Transcriptional repression by ApiAP2 factors is central to chronic toxoplasmosis

Tachyzoite to bradyzoite development in Toxoplasma is marked by major changes in gene expression resulting in a parasite that expresses a new repertoire of surface antigens hidden inside a modified parasitophorous vacuole called the tissue cyst. The factors that control this important life cycle transition are not well understood. Here we describe an important transcriptional repressor mechanism controlling bradyzoite differentiation that operates in the tachyzoite stage. The ApiAP2 factor, AP2IV-4, is a nuclear factor dynamically expressed in late S phase through mitosis/cytokinesis of the tachyzoite cell cycle. Remarkably, deletion of the AP2IV-4 locus resulted in the expression of a subset of bradyzoite-specific proteins in replicating tachyzoites that included tissue cyst wall components BPK1, MCP4, CST1 and the surface antigen SRS9. In the murine animal model, the mis-timing of bradyzoite antigens in tachyzoites lacking AP2IV-4 caused a potent inflammatory monocyte immune response that effectively eliminated this parasite and prevented tissue cyst formation in mouse brain tissue. Altogether, these results indicate that suppression of bradyzoite antigens by AP2IV-4 during acute infection is required for Toxoplasma to successfully establish a chronic infection in the immune-competent host

Reduced-Intensity/Reduced-Toxicity Conditioning Approaches Are Tolerated in XIAP Deficiency but Patients Fare Poorly with Acute GVHD

X-linked inhibitor of apoptosis (XIAP) deficiency is an inherited primary immunodeficiency characterized by chronic inflammasome overactivity and associated with hemophagocytic lymphohistiocytosis (HLH) and inflammatory bowel disease (IBD). Allogeneic hematopoietic cell transplantation (HCT) with fully myeloablative conditioning may be curative but has been associated with poor outcomes. Reports of reduced-intensity conditioning (RIC) and reduced-toxicity conditioning (RTC) regimens suggest these approaches are well tolerated, but outcomes are not well established. Retrospective data were collected from an international cohort of 40 patients with XIAP deficiency who underwent HCT with RIC or RTC. Thirty-three (83%) patients had a history of HLH, and thirteen (33%) patients had IBD. Median age at HCT was 6.5 years. Grafts were from HLA-matched (n = 30, 75%) and HLA-mismatched (n = 10, 25%) donors. There were no cases of primary graft failure. Two (5%) patients experienced secondary graft failure, and three (8%) patients ultimately received a second HCT. Nine (23%) patients developed grade II-IV acute GVHD, and 3 (8%) developed extensive chronic GVHD. The estimated 2-year overall and event-free survival rates were 74% (CI 55-86%) and 64% (CI 46-77%), respectively. Recipient and donor HLA mismatch and grade II-IV acute GVHD were negatively associated with survival on multivariate analysis with hazard ratios of 5.8 (CI 1.5-23.3, p = 0.01) and 8.2 (CI 2.1-32.7, p < 0.01), respectively. These data suggest that XIAP patients tolerate RIC and RTC with survival rates similar to HCT of other genetic HLH disorders. Every effort should be made to prevent acute GVHD in XIAP-deficient patients who undergo allogeneic HCT

Immune Response to Developmentally-Dependent Expression of Toxoplasma gondii Antigens

The switch from tachyzoite to bradyzoite enables the protozoan parasite, Toxoplasma gondii to establish latency in its host, and the immune response to the cyst stage of the parasite remains largely unknown. The work presented in this dissertation aim to establish the immune response to bradyzoite and cyst wall antigens.Macrophages are critical for controlling cyst burden, yet the receptors involved are unknown. I investigated the PRR, dectin1, in macrophage-cyst recognition, due to its role in macrophage responses to chitin in vitro, and fungal pathogens in vivo. We find that dectin1 is not required to control parasite burden or induce an inflammatory response in vivo. Contrary to our hypothesis, dectin1-/- mice display no differences in cyst numbers in the brain, but rather, dectin1 plays an indirect role by regulating inflammation.The immune system contributes to the parasites developmental switch in vivo, but parasite-specific factors are unclear. AP2 transcription factors contribute to developmental switching in other apicomplexans, and here, we examined AP2IV- 4 during Toxoplasma infection to determine if misexpression of bradyzoite antigens influences infection and cyst formation. We find that ΔAP2IV-4 parasites do not encyst in the brain. High dose infection with wild-type parasites results in 100% mortality by day 6 post-infection, whereas the PruΔAP2IV-4 mice survive. The response to PruΔAP2IV-4 is driven by inflammatory monocytes, which are known for their ability to control parasite replication.Despite the progress in the field, we know little about the growth and development of Toxoplasma cysts, and the cues for bradyzoite differentiation. Studying this in vivo is difficult, and studies have used artificial means to generate cysts. Thus, we need a system for studying this in vitro, that will facilitate our understanding of cyst development and host-parasite interactions. Here, I validate that cysts form spontaneously within neurons in vitro and neurons actively respond to parasite infection, but are poor antigen presenting cells.In this dissertation, I demonstrate that expression of stage-specific antigens shape the outcome of the immune response. Understanding this developmental switch in vitro and in vivo will reveal novel vaccination strategies or treatments targeted to latent stages of infection

