analysis revealed marked differences, characterized by a negative activation energy and negative enthalpy, between the reproductive phase of parasites within vertebrate cells and the vertebrate cells themselves. However, the thermodynamic parameters of parasites reproducing extracellularly in liquid medium and intracellularly were the same.     

Heterotrophic nutrition of the marine pennate diatom Navicula pavillardi Hustedt.

Navicula pavillardi Hustedt, a marine, littoral, pennate diatom, can grow in the dark on glutamate or on the complex organic supplements tryptone or yeast extract. Growth on glutamate in the dark took place without an initial lag phase, whereas growth on tryptone began only after a 2-day lag phase that could be abolished by the simultaneous presence of glucose. Lactate inhibited growth in the dark on glutamate, but not photoautotrophic growth. Relatively low concentrations of glutamine inhibited photoautotrophic growth. The observed doubling time for heterotrophic growth on glutamate or tryptone was about 70 h, compared with a doubling time of 24 h under optimal photoautotrophic conditions. Glucose did not decrease the doubling time in the dark on tryptone. The assimilation efficiency for glutamate was 41%. The estimated necessary uptake rate for glutamate to account for the observed heterotrophic doubling time on glutamate was close to those measured with isotope techniques. The kinetic parameters for glutamate uptake, which followed Michelis-Menten kinetics, were Ks = 0.018 mM, and Vmax = 7.0 X 10(-10) mumol per cell per minute. Although several amino acids served as sole nitrogen sources for photoautotrophic growth and were demonstrated by the use of isotope techniques to enter the cells, they could not be used as substrates for growth in the dark. Glucose was not taken up to a significant extent except by cells grown in the presence of tryptone. Lactate was taken up only by dark-grown cells. Results of preliminary studies on the metabolic fate of several uniformly labeled amino acids are presented.     

Hydroxyurea inhibits intracellular Toxoplasma gondii multiplication

Tachyzoites of Toxoplasma gondii multiply within the parasitophorous vacuole (PV) until the lysis of the host cell. This study was undertaken to evaluate the effect of hydroxyurea (a specific drug that arrests cell division at G1/S phase) on the multiplication of T. gondii tachyzoites in infected Vero cells. Infected host cells were treated with hydroxyurea for periods varying from 5 to 48 h, and the survival and morphology of the parasite were determined. Hydroxyurea arrested intracellular T. gondii multiplication in all periods tested. After 48 h of incubation with hydroxyurea, intracellular parasites were not easily observed in Vero cells. Ultrastructural observations showed that infected host cells treated with hydroxyurea for 24 h or more presented disrupted intracellular parasites within the PV. However, the host cells exhibited a normal morphology. Our observations suggest that hydroxyurea was able to interfere with the cycle of the intracellular parasite, leading to the complete destruction of the T. gondii without affecting the host cells.     

Synthesis of ribosomal and transfer ribonucleic acids in yeast during a nutritional shift-up.

The growth rate of Saccharomyces cerevisiae was increased by adding a mixture of amino acids to cultures containing proline as the sole nitrogen source. The transition from balanced growth in the basal medium (doubling time 4 h) to balanced growth in the enriched medium (doubling time 2 h) took about 2-5 h. The rate of RNA accumulation increased soon after the enrichment to almost its final value. This increase began after a short lag of 10 to 15 min, therefore synthesis of new RNA polymerase molecules may be required before stable RNA production can increase. The different stable RNA species were not stimulated at different times after the enrichment, but all increased continuosly throughout the transition. The rRNA species accumulated in a co-ordinate fashion at a rate faster than the rate of tRNA accumulation.     

Toxoplasma gondii in primary rat CNS cells: differential contribution of neurons, astrocytes, and microglial cells for the intracerebral development and stage differentiation.

