Susceptibility of Toxoplasma gondii to autophagy in human cells relies on multiple interacting parasite loci.

IMPORTANCE: Autophagy is a process used by cells to recycle organelles and macromolecules and to eliminate intracellular pathogens. Previous studies have shown that some stains of Toxoplasma gondii are resistant to autophagy-dependent growth restriction, while others are highly susceptible. Although it is known that autophagy-mediated control requires activation by interferon gamma, the basis for why parasite strains differ in their susceptibility is unknown. Our findings indicate that susceptibility involves at least five unlinked parasite genes on different chromosomes, including several secretory proteins targeted to the parasite-containing vacuole and exposed to the host cell cytosol. Our findings reveal that susceptibility to autophagy-mediated growth restriction relies on differential recognition of parasite proteins exposed at the host-pathogen interface, thus identifying a new mechanism for cell-autonomous control of intracellular pathogens.

Flecainide poisoning and prolongation of elimination due to alkalinization.

BACKGROUND: Flecainide is a 1C antidysrhythmic that is primarily used for ventricular tachycardia or premature ventricular contractions when other treatment is ineffective. It has a very narrow therapeutic window which may cause death in a double dose and requires inpatient initiation for cardiac monitoring. Despite established pharmacokinetic data from flecainide in therapeutic dosing, there is negligible data on flecainide toxicokinetics after an intentional overdose. Due to the inherent differences in pharmacokinetic and toxicokinetic principles, rarely can the peak effect or elimination half-life accurately be applied to the poisoned patient after an overdose. In overdose, flecainide can cause a variety of fatal dysrhythmias which may require sodium bicarbonate for stabilization but also may reduce the renal elimination of flecainide, meaning the life-saving treatment may prolong the time of toxicity. CASE REPORT: We present a case of an acute ingestion of flecainide with a known time of ingestion and known amount of ingestion who experienced subsequent life-threatening effects which required endotracheal intubation, sodium bicarbonate, aggressive electrolyte repletion, and multiple days in an intensive care unit. RESULTS: Serial serum and urine samples revealed a prolonged toxic serum concentration of flecainide. CONCLUSION: These results demonstrate the change in elimination kinetics of flecainide in the setting of urinary alkalization which is evident through prolonged morphologic changes present on serial electrocardiograms.

A Fatal Colchicine Ingestion With Antemortem Blood Concentration.

ABSTRACT: Although there are multiple therapeutic uses for colchicine, it is particularly dangerous in the setting of overdose due to an irreversible mechanism of action combined with a narrow therapeutic window. Colchicine is an antimitotic agent that binds tubulin and inhibits microtubule polymerization. This produces a predictable sequence of toxicity beginning with gastrointestinal effects with progression to multiorgan system dysfunction. Unfortunately, there are no specific antidotes for colchicine toxicity after organ injury has occurred, which can lead to tragic consequences. Despite the recognized toxicity, it is exceedingly rare to find a case in the medical literature with a confirmed time of ingestion, amount ingested, data from longitudinal examinations, and laboratory assessments, with a quantitative blood colchicine concentration. We present a case of acute colchicine overdose of 18 mg (approximately 0.25 mg/kg) with subsequent multiorgan failure and death with an antemortem blood colchicine concentration of 14 ng/mL at 18.5 hours after ingestion.

Disposition, outcomes, and lengths of stay due to bupropion overdose at a tertiary care center with a medical toxicology service.

INTRODUCTION: Bupropion is an antidepressant with unique mechanisms of action leading to a narrow therapeutic window. Parallel to increasing indications, there is an increasing number of overdoses and fatalities attributable to bupropion overdose. Due to the serious effects of a bupropion overdose including arrhythmias and early or delayed seizures, these patients necessitate prolonged monitoring with high levels of medical care. In the setting of a tertiary care center with a medical toxicology consult service, our institution is heavily relied upon to manage these patients. This study was performed to provide clarity on the resources used, lengths-of-stay, and treatments provided for these patients. METHODS: All patients at a tertiary care center with an oral bupropion overdose and a medical toxicology consult less than 24 h after the ingestion were included between July 15, 2017 and October 14, 2021. Chart review was performed to determine lengths-of-stay, the unit of disposition, treatments provided, and outcomes. RESULTS: A total of 73 cases were identified with 36 bupropion-only ingestions. Most cases were transferred from outside facilities, developed seizures, had QRS prolongation; and almost a third required intubation. The vast majority were admitted to an ICU and received GABA-A agonists. A median of 1.47 days per case was spent in the ED or ICU. There was an average of 41.9 ED or ICU bed-days per year and 68.5 non-psychiatric bed-days per year occupied by a patient after a bupropion overdose at a single center. CONCLUSIONS: Bupropion overdose necessitates high resource utilization which we believe will increase with the expanding indications for its use.

