Evidence for Finely-Regulated Asynchronous Growth of Toxoplasma gondii Cysts Based on Data-Driven Model Selection

Toxoplasma gondii establishes a chronic infection by forming cysts preferentially in the brain. This chronic infection is one of the most common parasitic infections in humans and can be reactivated to develop life-threatening toxoplasmic encephalitis in immunocompromised patients. Host-pathogen interactions during the chronic infection include growth of the cysts and their removal by both natural rupture and elimination by the immune system. Analyzing these interactions is important for understanding the pathogenesis of this common infection. We developed a differential equation framework of cyst growth and employed Akaike Information Criteria (AIC) to determine the growth and removal functions that best describe the distribution of cyst sizes measured from the brains of chronically infected mice. The AIC strongly support models in which T. gondii cysts grow at a constant rate such that the per capita growth rate of the parasite is inversely proportional to the number of parasites within a cyst, suggesting finely-regulated asynchronous replication of the parasites. Our analyses were also able to reject the models where cyst removal rate increases linearly or quadratically in association with increase in cyst size. The modeling and analysis framework may provide a useful tool for understanding the pathogenesis of infections with other cyst producing parasites.     

Development of high-throughput methods to quantify cysts of Toxoplasma gondii

Toxplasma is a protozoan parasite, which forms persistent cysts in tissues of chronically infected animals and humans. Cysts can reactivate leading to severe pathologies. They also contribute to the transmission of Toxoplasma infection in humans by ingestion of undercooked meat. Classically, the quantification of cyst burden in tissues uses microscopy methods, which are laborious and time consuming. Here, we have developed automated protocols to quantify cysts, based on flow cytometry or high-throughput microscopy. Brains of rodents infected with cysts of Prugniaud strain were incubated with the FITC-Dolichos biflorus lectin and analyzed by flow cytometry and high-throughput epifluorescence microscopy. The comparison of cyst counts by manual epifluorescence microscopy to flow cytometry or to high-throughput epifluorescence microscopy revealed a good correlation (r = 0.934, r = 0.993, P < 0.001 respectively). High-throughput epifluorescence microscopy was found to be more specific and sensitive than flow cytometry and easier to use for large series of samples. This reliable and easy protocol allow the specific detection of Toxoplasma cysts in brain, even at low concentrations; it could be a new way to detect them in water and in contaminate food.     

A pathway to cure chronic infection with Toxoplasma gondii through immunological intervention

Toxoplasma gondii, an obligate intracellular protozoan parasite, can establish a chronic infection in the brain by forming tissue cysts. This chronic infection is widespread in humans worldwide including developed countries, with up to one third of the population being estimated to be infected with this parasite. Diagnosis of this chronic infection is usually conducted by serological detection of IgG antibodies against this parasite. Since infected individuals remain positive for these antibodies for years, it has generally been considered that this infection is a lifelong infection. It is also often considered that this chronic infection is “latent” or “quiescent”. However, recent discovery of the capability of perforin-dependent, CD8⁺ T cell-mediated immune responses to eliminate T. gondii cysts in collaboration with phagocytes illustrated dynamic interplays between T. gondii cysts and host immune system during this chronic infection. Importantly, the cytotoxic T cell-mediated protective immunity is able to remove mature cysts of the parasite. It is now clear that chronic T. gondii infection is not “latent” or “quiescent”. Elucidating the mechanisms of the dynamic host-pathogen interactions between the anti-cyst protective immunity and T. gondii cysts and identifying the pathway to appropriately activate anti-cyst CD8⁺ cytotoxic T cells would be able to open a door for eradicating T. gondii cysts and curing chronic infection with this parasite.     