The central nervous system (CNS) of the intermediate host plays a central role in the lifelong persistence of Toxoplasma gondii as well as in the pathogenesis of congenital toxoplasmosis and reactivated infection in immunocompromised patients. In order to analyze the parasite-host interaction within the CNS, the host cell invasion, the intracellular replication, and the stage conversion from tachyzoites to bradyzoites was investigated in mixed cultures of dissociated CNS cells from cortices of Wistar rat embryos. Two days post infection (p.i.) with T. gondii tachyzoites, intracellular parasites were detected within neurons, astrocytes, and microglial cells as assessed by double immunofluorescence and confocal microscopy. Quantitative analyses revealed that approximately 10% of neurons and astrocytes were infected with T. gondii, while 30% of the microglial cells harbored intracellular parasites. However, the replication of T. gondii within microglial cells was considerably diminished, since 93% of the parasitophorous vacuoles (PV) contained only one to two parasites which often appeared degenerated. This toxoplasmacidal activity was not abrogated after treatment with NO synthase inhibitors or neutralization of IFN-gamma production. In contrast, 30% of the PV in neurons and astrocytes harbored clearly proliferating parasites with at least four to eight parasites per vacuole. Four days p.i. with tachyzoites of T. gondii, bradyzoites were detected within neurons, astrocytes, and microglial cells of untreated cell cultures. However, the majority of bradyzoite-containing vacuoles were located in neurons. Spontaneous differentiation to the bradyzoite stage was not inhibited after addition of NO synthase inhibitors or neutralization of IFN-gamma. In conclusion, our results indicate that intracerebral replication of T. gondii as well as spontaneous conversion from the tachyzoite to the bradyzoite stage is sustained predominantly by neurons and astrocytes, whereas microglial cells may effectively inhibit parasitic growth within the CNS.     

Susceptibility of Eimeria bovis and Toxoplasma gondii to oxygen intermediates and a new mathematical model for parasite killing

Eimeria bovis and Toxoplasma gondii differ in their susceptibility to macrophages activated by lymphokines. Interferon-gamma can activate macrophages to totally inhibit E. bovis sporozoite development, whereas growth of T. gondii tachyzoites in macrophages is not totally affected. The susceptibility of these parasites to oxygen intermediates and their ability to evade the oxidative burst by macrophages were investigated in cell-free systems. Using a logistic model to assess growth inhibition, T. gondii growth was impaired by 50% at 10(-4.25) M (56 microM) H2O2, with 30 min as the optimum time for measuring inhibition. Preliminary results indicate that T. gondii follows mode-one and mode-two killing with relation to time after exposure to H2O2, implying a role for OH. and the induction of a DNA repair mechanism. The same model was used to assess inhibition of E. bovis growth that was more susceptible, being inhibited to 50% by 10(-5) M (10 microM) H2O2. Both parasites were susceptible to the effects of xanthine-xanthine oxidase that releases a full complement of oxygen intermediates (H2O2, OH., (1)O2, and O2-). Adding quenchers or scavengers to the system confirmed that T. gondii was susceptible to products of the interaction of O2- and H2O2 (OH. and (1)O2), and that E. bovis sporozoites were at least partially susceptible to H2O2 and O2-, but extremely susceptible to OH.. These data were supported by studies on scavenging enzymes present in the parasites. Toxoplasma gondii was rich in superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPO), and E. bovis had less catalase and SOD.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth of and competition between Neospora caninum and Toxoplasma gondii in vitro

Competitive interactions between Neospora caninum and Toxoplasma gondii were studied because both species appear to have identical ecological niches in vitro. Tachyzoites of N. caninum (NC-1 isolate) and T. gondii (RH isolate) were compared in three in vitro studies: (1) rate of penetration of host cells; (2) generation time; and (3) competition between the two species when grown together in the same flask and allowed to compete for space. When tachyzoites of the two species were inoculated onto human foreskin fibroblasts, 3.24-times more N. caninum tachyzoites penetrated cells by 1 h p.i. At 3 h p.i., there were 2.87-times more N. caninum intracellular tachyzoites than T. gondii tachyzoites. The generation times for N. caninum (NC-1 isolate) and T. gondii (RH isolate) were approximately 14-15 h and 8-10 h, respectively. Before exponential growth occurred, both species displayed a lag period, which was 10-12 h for N. caninum and 8-10 h for T. gondii. To observe competition, equal numbers of tachyzoites of each species were mixed and inoculated into flasks of host cells, and the monolayers were allowed to proceed to >90% lysis before the next transfer. Competition was analysed for 31 days by labelling samples of each flask with a species-specific monoclonal antibody and determining the ratio of each species. In all trials, T. gondii outcompeted N. caninum. By 4 days p.i., 70% of the tachyzoites were T. gondii; this percentage increased to 97% by 23 days p.i. When the starting inoculum contained 75% N. caninum and 25% T. gondii tachyzoites, T. gondii was still competitively superior. When infected monolayers that were labelled with T. gondii-specific antibodies were examined, it was noted that both species can occupy and undergo endodyogeny in the same host simultaneously.     