Immune Response to Developmentally-Dependent Expression of Toxoplasma gondii Antigens

The switch from tachyzoite to bradyzoite enables the protozoan parasite, Toxoplasma gondii to establish latency in its host, and the immune response to the cyst stage of the parasite remains largely unknown. The work presented in this dissertation aim to establish the immune response to bradyzoite and cyst wall antigens.Macrophages are critical for controlling cyst burden, yet the receptors involved are unknown. I investigated the PRR, dectin1, in macrophage-cyst recognition, due to its role in macrophage responses to chitin in vitro, and fungal pathogens in vivo. We find that dectin1 is not required to control parasite burden or induce an inflammatory response in vivo. Contrary to our hypothesis, dectin1-/- mice display no differences in cyst numbers in the brain, but rather, dectin1 plays an indirect role by regulating inflammation.The immune system contributes to the parasites developmental switch in vivo, but parasite-specific factors are unclear. AP2 transcription factors contribute to developmental switching in other apicomplexans, and here, we examined AP2IV- 4 during Toxoplasma infection to determine if misexpression of bradyzoite antigens influences infection and cyst formation. We find that ΔAP2IV-4 parasites do not encyst in the brain. High dose infection with wild-type parasites results in 100% mortality by day 6 post-infection, whereas the PruΔAP2IV-4 mice survive. The response to PruΔAP2IV-4 is driven by inflammatory monocytes, which are known for their ability to control parasite replication.Despite the progress in the field, we know little about the growth and development of Toxoplasma cysts, and the cues for bradyzoite differentiation. Studying this in vivo is difficult, and studies have used artificial means to generate cysts. Thus, we need a system for studying this in vitro, that will facilitate our understanding of cyst development and host-parasite interactions. Here, I validate that cysts form spontaneously within neurons in vitro and neurons actively respond to parasite infection, but are poor antigen presenting cells.In this dissertation, I demonstrate that expression of stage-specific antigens shape the outcome of the immune response. Understanding this developmental switch in vitro and in vivo will reveal novel vaccination strategies or treatments targeted to latent stages of infection

Targeted Transcriptomic Analysis of C57BL/6 and BALB/c Mice During Progressive Chronic Toxoplasma gondii Infection Reveals Changes in Host and Parasite Gene Expression Relating to Neuropathology and Resolution.

Toxoplasma gondii is a resilient parasite that infects a multitude of warm-blooded hosts and results in a lifelong chronic infection requiring continuous responses by the host. Chronic infection is characterized by a balanced immune response and neuropathology that are driven by changes in gene expression. Previous research pertaining to these processes has been conducted in various mouse models, and much knowledge of infection-induced gene expression changes has been acquired through the use of high throughput sequencing techniques in different mouse strains and post-mortem human studies. However, lack of infection time course data poses a prominent missing link in the understanding of chronic infection, and there is still much that is unknown regarding changes in genes specifically relating to neuropathology and resulting repair mechanisms as infection progresses throughout the different stages of chronicity. In this paper, we present a targeted approach to gene expression analysis during T. gondii infection through the use of NanoString nCounter gene expression assays. Wild type C57BL/6 and BALB/c background mice were infected, and transcriptional changes in the brain were evaluated at 14, 28, and 56 days post infection. Results demonstrate a dramatic shift in both previously demonstrated and novel gene expression relating to neuropathology and resolution in C57BL/6 mice. In addition, comparison between BALB/c and C57BL/6 mice demonstrate initial differences in gene expression that evolve over the course of infection and indicate decreased neuropathology and enhanced repair in BALB/c mice. In conclusion, these studies provide a targeted approach to gene expression analysis in the brain during infection and provide elaboration on previously identified transcriptional changes and also offer insights into further understanding the complexities of chronic T. gondii infection