Bicyclic azetidines target acute and chronic stages of Toxoplasma gondii by inhibiting parasite phenylalanyl t-RNA synthetase.

Toxoplasma gondii commonly infects humans and while most infections are controlled by the immune response, currently approved drugs are not capable of clearing chronic infection in humans. Hence, approximately one third of the world's human population is at risk of reactivation, potentially leading to severe sequelae. To identify new candidates for treating chronic infection, we investigated a series of compounds derived from diversity-oriented synthesis. Bicyclic azetidines are potent low nanomolar inhibitors of phenylalanine tRNA synthetase (PheRS) in T. gondii, with excellent selectivity. Biochemical and genetic studies validate PheRS as the primary target of bicyclic azetidines in T. gondii, providing a structural basis for rational design of improved analogs. Favorable pharmacokinetic properties of a lead compound provide excellent protection from acute infection and partial protection from chronic infection in an immunocompromised mouse model of toxoplasmosis. Collectively, PheRS inhibitors of the bicyclic azetidine series offer promise for treatment of chronic toxoplasmosis.

ISG15 Connects Autophagy and IFN-γ-Dependent Control of Toxoplasma gondii Infection in Human Cells.

The intracellular protozoan parasite Toxoplasma gondii is capable of infecting most nucleated cells, where it survives in a specially modified compartment called the parasitophorous vacuole (PV). Interferon gamma (IFN-γ) is the major cytokine involved in activating cell-autonomous immune responses to inhibit parasite growth within this intracellular niche. In HeLa cells, IFN-γ treatment leads to ubiquitination of susceptible parasite strains, recruitment of the adaptors p62 and NDP52, and engulfment in microtubule-associated protein 1 light chain 3 (LC3)-positive membranes that restrict parasite growth. IFN-γ-mediated growth restriction depends on core members of the autophagy (ATG) pathway but not the initiation or degradative steps in the process. To explore the connection between these different pathways, we used permissive biotin ligation to identify proteins that interact with ATG5 in an IFN-γ-dependent fashion. Network analysis of the ATG5 interactome identified interferon-stimulated gene 15 (ISG15), which is highly upregulated by IFN treatment, as a hub connecting the ATG complex with other IFN-γ-induced genes, suggesting that it forms a functional link between the pathways. Deletion of ISG15 resulted in impaired recruitment of p62, NDP52, and LC3 to the PV and loss of IFN-γ-restricted parasite growth. The function of ISG15 required conjugation, and a number of ISGylated targets overlapped with the IFN-γ-dependent ATG5 interactome, including the adapter p62. Collectively, our findings establish a role for ISG15 in connecting the ATG pathway with IFN-γ-dependent restriction of T. gondii in human cells.IMPORTANCE Interferon(s) provide the primary defense against intracellular pathogens, a property ascribed to their ability to upregulate interferon-stimulated genes. Due to the sequestered niche occupied by Toxoplasma gondii, the host has elaborated intricate ways to target the parasite within its vacuole. One such mechanism is the recognition by a noncanonical autophagy pathway that envelops the parasite-containing vacuole and stunts growth in human cells. Remarkably, autophagy-dependent growth restriction requires interferon-γ, yet none of the classical components of autophagy are induced by interferon. Our studies draw a connection between these pathways by demonstrating that the antiviral protein ISG15, which is normally upregulated by interferons, links the autophagy-mediated control to ubiquitination of the vacuole. These findings suggest a similar link between interferon-γ signaling and autophagy that may underlie defense against other intracellular pathogens.