An enzyme-release assay for the assessment of the lytic activities of complement or antimicrobial peptides on extracellular Toxoplasma gondii

A method is described which allows the evaluation of the membrane lytic activity of either complement or antimicrobial peptides against the extracellular stage of the human protozoan parasite Toxoplasma gondii. The assay is based on lacZ transgenic parasites, determining the activity of released cytoplasmic beta-galactosidase into the culture supernatant upon membrane disintegration. This method was used to evaluate the lytic activities of (i) complement which is a natural defense mechanism in infected hosts against extracellular parasites, and (ii) antimicrobial peptides which have not been evaluated against T. gondii before. The results show that the assay provides a simple and convenient way to assess the membrane lytic activity of such compounds and that T. gondii, like other protozoan parasites, is vulnerable to the membrane-lytic effect of antimicrobial peptides.     

Functions of the various IgG Fc receptors in mediating killing of Toxoplasma gondii.

The three types of IgG FcR (Fc gamma RI, Fc gamma RII, Fc gamma RIII) on human leukocytes play an important role in elimination of antibody-coated infectious agents. To further understand the role of the different Fc gamma R in mediating this killing, we examined the ability of human myeloid and lymphoid cells to kill the protozoan Toxoplasma gondii in the presence of antitoxoplasma IgG or bispecific antibodies. Although human myeloid cells (monocytes, macrophages, neutrophils, and eosinophils) all lysed unsensitized T. gondii, killing by these cells was significantly enhanced by opsonization with antitoxoplasma rabbit IgG. Human lymphocytes, however, did not lyse T. gondii unless the parasites were coated with antibody. The role of antibody and Fc gamma R in mediating ADCC of T. gondii was then examined using bispecific antibodies made by chemically cross-linking Fab fragments of antitoxoplasma antibodies to Fab fragments of antibodies specific for human leukocyte surface Ag, including Fc gamma R. Thus, simultaneous binding of these bispecifics to parasites and effector cells allowed an evaluation of killing when T. gondii were targeted to each Ag independently. Bispecifics which targeted T. gondii to Fc gamma RI, II or III enhanced lysis by monocytes. However, similar results were obtained with bispecifics targeting T. gondii to non-Fc gamma R Ag (CD11b or beta 2-microglobulin) on monocytes. Likewise, polymorphonuclear leukocytes mediated significantly more lysis in the presence of bispecifics linking T. gondii to Fc gamma RII, Fc gamma RIII, or the two non-Fc gamma R Ag CD11b and beta 2-microglobulin. Thus, although human myeloid cells did not require antibody-Fc gamma R triggering to kill T. gondii, antibody appeared to enhance lysis by capturing and directing the parasites to the effector cell surface. Human lymphocytes, in contrast, mediated significant lysis of T. gondii only in the presence of bispecifics targeting T. gondii to Fc gamma RIII, indicating a requirement for specific triggering of Fc gamma RIII for killing by large granular lymphocytes. Consequently, using bispecifics to compare targeting to specific Ag, both non-Fc gamma R and Fc gamma R, allowed determination of the role of antibody-Fc gamma R interactions in T. gondii killing. In addition, these studies demonstrate the potential of bispecifics in determining the role of specific Ag in killing of or infection by pathogens.     

Laser scanning cytometer-based assays for measuring host cell attachment and invasion by the human pathogen Toxoplasma gondii.