Targeted Transcriptomic Analysis of C57BL/6 and BALB/c Mice During Progressive Chronic Toxoplasma gondii Infection Reveals Changes in Host and Parasite Gene Expression Relating to Neuropathology and Resolution

Toxoplasma gondii is a resilient parasite that infects a multitude of warm-blooded hosts and results in a lifelong chronic infection requiring continuous responses by the host. Chronic infection is characterized by a balanced immune response and neuropathology that are driven by changes in gene expression. Previous research pertaining to these processes has been conducted in various mouse models, and much knowledge of infection-induced gene expression changes has been acquired through the use of high throughput sequencing techniques in different mouse strains and post-mortem human studies. However, lack of infection time course data poses a prominent missing link in the understanding of chronic infection, and there is still much that is unknown regarding changes in genes specifically relating to neuropathology and resulting repair mechanisms as infection progresses throughout the different stages of chronicity. In this paper, we present a targeted approach to gene expression analysis during T. gondii infection through the use of NanoString nCounter gene expression assays. Wild type C57BL/6 and BALB/c background mice were infected, and transcriptional changes in the brain were evaluated at 14, 28, and 56 days post infection. Results demonstrate a dramatic shift in both previously demonstrated and novel gene expression relating to neuropathology and resolution in C57BL/6 mice. In addition, comparison between BALB/c and C57BL/6 mice demonstrate initial differences in gene expression that evolve over the course of infection and indicate decreased neuropathology and enhanced repair in BALB/c mice. In conclusion, these studies provide a targeted approach to gene expression analysis in the brain during infection and provide elaboration on previously identified transcriptional changes and also offer insights into further understanding the complexities of chronic T. gondii infection.</jats:p

Exploring parental feeding practices and food choices during the cost-of-living crisis