Primary brain cell infection by Toxoplasma gondii reveals the extent and dynamics of parasite differentiation and its impact on neuron biology

Toxoplasma gondii is a eukaryotic parasite that forms latent cysts in the brain of immunocompetent individuals. The latent parasite infection of the immune-privileged central nervous system is linked to most complications. With no drug currently available to eliminate the latent cysts in the brain of infected hosts, the consequences of neurons'' long-term infection are unknown. It has long been known that T. gondii specifically differentiates into a latent form (bradyzoite) in neurons, but how the infected neuron responds to the infection remains to be elucidated. We have established a new in vitro model resulting in the production of mature bradyzoite cysts in brain cells. Using dual, host and parasite RNA-seq, we characterized the dynamics of differentiation of the parasite, revealing the involvement of key pathways in this process. Moreover, we identified how the infected brain cells responded to the parasite infection revealing the drastic changes that take place. We showed that neuronal-specific pathways are strongly affected, with synapse signalling being particularly affected, especially glutamatergic synapse signalling. The establishment of this new in vitro model allows investigating both the dynamics of parasite differentiation and the specific response of neurons to long-term infection by this parasite.     

T-cell production of matrix metalloproteinases and inhibition of parasite clearance by TIMP-1 during chronic Toxoplasma infection in the brain

Chronic infection with the intracellular protozoan parasite Toxoplasma gondii leads to tissue remodelling in the brain and a continuous requirement for peripheral leucocyte migration within the CNS (central nervous system). In the present study, we investigate the role of MMPs (matrix metalloproteinases) and their inhibitors in T-cell migration into the infected brain. Increased expression of two key molecules, MMP-8 and MMP-10, along with their inhibitor, TIMP-1 (tissue inhibitor of metalloproteinases-1), was observed in the CNS following infection. Analysis of infiltrating lymphocytes demonstrated MMP-8 and -10 production by CD4+ and CD8+ T-cells. In addition, infiltrating T-cells and CNS resident astrocytes increased their expression of TIMP-1 following infection. TIMP-1-deficient mice had a decrease in perivascular accumulation of lymphocyte populations, yet an increase in the proportion of CD4+ T-cells that had trafficked into the CNS. This was accompanied by a reduction in parasite burden in the brain. Taken together, these findings demonstrate a role for MMPs and TIMP-1 in the trafficking of lymphocytes into the CNS during chronic infection in the brain.     

MyD88 expression by CNS-resident cells is pivotal for eliciting protective immunity in brain abscesses

MyD88 KO (knockout) mice are exquisitely sensitive to CNS (central nervous system) infection with Staphylococcus aureus, a common aetiological agent of brain abscess, exhibiting global defects in innate immunity and exacerbated tissue damage. However, since brain abscesses are typified by the involvement of both activated CNS-resident and infiltrating immune cells, in our previous studies it has been impossible to determine the relative contribution of MyD88-dependent signalling in the CNS compared with the peripheral immune cell compartments. In the present study we addressed this by examining the course of S. aureus infection in MyD88 bone marrow chimaera mice. Interestingly, chimaeras where MyD88 was present in the CNS, but not bone marrow-derived cells, mounted pro-inflammatory mediator expression profiles and neutrophil recruitment equivalent to or exceeding that detected in WT (wild-type) mice. These results implicate CNS MyD88 as essential in eliciting the initial wave of inflammation during the acute response to parenchymal infection. Microarray analysis of infected MyD88 KO compared with WT mice revealed a preponderance of differentially regulated genes involved in apoptotic pathways, suggesting that the extensive tissue damage characteristic of brain abscesses from MyD88 KO mice could result from dysregulated apoptosis. Collectively, the findings of the present study highlight a novel mechanism for CNS-resident cells in initiating a protective innate immune response in the infected brain and, in the absence of MyD88 in this compartment, immunity is compromised.     

Inducible nitric oxide synthase in innate immune cells is important for restricting cyst formation of Toxoplasma gondii in the brain but not required for the protective immune process to remove the cysts

Significantly larger numbers of Toxoplasma gondii cysts were detected in the brains of RAG1^?/?NOS2^?/? than RAG1^?/? mice following infection. In contrast, the cyst numbers markedly decreased in a same manner in both strains of mice after receiving CD8⁺ immune T cells. Thus, NOS2-mediated innate immunity is important for inhibiting formation of cysts in the brain but not required for the T cell-initiated cyst removal, which is associated with phagocyte accumulation. Treatment with chloroquine, an inhibitor of endolysosomal acidification, partially but significantly inhibited the T cell-mediated cyst removal, suggesting that phagosome–lysosome fusion could be involved in the T. gondii cyst elimination.     