Data on hydroxychloroquine interference with urine laboratory testing.

Hydroxychloroquine is a medication used to treat rheumatoid arthritis, systemic lupus erythematosus, and other autoimmune disorders. Previous studies have shown that hydroxychloroquine and the structurally related drug chloroquine have the potential to interfere with some common urine chemistry tests, especially at high concentrations. In the related research article, we observed suspected interference with urine drug of abuse testing in a patient who ingested approximately 12 g of hydroxychloroquine in an acute overdose, with urine hydroxychloroquine concentrations exceeding 500 mg/L. This case prompted a more detailed investigation of the effects of hydroxychloroquine spiked into pooled de-identified urine specimens from a hospital clinical laboratory. The data in this article provides the raw data for 24 urine assays that were investigated. The analyzed data is provided in the tables included in this article. The dataset reported is related to the research article entitled "Diagnostic Pitfalls and Laboratory Test Interference After Hydroxychloroquine Intoxication: A Case Report" [1].

Diagnostic pitfalls and laboratory test interference after hydroxychloroquine intoxication: A case report.

Hydroxychloroquine is a medication used to treat autoimmune conditions. Overdoses of hydroxychloroquine are uncommon, with most recommendations on monitoring drawing from experience with more common overdoses of the related drug chloroquine. We present a case of an adolescent with intentional overdose of approximately 12 g of hydroxychloroquine. The prominent clinical features were hypokalemia and widened QRS and QT intervals on the electrocardiogram. Therapy included epinephrine by intravenous drip and bicarbonate infusions along with supportive care and cardiac monitoring. The patient recovered without sequelae. Urine drug testing showed an absorbance alarm for one of the components of the institution drug of abuse screening panel, an oxycodone screen using an enzyme immunoassay. Analysis of two urine specimens collected during the hospitalization revealed hydroxychloroquine concentrations of greater than 500 mg/L (approximately 7.5 h after ingestion) and 130 mg/L (approximately 14 h after ingestion). Only the urine with greater than 500 mg/L hydroxychloroquine produced absorbance alarms on the drug of abuse testing. We separately analyzed the impact on 24 urine assays of varying concentrations of hydroxychloroquine spiked into de-identified pooled urine samples. For 6 of the assays (buprenorphine, cotinine, oxycodone, and tetrahydrocannabinol qualitative drug screens; microalbumin and urine myoglobin quantitative assays), hydroxychloroquine produced significant bias and/or instrument alarms. Overall, our study demonstrates that urine concentrations of hydroxychloroquine can reach very high concentrations (exceeding 500 mg/L) following overdose, with the potential to interfere with a range of urine assays including drug of abuse screening and microalbumin. Similar to previous reports, hydroxychloroquine overdose can produce hypokalemia and electrocardiographic abnormalities.

Protozoan persister-like cells and drug treatment failure.

Antimicrobial treatment failure threatens our ability to control infections. In addition to antimicrobial resistance, treatment failures are increasingly understood to derive from cells that survive drug treatment without selection of genetically heritable mutations. Parasitic protozoa, such as Plasmodium species that cause malaria, Toxoplasma gondii and kinetoplastid protozoa, including Trypanosoma cruzi and Leishmania spp., cause millions of deaths globally. These organisms can evolve drug resistance and they also exhibit phenotypic diversity, including the formation of quiescent or dormant forms that contribute to the establishment of long-term infections that are refractory to drug treatment, which we refer to as 'persister-like cells'. In this Review, we discuss protozoan persister-like cells that have been linked to persistent infections and discuss their impact on therapeutic outcomes following drug treatment.

Accidental intoxications in toddlers: lack of cross-reactivity of vilazodone and its urinary metabolite M17 with drug of abuse screening immunoassays.