This study is part of an undergraduate dissertation and includes 4 topics. Therefore several predictors of parental feeding practices and food choices are investigated and presented below. Topic 1. Family characteristics as predictors of parental control: Previous research has identified a positive relationship between a parent having obesity and their child having a BMI classified as overweight or obese (Nielsen et al., 2021). Attributing this relationship to either genetics or environment is difficult as it is likely an interaction of both factors. For example, Herle et al. (2020) suggested that genetics affect how we engage with our environment, influencing our eating behaviours. Environmental factors which bear influence on a child’s weight status include family income and educational attainment (Brophy et al., 2009); however, the present study focuses on parental feeding practices. Feeding practices are the focal point of this study as parents’ influence on the child’s diet is key, and a direct contributor to the child’s weight (Golan and Crow, 2004). Specifically, overt and covert feeding control are under investigation. Ogden and colleagues (2006) defined overt feeding control practices as diet-management strategies the child can perceive and covert practices as diet-management strategies that the child cannot perceive. Parent and child characteristics can predict the type of feeding practice that a parent uses (Brown et al., 2008), thus the factors which influence control type are of interest in this study. Brown and colleagues (2008) found that parents with a lower BMI are more likely to use both overt and covert feeding control practices; however, to date no evidence has established why parental BMI bears such an influence on feeding control strategies. Therefore, the present study aims to replicate prior findings (Brown et al., 2008), and incorporate a qualitative strand to bridge the gap in our knowledge and allow a better understanding of why parental BMI influences feeding control. The qualitative question will aim to gather parents' opinions on why they choose to feed their child in the way that they do, which we hope will contribute novel findings to this area of research. Topic 2. Parental feeding practices during the cost-of-living crisis: Caregivers play a key role in influencing children’s food-related behaviours (Scaglioni et al., 2018). One way in which this is achieved is via parental feeding practices. (Savage et al., 2007). Parental feeding practices refer to the use of multiple strategies and actions, used to support the development of children’s food-related preferences (Loth et al., 2013). Research elucidates four common domains of parental feeding practices. These domains are: (1) control, using pressure and restriction (Wehrly et al., 2014), (2) encouragement, praise given to motivate food-related behaviours (Wardle et al, 2002), (3) instrumental feeding, food used to reward or punish behaviour (Mason, 2015) and (4) emotional feeding, food used to regulate emotions (Wardle et al., 2002). These parental feeding practices influence the development of different food-related behaviours (Raijmakers et al., 2014). For example, emotional feeding has been associated with a rise in high-fat and calorie-dense food consumption (Rodgers et al., 2013). These have been linked to unhealthy weight gain and dietary habits in children (Blissett et al., 2010) Furthermore, parental characteristics such as socioeconomic status, described as the social positioning of individuals, has been demonstrated to greatly alter the types of parental feeding practices employed (Warkentin et al., 2018). For instance, high-income households have frequently reported less engagement in pressure-to-eat feeding practices, such as urging children to eat more (Ogden et al., 2006). Instead, many caregivers from high-income families disclose that they adopt the use of restrictive feeding practices by limiting the availability of unhealthy foods (Fisher &amp; Birch, 1999). Compared to high-income families, research has highlighted how low-income families report the use of emotional and indulgent feeding practices (Wehrly et al., 2014). These feeding practices employed by low-income families focus on regulating children’s emotions with food whilst the introduction of food-related rules is limited (Hughes et al., 2021). These findings suggest that low-income families adopt less supportive feeding practices which have been linked with a range of negative health implications compared with high-income families. Lower-income households have further reported heightened levels of stress due to limited access to food and money (Gundersen &amp; Ziliak, 2015). Different types of stress have been shown to impact the type of parental feeding practices used (Jansen et al., 2021). Research investigating monetary uncertainty (Luo et al., 2022) and psychological stress (Hughes et al., 2015) clearly demonstrated a negative relationship with the adoption of supportive parental feeding strategies, such as encouragement. Berge et al., (2020) found that increased levels of monetary stress were associated with increased use of control. Furthermore, households that have restricted access to nutritionally adequate food have reported the use of unsupportive feeding practices, including coercion, control (Adams et al., 2020) and indulgent feeding (Hughes et al., 2008). These findings support the idea that monetary stress is negatively associated with the use of healthy and supportive feeding practices. Qualitative research has extended these findings and highlighted the idea that many individuals felt they had to adjust their feeding practices to compensate and be able to cope with momentary factors that increase stress (Loth et al., 2022). In addition, stress-inducing factors have also been shown to influence the type of parental feeding practices used (Jansen et al., 2021). One example of a stress-induced change is the pandemic. The pandemic led to a worldwide economic fallout, resulting in millions of individuals losing their jobs, and presenting new challenges for many regarding the ability to buy groceries and pay bills. Research conducted during the pandemic revealed that many caregivers altered their feeding practices in response to the uncertainty they faced (Adams et al., 2020). Several studies found that individuals used controlling food-related practices to compensate for heightened levels of stress and limited access to essential items, including groceries (Loth et al., 2022; Berge et al., 2017). This is of particular concern as controlling feeding strategies have been linked with reduced nutritional and increased calorific intake, and childhood obesity (Savage et al., 2007; Fisher &amp; Birch, 1999). These findings are of particular interest as the United Kingdom is currently experiencing a cost-of-living crisis (Francis-Devine et al., 2022); the price of essential items rising quicker than average household incomes (Keith Neal., 2022). Low-income families have revealed that this current situation is adding to pre-existing stress of budgetary pressure and food insecurity (Patrick &amp; Pybus, 2022). However, the impact of the cost-of-living crisis on parental feeding practices remains unexplored at this current time. There is a large body of quantitative and qualitative research which has examined the associations between stress and socioeconomic status on parental feeding practices. However, the implementation of mixed-method approaches exploring these associations across the literature is limited. The current study will adopt a