Chronic Toxoplasma infection modifies the structure and the risk of host behavior

The intracellular parasite Toxoplasma has an indirect life cycle, in which felids are the definitive host. It has been suggested that this parasite developed mechanisms for enhancing its transmission rate to felids by inducing behavioral modifications in the intermediate rodent host. For example, Toxoplasma-infected rodents display a reduction in the innate fear of predator odor. However, animals with Toxoplasma infection acquired in the wild are more often caught in traps, suggesting that there are manipulations of intermediate host behavior beyond those that increase predation by felids. We investigated the behavioral modifications of Toxoplasma-infected mice in environments with exposed versus non-exposed areas, and found that chronically infected mice with brain cysts display a plethora of behavioral alterations. Using principal component analysis, we discovered that most of the behavioral differences observed in cyst-containing animals reflected changes in the microstructure of exploratory behavior and risk/unconditioned fear. We next examined whether these behavioral changes were related to the presence and distribution of parasitic cysts in the brain of chronically infected mice. We found no strong cyst tropism for any particular brain area but found that the distribution of Toxoplasma cysts in the brain of infected animals was not random, and that particular combinations of cyst localizations changed risk/unconditioned fear in the host. These results suggest that brain cysts in animals chronically infected with Toxoplasma alter the fine structure of exploratory behavior and risk/unconditioned fear, which may result in greater capture probability of infected rodents. These data also raise the possibility that selective pressures acted on Toxoplasma to broaden its transmission between intermediate predator hosts, in addition to felid definitive hosts.     

Toxoplasma gondii: Flat-mounting of retina as a new tool for the observation of ocular infection in mice

Ocular toxoplasmosis is the principal cause of posterior uveitis and a leading cause of blindness. Animal models are required to improve our understanding of the pathogenesis of this disease. The method currently used for the detection of retinal cysts in animals involves the observation, under a microscope, of all the sections from infected eyes. However, this method is time-consuming and lacks sensitivity. We have developed a rapid, sensitive method for observing retinal cysts in mice infected with Toxoplasma gondii. This method involves combining the flat-mounting of retina - a compromise between macroscopic observation and global analysis of this tissue - and the use of an avirulent recombinant strain of T. gondii expressing the Escherichia coli β-galactosidase gene, visually detectable at the submacroscopic level. Single cyst unilateral infection was found in six out of 17 mice killed within 28 days of infection, whereas a bilateral infection was found in only one mouse. There was no correlation between brain cysts number and ocular infection.     

Aging and the immune response. I. Antibody formation and chronic infection in Toxoplasma gondii-infected mice

Studies were performed to determine the effect of aging on the antibody response and cyst formations after infection with a relatively avirulent strain of the intracellular parasite Toxoplasma gondii. When compared with young mice (4 months), aged mice showed a significant decrease in the magnitude of humoral immune response to infection. This decrease was observed at the peak of the acute infection and also during chronic infection. Evaluation of the presence of Toxoplasma cysts, a measure of the latent infection, revealed that the numbers of tissue cysts present 11 weeks after infection increased with the age of the mice at time of infection. The larger numbers of cysts in older mice which had received the same inoculum size of T. gondii as young mice, together with our previous observations of increased susceptibility of these older mice to T. gondii, suggest that an age-related decrease in the early immune response to this infection allows an increased multiplication of the organism in vivo, leading to increased cyst numbers or death.     