BACKGROUND: Vilazodone is an FDA approved medication used to treat major depressive disorder. The authors describe two cases of accidental vilazodone exposure in toddlers who presented with symptoms similar to amphetamine exposure and also with unexplained positive amphetamine urine immunoassay drug screens. Given a lack of published data on cross-reactivity of vilazodone and its metabolites with drug of abuse screening tests, the authors investigated drug of abuse immunoassay cross-reactivity of vilazodone and metabolites using computational and empirical approaches. METHODS: To ascertain the likelihood that vilazodone would cross-react with drug of abuse screening immunoassays, the authors assessed the two-dimensional (2D) similarity of the vilazodone parent molecule and known metabolites to an array of antigenic targets for urine immunoassay drug screens. To facilitate studies of the commercially unavailable M17 metabolite, it was prepared synthetically through a novel scheme. Urine and serum were spiked with vilazodone and M17 into urine (200-100,000 ng/mL) and serum (20-2000 ng/mL) samples and tested for cross-reactivity. RESULTS: Computational analysis using 2D similarity showed that vilazodone and metabolites have generally low similarity to antigenic targets of common drug of abuse screening immunoassays, predicting weak or no cross-reactivity. The M17 metabolite had 2D similarity to amphetamines and tricyclic antidepressants in a range similar to some other compounds exhibiting weak cross-reactivity on these immunoassays. Cross-reactivity testing was therefore performed on two different urine amphetamines immunoassays and a serum tricyclic antidepressant immunoassay. However, actual testing of cross reactivity for vilazodone and the M17 metabolite did not detect cross-reactivity for any urine amphetamines screen at concentrations up to 100,000 ng/mL and for a serum tricyclic antidepressants assays at concentrations up to 2000 ng/mL. CONCLUSION: While the vilazodone metabolite M17 has weak 2D structural similarity to amphetamines and tricyclic antidepressants, the current study did not demonstrate any experimental cross-reactivity with two different urine amphetamines immunoassays and a serum tricyclic antidepressant immunoassay. Vilazodone ingestions in young children present a diagnostic challenge in their similarity to amphetamine ingestions and the lack of routine laboratory tests for vilazodone. Further work is needed to understand the metabolic profile for vilazodone in children versus adults.

Discovery of Selective Toxoplasma gondii Dihydrofolate Reductase Inhibitors for the Treatment of Toxoplasmosis.

A safer treatment for toxoplasmosis would be achieved by improving the selectivity and potency of dihydrofolate reductase (DHFR) inhibitors, such as pyrimethamine (1), for Toxoplasma gondii DHFR ( TgDHFR) relative to human DHFR ( hDHFR). We previously reported on the identification of meta-biphenyl analog 2, designed by in silico modeling of key differences in the binding pocket between TgDHFR and hDHFR. Compound 2 improves TgDHFR selectivity 6.6-fold and potency 16-fold relative to 1. Here, we report on the optimization and structure-activity relationships of this arylpiperazine series leading to the discovery of 5-(4-(3-(2-methoxypyrimidin-5-yl)phenyl)piperazin-1-yl)pyrimidine-2,4-diamine 3. Compound 3 has a TgDHFR IC50 of 1.57 ± 0.11 nM and a hDHFR to TgDHFR selectivity ratio of 196, making it 89-fold more potent and 16-fold more selective than 1. Compound 3 was highly effective in control of acute infection by highly virulent strains of T. gondii in the murine model, and it possesses the best combination of selectivity, potency, and prerequisite drug-like properties to advance into IND-enabling, preclinical development.

Transaminase and Creatine Kinase Ratios for Differentiating Delayed Acetaminophen Overdose from Rhabdomyolysis.