mixed-methods design which will aim to provide us with a better understanding of these associations by combining both quantitative and qualitative approaches to yield stronger and richer data regarding this relationship. Moreover, the publication of research to date investigating the impact of the cost-of-living crisis is limited. Therefore, the current study aims to understand these relationships in relation to the cost-of-living crisis, in more detail by using both methodologies together along with an additional exploratory section, which has yet to be done. Topic 3. Exploring same sex modelling in meat consumption: The threat posed by climate change has made finding sustainable alternatives to our current behaviours increasingly urgent. The answer may lie in our diets. Tilman and Clark (2014) found a shift to plant-based diets could significantly reduce greenhouse gas emissions. Furthermore, Wynes and Nicholas (2017) report switching to a plant-based diet is four times more effective than recycling. Unfortunately, current masculine ‘ideals’ upheld by society provide a unique challenge to the plant-based diet solution. Literature has explored the links between meat eating and masculinity, consistently showing men consume more meat and view vegetarian diets as feminine (Modlinska et al., 2020). Historically, meat has been a symbol of power and wealth and women, who were viewed as second-class citizens, were made to eat ‘second-class’ foods such as vegetables and grains (Adams, 1990/2015). In contemporary society, manhood must be earned through the constant acting out of superior masculine behaviours over inferior feminine ones (Connell, 2005). Because omnivorous diets are consistently rated as more masculine that vegetarian diets (Rothgerber, 2013; Ruby &amp; Heine, 2011), it has been proposed that men eat more meat as it allows them to prove their manhood by making the more masculine choice. A potential explanation for how the associations between meat and masculinity are upheld in society lies in the social cognitive theory. This theory states individuals learn by observing modelled behaviour, then transforming this into a memory representation in the form of rules of appropriate conduct. These rules instruct the subsequent behaviours of the learner and new behaviours are reinforced if their consequences are beneficial (Bandura, 1986). A potential mediating factor for observational learning is the model’s gender, as observers are more likely to pay attention to and learn about conduct that is personally relevant to them, so they are more likely to focus on a same-sex model (Bussey &amp; Bandura, 1999). Research supports this, as studies have found children are more likely to imitate the behaviours of models that match their gender, but this effect is only significant under conditions where the imitated behaviour is sex-linked (Perry &amp; Bussey, 1979). As demonstrated above, meat eating is consistently associated with being more masculine, so it is a sex-linked behaviour. Therefore, we could predict children are more likely to model their meat-eating behaviours from the parent of the same gender, in order to fit in with gender roles. There is already evidence of gender roles influencing the same-sex modelling of eating behaviours, as maternal restricted eating is more predictive of daughter’s restricted eating than sons (Zarychta et al., 2019). Sociocultural models explain this by stating that culturally, there is a higher pressure on women to live up to a thin ‘ideal’, making restricted eating characteristics more common among women than men (Cash et al., 2004; Striegel-Moore &amp; Bulik, 2007). Because restricted eating is gender-typed in this way, it provides evidence that behaviours linking to one specific gender are more likely to be modelled by parent child pairs of the same gender. This demonstrates gender may be a mediating factor in sex-linked eating behaviours but this has not yet been investigated in the case of meat consumption, despite the potential insight it could provide into challenges to living sustainably. This study aims to be the first to establish the relationship between same-sex modelling and meat consumption. Topic 4. Exploring the relationship between attachment style, eating habits and stress: Understanding the role of attachment in relationships is key, as it allows us to predict certain characteristics such as security (Fraley &amp; Roisman, 2019), happiness in relationships (Momeni et al, 2022) and quality of life (Darban et al, 2020). Knowing what attachment style an individual is can help them to learn what to change in order to form stronger relationships and what to avoid. Attachment style can be defined as the way in which an individual acts in a close relationship. Anxious attachment style describes individuals who have a constant fear of abandonment and are typically worried about their relationship (Smyth et al, 2015). Conversely, avoidant attachment style describes those who have a fear of close relationships, causing them to avoid any intimacy (Caravello &amp; Gabriel, 2006). One behaviour which has been found to link with attachment style is disinhibition, which refers to a loss of control when eating and a tendency to overeat (Maayan et al, 2012). Research has found disinhibition accounting for one third of the link between BMI and obesity (Brunner et al, 2021). According to previous literature, a positive relationship exists between disinhibition and anxious attachment style, such that the more anxiously attached an individual is, the more likely they are to disinhibit (Wilkinson et al, 2010). This positive relationship is due to external affect regulation, which is when individuals control their emotions through external means. As anxiously attached individuals have a lack of internal control on their emotions (Wilkinson et al, 2010), food can be used as an alternative in order to soothe or distract themselves from their emotions (Mikulincer, 1998).The primary aim of this study is therefore to replicate the current literature which shows the relationship between anxiously attached individuals and disinhibition. This is important as there is little research looking into this relationship, so it is important to ensure the findings are valid. Additionally, Not only has previous literature shown the influence of attachment styles on emotional control (Feeney, 1995), but there has also been research showing a relationship with stress and attachment style, due to stress being an emotional response (Kidd et al, 2011). The cost-of-living crisis is the current economic crisis in the UK which can cause high levels of stress for individuals, due to an increase in price of daily essentials and bills. Stress levels during the cost-of-living crisis can act as a measure of current stress levels, and this measure can aid the secondary aim which will be to investigate how avoidantly attached and anxiously attached individuals differ in perceived stress levels during this cost-of-living crisis. There is evidence to suggest that anxiously attached individuals may report higher stress levels (Maunder et al, 2006) due to these individuals displaying stress to try to increase their proximity to those around them (Bowlby, 1979). A relationship between stress levels and snacking behaviour also exists, such that higher stress levels can increase snack consumption (Kim &amp; Jeong, 2007). Therefore, anxiously attached individuals may engage in more frequent snacking in the past week (due to the cost-of-living crisis) compared to six months ago due to stress increasing snack consumption