BACKGROUND: Toxoplasma gondii is among the most common protozoan parasites of humans. Both attachment to and invasion of host cells by T. gondii are necessary for infection, yet little is known about the molecular mechanisms underlying these processes. T. gondii's etiological importance and its role as a model organism for studying invasion in related parasites necessitate a means to quantitatively assay host cell attachment and invasion. METHODS: We present here Laser Scanning Cytometer (LSC)-based assays of T. gondii invasion and attachment. The invasion assay involves automated counting of invaded and non-invaded parasites, differentially labeled with distinct fluorochromes. The attachment assay compares the relative binding of differentially labeled parasites. The assays were evaluated using treatments known to decrease invasion or attachment. RESULTS: The LSC-based assays are robust and reproducible, remove operator bias, and significantly increase the sample size that can be feasibly counted compared to other currently available microscope-based methods. In the first application of the new assays, we have shown that parasites attach to fixed and unfixed host cells using different mechanisms. CONCLUSIONS: The LSC-based assays represent useful new methods for quantitatively measuring attachment and invasion by T. gondii, and can be readily adapted to study similar processes in other host-pathogen systems.     

Antibody dependent cell-mediated cytotoxicity against Trypanosoma cruzi.

This paper describes in vitro antibody dependent cytotoxicity against Trypanosoma cruzi epimastigotes by normal mouse splenic lymphocytes. Cytotoxicity was expressed as the percentage reduction in the number of motile parasites upon incubation with lymphocytes at 37 degrees C in a defined medium. Failure of the non-motile parasites to regain motility and their ensuing degeneration of 28 degrees C in liver infusion tryptose (LIT) medium confirmed loss of motility as a criterion of cytotoxicity. Incubation of T. cruzi cruzi at 37 degrees C for 18 h in a defined medium per se did not interfere with motility but was followed by a lag phase of the growth curve in LIT medium at 28 degrees C. The lag phase was prolonged for T. cruzi which had previously been incubated at 37 degrees C in the absence of cells.     

Kinetics of the initial rounds of cell division of Toxoplasma gondii

RH strain Toxoplasma gondii tachyzoites that had naturally lysed their host cells were allowed to infect new host cells for a limited amount of time; subsequent parasite cell divisions were observed closely. On the basis of 4 independent trials, the estimated time to first cell division was 9.8 hr postinfection (PI) and was quite variable (95% confidence interval [CI]: 3.1-16.5 hr PI). The estimated time to second cell division was 14.9 hr PI and was less variable (95% CI: 12.1-17.7 hr PI). Few parasites divided before 6 hr PI in these 4 trials. When tachyzoites were derived by forced lysis (scraping an infected host cell culture and passing it through 27-gauge needles), the first parasite cell division occurred much more rapidly than had been observed in any of the trials with parasites derived by natural lysis. When parasites derived by forced lysis were held away from host cells for 3 hr PI, the first cell division was delayed in a manner similar to that seen in parasites derived by natural lysis. No differences were observed in the timing of the second cell division of parasites derived by forced lysis whether or not they had been held away from cells. These studies demonstrate that the conditions to which tachyzoites are exposed during transit from one host cell to another can affect the kinetics of parasite cell division in the new host cell.     

Comprehension of attachment and multiplication properties by observing individual cell behaviors in anchorage-dependent culture

The behavioral properties of cell attachment and division were characterized by direct observation of individual cells in the culture of murine fibroblasts. At the cell attachment stage in the culture for early 10 h, the extent of cell spreading, which was defined as a ratio of the projected area of each cell against its saturated value, had a relatively broad distribution at 0.25 h, and it shifted to a higher level with elapsed time up to 10 h with narrowing in the distribution. The critical value of the extent of cell spreading was determined to be 0.54 as a threshold at which a cell is assumed to complete its adhesion to culture surface. The ratio of the number of cells with the extent of cell spreading over 0.54 against the total number of examined cells fairly followed the profile of cell adhesion which was obtained by measuring the number of adherent cells on culture surface.