Induction of IL-10-producing regulatory B cells following Toxoplasma gondii infection is important to the cyst formation

Toxoplasma gondii is a protozoan parasite that infects humans and animals via congenital or postnatal routes. During parasite infection, IL-10-producing Bregs are stimulated as part of the parasite-induced host immune responses that favor infection. In this study, we investigated whether T. gondii infection induces immune regulatory cells including IL-10-producing CD1d^highCD5⁺ regulatory B cells (Bregs) and whether Breg induction is critical for the development of chronic infection of T. gondii. Furthermore, B cell-deficient (μMT) mice revealed that the IL-10-producing B cells might be associated with the development of chronic T. gondii infection. To better understand the mechanism underlying the accumulation of IL-10-producing B cells upon T. gondii infection, we determined the effect of products released by T. gondii on the induction and differentiation of IL-10-producing B cells during the acute stage of infection using transgenic green fluorescent protein (GFP)-expressing T. gondii strain. We demonstrated that products secreted at the stage of cell lysis by fully replicated tachyzoites induced the differentiation of naive B cells to IL-10-producing Bregs. Our results indicated that the downregulation of the immune response via Bregs during T. gondii infection is related to cyst formation in the host brain and to the establishment of chronic infection.     

Toxoplasma gondii: inconsistent dissemination patterns following oral infection in mice

Since Toxoplasma gondii is transmitted in the wild through the ingestion of infective cysts, oral infection is a preferred model for studying the natural mode of parasite dissemination and pathogenesis. Using luciferase-expressing strains of T. gondii and in vivo imaging, we observed different patterns of disease progression in mice depending of the method of oral infection. Oral gavage of infective cysts (e.g., bradyzoites) resulted in an inconsistent pattern of parasite dissemination; in the majority (20/29) of infected mice, luciferase-derived signal (indicating high numbers of Toxoplasma tachyzoites) was first observed in the right chest area. At later time points this signal spread to other parts of the mouse, including the abdominal area. In the remaining mice (9/29), parasites were first observed replicating in the abdominal area, as might be expected. In contrast, when mice were infected naturally (either via ingestion of whole brains from previously infected mice or brain cyst homogenate-soaked bread), parasites were first observed replicating in the abdominal area in all mice examined (10/10). Based on the inconsistency of infections initiated with oral gavage, it is recommended that natural feeding be used to infect mice when a consistent oral infection is desired.     

Mechanisms of innate resistance to Toxoplasma gondii infection.

The interaction of protozoan parasites with innate host defences is critical in determining the character of the subsequent infection. The initial steps in the encounter of Toxoplasma gondii with the vertebrate immune system provide a striking example of this important aspect of the host-parasite relationship. In immuno-competent individuals this intracellular protozoan produces an asymptomatic chronic infection as part of its strategy for transmission. Nevertheless, T. gondii is inherently a highly virulent pathogen. The rapid induction by the parasite of a potent cell-mediated immune response that both limits its growth and drives conversion to a dormant cyst stage explains this apparent paradox. Studies with gene-deficient mice have demonstrated the interleukin-12 (IL-12)-dependent production of interferon gamma (IFN-gamma) to be of paramount importance in controlling early parasite growth. However, this seems to be independent of nitric oxide production as mice deficient in inducible nitric oxide synthase (iNOS) and tumour necrosis factor receptor were able to control early growth of T. gondii, although, they later succumbed to infection. Nitric oxide does, however, seem to be important in controlling persistent infection; treating chronic infection with iNOS metabolic inhibitors results in disease reactivation. Preliminary evidence implicates neutrophils in effector pathways against this parasite distinct from that described for macrophages. Once initiated, IL-12-dependent IFN-gamma production in synergy with other proinflammatory cytokines can positively feed back on itself to induce ''cytokine shock''. Regulatory cytokines, particularly IL-10, are essential to down-regulate inflammation and limit host pathology.     

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma establishes a lifelong persistent infection in the brain. While this brain infection is asymptomatic in most immunocompetent people, in the developing fetus or immunocompromised individuals such as acquired immune deficiency syndrome (AIDS) patients, this predilection for and persistence in the brain can lead to devastating neurologic disease. Thus, it is clear that the brain-Toxoplasma interaction is critical to the symptomatic disease produced by Toxoplasma, yet we have little understanding of the cellular or molecular interaction between cells of the central nervous system (CNS) and the parasite. In the mouse model of CNS toxoplasmosis it has been known for over 30 years that neurons are the cells in which the parasite persists, but little information is available about which part of the neuron is generally infected (soma, dendrite, axon) and if this cellular relationship changes between strains. In part, this lack is secondary to the difficulty of imaging and visualizing whole infected neurons from an animal. Such images would typically require serial sectioning and stitching of tissue imaged by electron microscopy or confocal microscopy after immunostaining. By combining several techniques, the method described here enables the use of thick sections (160 µm) to identify and image whole cells that contain cysts, allowing three-dimensional visualization and analysis of individual, chronically infected neurons without the need for immunostaining, electron microscopy, or serial sectioning and stitching. Using this technique, we can begin to understand the cellular relationship between the parasite and the infected neuron.     