A role for dectin-1 in the CNS during chronic <i>Toxoplasma gondii</i> infection (MPF5P.739)

Abstract The latent stage of T.gondii infection is characterized by the formation of tissue cysts within the CNS. These tissue cysts are formed by manipulation of the parasitophorous vacuole, and chitin is a structural component of the cyst wall. Our lab has shown that Toxoplasma cysts induce an M2 phenotype in macrophages in a contact-dependent manner. Upon contact, these macrophages actively secrete the mammalian chitinase, AMCase, to break down the cyst wall. We have previously demonstrated that AMCase production by macrophages is essential for control of cyst burden in the brain during T. gondii infection. The molecular interactions involved in macrophage-cyst recognition, and the signaling pathway for AMCase production are as yet unknown. Although chitin recognition has been poorly defined, studies suggest dectin-1 may be a receptor. Here, we present data on a role for dectin-1 during T. gondii infection. Dectin-1 is upregulated in the brain in response to infection and is expressed on arginase-1+, M2 macrophages. Consistent with dectin-1 expression on M2 macrophages, dectin-1 is not required to control parasite burden or induce an inflammatory immune response in vivo. However, dectin-1 deficient mice do exhibit trends towards decreased cyst numbers in the brain and a concomitant increase in AMCase transcripts and AMCase activity ex vivo. Thus, this data suggests that dectin-1 is modulating the immune response and chitinase production during chronic Toxoplasma infection.</jats:p

Transcriptional repression by ApiAP2 factors is central to chronic toxoplasmosis

AbstractBradyzoite differentiation is marked by major changes in gene expression resulting in a parasite that expresses a new repertoire of surface antigens hidden inside a modified parasitophorous vacuole called the tissue cyst. The factors that control this important life cycle transition are not well understood. Here we describe an importantToxoplasmatranscriptional repressor mechanism controlling bradyzoite differentiation that operates exclusively in the tachyzoite stage. The ApiAP2 factor, AP2IV-4, is a nuclear factor dynamically expressed in late S phase through mitosis/cytokinesis of the tachyzoite cell cycle. Remarkably, deletion of the AP2IV-4 locus resulted in the increased expression of bradyzoite mRNAs in replicating tachyzoites, and in two different genetic lineages we confirmed the misexpression of tissue cyst wall components (e.g. BPK1, MCP4, CST1) and the bradyzoite surface antigen SRS9 in the tachyzoite stage. In the murine animal model, the loss of AP2IV-4 had profound biological consequences. Type II prugniaud strain parasites lacking AP2IV-4 were unable to form tissue cysts in brain tissue and the absence of this factor also recruited a potent immune response characterized by increases inflammatory monocytes, IFN-γ and higher numbers of both CD8+ and CD4+ T-cells. Altogether, these results indicate that suppression of bradyzoite antigens by AP2IV-4 during acute infection is required forToxoplasmato establish a chronic infection in the immune-competent host.Author SummaryTheToxoplasmabiology that underlies the establishment of a chronic infection is developmental conversion of the acute tachyzoite stage into the latent bradyzoite-tissue cyst stage. Despite the important clinical consequences of this developmental pathway, the molecular basis of the switch mechanisms that control formation of the tissue cyst is still poorly understood. A fundamental feature of tissue cyst formation is the expression of bradyzoite-specific genes. Here we show the transcription factor AP2IV-4 directly silences bradyzoite mRNA and protein expression in the acute tachyzoite stage demonstrating that developmental control of tissue cyst formation is as much about when not to express bradyzoite genes as it is about when to activate them. Loosing the suppression of bradyzoite gene expression in the acute tachyzoite stage caused by deleting AP2IV-4 blocked the establishment of chronic disease in healthy animals through the pre-arming of the immune system suggesting a possible strategy for preventing chronicToxoplasmainfections.</jats:sec