At the cell growth stage in the culture for 20–64 h, doubling time of cell population increased gradually as the culture progressed toward confluence. Generation times (or cell-dividing spans) of individual cells, however, did not show a discriminating dependency on cell concentration and culture time. To clarify the influence of local congestion on the cell division, the generation time was formulated as a function of the number of contact cells around each target cell. Applying the cell placement growth model to estimating the extent of contact inhibition, the reciprocal value of doubling time could be correlated with the average of reciprocal generation times, implying that the doubling time on a cell-population basis is explained by considering the variation in dividing spans of individual cells affected by local contact environment.     

Toxoplasma gondii actively remodels the microtubule network in host cells

Toxoplasma gondii infection triggers host microtubule rearrangement and organelle recruitment around the parasite vacuole. Factors affecting initial stages of microtubule remodeling are unknown. To illuminate the mechanism, we tested the hypothesis that the parasite actively remodels host microtubules. Utilizing heat-killed parasites and time-lapse analysis, we determined microtubule rearrangement requires living parasites and is time dependent. We discovered a novel aster of microtubules (MTs) associates with the vacuole within 1h of infection. This aster lacks the concentrated foci of gamma (gamma)-tubulin normally associated with MT nucleation sites. Unexpectedly, vacuole enlargement does not correlate with an increase in MT staining around the vacuole. We conclude microtubule remodeling does not result from steric constraints. Using nocodazole washout studies, we demonstrate the vacuole nucleates host microtubule growth in-vivo via gamma-tubulin-associated sites. Moreover, superinfected host cells display multiple gamma-tubulin foci. Microtubule dynamics are critical for cell cycle control in uninfected cells. Using non-confluent monolayers, we show host cells commonly fail to finish cytokinesis resulting in larger, multinucleated cells. Our data suggest intimate interactions between T. gondii and host microtubules result in suppression of cell division and/or cause a mitotic defect, thus providing a larger space for parasite duplication.     

Induction of suppressor of cytokine signaling-1 by Toxoplasma gondii contributes to immune evasion in macrophages by blocking IFN-gamma signaling

Toxoplasma gondii is an intracellular parasite that survives and multiplies in professional phagocytes such as macrophages. Therefore, T. gondii has to cope with the panel of antimicrobial host immune mechanisms, among which IFN-gamma plays a crucial role. We report in this study that in vitro infection of murine macrophages with viable, but not with inactivated, parasites results in inhibition of IFN-gamma signaling within the infected cells. Thus, infection of RAW264.7 macrophages with tachyzoites inhibited IFN-gamma-induced STAT-1 tyrosine phosphorylation, mRNA expression of target genes, and secretion of NO. These effects were dependent on direct contact of the host cells with living parasites and were not due to secreted intermediates. In parallel, we report the induction of suppressor of cytokine signaling-1 (SOCS-1), which is a known feedback inhibitor of IFN-gamma receptor signaling. SOCS-1 was induced directly by viable parasites. SOCS overexpression in macrophages did not affect tachyzoite proliferation per se, yet abolished the inhibitory effects of IFN-gamma on parasite replication. The inhibitory effects of T. gondii on IFN-gamma were diminished in macrophages from SOCS-1-/- mice. The results suggest that induction of SOCS proteins within phagocytes due to infection with T. gondii contributes to the parasite's immune evasion strategies.     

In vitro multiplication of Toxoplasma gondii and Trypanosoma cruzi in mouse, rat, and hamster astrocytes

The infection and multiplication of Toxoplasma gondii and Trypanosoma cruzi were compared in primary cultures of white rat, mouse and hamster astrocytes. These cells were cultured on cover slides and infected with T. gondii tachyzoites or T. cruzi blood trypomastigotes. Results show that hamster astrocytes are more susceptible to the multiplication of both parasites than rat and mouse cells. There was no statistical difference between the T. gondii infection in rat and mouse astrocytes (p < 0.05), and this suggests an important role of other mechanisms or cells in the white rat natural resistance to this parasite. Because the hamster astrocytes are less resistant to these parasites multiplication and not necessarily to the invasion, any difference observed could be due to an intracellular effect: hamster brain astrocytes favor survival and multiplication of these parasites.