Releasing latent Toxoplasma gondii cysts from host cells to the extracellular environment induces excystation

Toxoplasma gondii, an obligate intracellular protozoan parasite, infects a wide variety of mammals and birds. Although T. gondii infects the brain and muscles in its latent cyst form containing bradyzoite stage parasites during chronic infection, when a chronically infected host becomes immunodeficient or is preyed upon by a predator, the latent cyst undergoes excystation. However, it is not yet known how T. gondii recognises the triggers of excystation in the microenvironment surrounding the cyst. In this study, we incubated T. gondii cysts from host cells in several solutions containing a variety of ionic compositions. Excystation occurred in a solution with an ionic composition which mimicked that of the extracellular environment. However, excystation did not occur in a solution that mimicked the intracellular environment. We also found that the specific Na⁺/K⁺ ratio and the presence of Ca²⁺, mimicking the extracellular environment, are required to trigger excystation. To examine whether the stage conversion of bradyzoite to tachyzoite occurs prior to egress, we constructed a gene-modified T. gondii strain expressing a green fluorescent protein specifically in the tachyzoite stage. During the process of cyst reactivation of this strain, green fluorescence was detected prior to excystation. This suggests that stage conversion from bradyzoite to tachyzoite occurs prior to cyst disruption. These results indicate that T. gondii bradyzoites monitor the ionic composition of their surroundings to recognise their expulsion from host cells, to effectively time their excystation and stage conversion.     

The immune system utilizes two distinct effector mechanisms of T cells depending on two different life cycle stages of a single pathogen,Toxoplasma gondii,to control its cerebral infection

Toxoplasma gondii takes two different life cycle stages within intermediate hosts including humans. Tachyzoites proliferate during the acute stage, and they transform into cysts to establish a chronic infection preferentially in the brain. IFN-γ production by infiltrated CD4⁺ and CD8⁺ T cells is required for the prevention of cerebral tachyzoite growth. IFN-γ production by brain-resident cells, most likely microglia, plays a key first line defense role to facilitate both innate and T cell-mediated protective immunity to control the tachyzoite growth. IFN-γ produced by brain-resident cells activates cerebral expression of IFN-dependent effector molecules to suppress tachyzoite growth during the early stage of infection. Their IFN-γ production also induces an expression of CXCL9 and CXCL10 chemokines to recruit immune T cells into the brain, and upregulates cerebral expression of MHC class I and II molecules for antigen presentation to the recruited T cells to activate their IFN-γ production. CD8⁺ T cells also have the activity to remove T. gondii cysts from the brains of infected hosts. Of interest, the anti-cyst activity of CD8⁺ T cells does not require their IFN-γ but does require perforin. Notably, we discovered that CD8⁺ cytotoxic T cells penetrate in the cysts in a perforin-mediated manner, which induces morphological deterioration and destruction of the cysts and an accumulation of microglia and macrophages for their elimination. Thus, the immune system employs two distinct effector mechanisms mediated by IFN-γ or perforin depending on two different life cycle stages of a single pathogen, T. gondii, to control its cerebral infection.     

Methods to produce and safely work with large numbers of Toxoplasma gondii oocysts and bradyzoite cysts

Two major obstacles to conducting studies with Toxoplasma gondii oocysts are the difficulty in reliably producing large numbers of this life stage and safety concerns because the oocyst is the most environmentally resistant stage of this zoonotic organism. Oocyst production requires oral infection of the definitive feline host with adequate numbers of T. gondii organisms to obtain unsporulated oocysts that are shed in the feces for 3-10 days after infection. Since the most successful and common mode of experimental infection of kittens with T. gondii is by ingestion of bradyzoite tissue cysts, the first step in successful oocyst production is to ensure a high bradyzoite tissue cyst burden in the brains of mice that can be used for the oral inoculum. We compared two methods for producing bradyzoite brain cysts in mice, by infecting them either orally or subcutaneously with oocysts. In both cases, oocysts derived from a low passage T. gondii Type II strain (M4) were used to infect eight-ten week-old Swiss Webster mice. First the number of bradyzoite cysts that were purified from infected mouse brains was compared. Then to evaluate the effect of the route of oocyst inoculation on tissue cyst distribution in mice, a second group of mice was infected with oocysts by one of each route and tissues were examined by histology. In separate experiments, brains from infected mice were used to infect kittens for oocyst production. Greater than 1.3 billion oocysts were isolated from the feces of two infected kittens in the first production and greater than 1.8 billion oocysts from three kittens in the second production. Our results demonstrate that oral delivery of oocysts to mice results in both higher cyst loads in the brain and greater cyst burdens in other tissues examined as compared to those of mice that received the same number of oocysts subcutaneously. The ultimate goal in producing large numbers of oocysts in kittens is to generate adequate amounts of starting material for oocyst studies. Given the potential risks of working with live oocysts in the laboratory, we also tested a method of oocyst inactivation by freeze-thaw treatment. This procedure proved to completely inactivate oocysts without evidence of significant alteration of the oocyst molecular integrity.     

Toxoplasma gondii: determination of the onset of chronic infection in mice and the in vitro reactivation of brain cysts

Toxoplasma gondii is an intra-cellular parasite that infects humans through vertical and horizontal transmission. The cysts remain dormant in the brain of infected humans and can reactivate in immunocompromised hosts resulting in acute toxoplasmic encephalitis which may be fatal. We determined the onset and progression of brain cysts generation in a mouse model following acute toxoplasmosis as well as the ability of brain cysts to reactivate in vitro. Male Balb/c mice, (uninfected control group, n = 10) were infected orally (study group, n = 50) with 1000 tachyzoites of T. gondii (ME49 strain) and euthanized at 1, 2, 4, 8 and 16 weeks post infection. Brain tissue was harvested, homogenized, stained and the number of brain cysts counted. Aliquots of brain homogenate with cysts were cultured in vitro with confluent Vero cells and the number of cysts and tachyzoites counted after 1 week. Brain cysts but not tachyzoites were detected at week 2 post infection and reached a plateau by week 4. In vitro Vero cells culture showed similar pattern for cysts and tachyzoites and reactivation of cyst in vitro was not influenced by the age of the brain cysts.     

Consumption of a nectar alkaloid reduces pathogen load in bumble bees

Diet has a significant effect on pathogen infections in animals and the consumption of secondary metabolites can either enhance or mitigate infection intensity. Secondary metabolites, which are commonly associated with herbivore defense, are also frequently found in floral nectar. One hypothesized function of this so-called toxic nectar is that it has antimicrobial properties, which may benefit insect pollinators by reducing the intensity of pathogen infections. We tested whether gelsemine, a nectar alkaloid of the bee-pollinated plant Gelsemium sempervirens, could reduce pathogen loads in bumble bees infected with the gut protozoan Crithidia bombi. In our first laboratory experiment, artificially infected bees consumed a daily diet of gelsemine post-infection to simulate continuous ingestion of alkaloid-rich nectar. In the second experiment, bees were inoculated with C. bombi cells that were pre-exposed to gelsemine, simulating the direct effects of nectar alkaloids on pathogen cells that are transmitted at flowers. Gelsemine significantly reduced the fecal intensity of C. bombi 7 days after infection when it was consumed continuously by infected bees, whereas direct exposure of the pathogen to gelsemine showed a non-significant trend toward reduced infection. Lighter pathogen loads may relieve bees from the behavioral impairments associated with the infection, thereby improving their foraging efficiency. If the collection of nectar secondary metabolites by pollinators is done as a means of self-medication, pollinators may selectively maintain secondary metabolites in the nectar of plants in natural populations.