INTRODUCTION: Rhabdomyolysis and delayed acetaminophen hepatotoxicity may be associated with elevated serum transaminase values. Establishing the cause of elevated transaminases may be especially difficult because of limited or inaccurate histories of acetaminophen ingestion. We hypothesized that the comparative ratios of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatine kinase (CK) can differentiate acetaminophen hepatotoxicity from rhabdomyolysis. METHODS: A retrospective chart review of patients in four hospitals from 2006 to 2011 with a discharge diagnosis of acetaminophen toxicity or rhabdomyolysis was performed. Subjects were classified into three groups: rhabdomyolysis, acetaminophen overdose (all), and acetaminophen overdose with undetectable serum acetaminophen concentrations [acetaminophen(delayed)]. Ratios of AST, ALT, and CK were compared using non-parametric statistical methods. RESULTS: 1,353 subjects were identified and after applying our exclusion criteria there were 160 in the rhabdomyolysis group, 68 in the acetaminophen overdose (all) group, and 29 in the acetaminophen (delayed) group. The AST/ALT ratio for the rhabdomyolysis group was 1.66 (Interquartile range: 1.18-2.22), for the acetaminophen overdose (all) group was 1.38 (1.08-1.69, statistically lower than the rhabdomyolysis group, p = 0.018), and for the acetaminophen (delayed)group was 1.30 (1.06-1.63, p = 0.037). CK/AST ratios were 21.3 (12.8-42.2), 5.49 (2.52-15.1, p < 0.001), and 3.80 (1.43-13.8, p < 0.001) respectively. CK/ALT ratios were 37.1 (16.1-80.0), 5.77 (2.79-25.2, p < 0.001), and 5.03 (2.20-17.4, p < 0.001) respectively. Increasing CK to transaminase ratio cutoffs resulted in increasing test sensitivity but lower specificity. CONCLUSION: AST/ALT, CK/AST and CK/ALT ratios are significantly larger in rhabdomyolysis when compared to patients with acetaminophen toxicity. This result suggests that the ratios could be used to identify patients with rhabdomyolysis who otherwise might have been diagnosed as delayed acetaminophen toxicity. Such patients may not require treatment with N-acetylcysteine, resulting in cost savings and improved resource utilization.

High Throughput Screen Identifies Interferon γ-Dependent Inhibitors of Toxoplasma gondii Growth.

Toxoplasma gondii is an obligate intracellular parasite capable of causing severe disease due to congenital infection and in patients with compromised immune systems. Control of infection is dependent on a robust Th1 type immune response including production of interferon gamma (IFN-γ), which is essential for control. IFN-γ activates a variety of antimicrobial mechanisms in host cells, which are then able to control intracellular parasites such as T. gondii. Despite the effectiveness of these pathways in controlling acute infection, the immune system is unable to eradicate chronic infections that can persist for life. Similarly, while antibiotic treatment can control acute infection, it is unable to eliminate chronic infection. To identify compounds that would act synergistically with IFN-γ, we performed a high-throughput screen of diverse small molecule libraries to identify inhibitors of T. gondii. We identified a number of compounds that inhibited parasite growth in vitro at low µM concentrations and that demonstrated enhanced potency in the presence of a low level of IFN-γ. A subset of these compounds act by enhancing the recruitment of light chain 3 (LC3) to the parasite-containing vacuole, suggesting they work by an autophagy-related process, while others were independent of this pathway. The pattern of IFN-γ dependence was shared among the majority of analogs from 6 priority scaffolds, and analysis of structure activity relationships for one such class revealed specific stereochemistry associated with this feature. Identification of these IFN-γ-dependent leads may lead to development of improved therapeutics due to their synergistic interactions with immune responses.

Evaluation of Current and Emerging Antimalarial Medicines for Inhibition of Toxoplasma gondii Growth in Vitro.

Toxoplasma gondii is a common zoonotic infection of humans, and estimates indicate that 1-2 billion people are chronically infected. Although largely asymptomatic, chronic infection poses risk of serious disease due to reactivation should immunity decline. Current therapies for toxoplasmosis only control acute infection caused by actively proliferating tachyzoites but do not eradicate the chronic tissue cyst stages. As well, there are considerable adverse side effects of the most commonly used therapy of combined sulfadiazine and pyrimethamine. Targeting the folate pathway is also an effective treatment for malaria, caused by the related parasites Plasmodium spp., suggesting common agents might be used to treat both infections. Here, we evaluated currently approved and newly emerging medicines for malaria to determine if such compounds might also prove useful for treating toxoplasmosis. Surprisingly, the majority of antimalarial compounds being used currently or in development for treatment of malaria were only modestly effective at inhibiting in vitro growth of T. gondii tachyzoites. These findings suggest that many essential processes in P. falciparum that are targeted by antimalarial compounds are either divergent or nonessential in T. gondii, thus limiting options for repurposing of current antimalarial medicines for toxoplasmosis.

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.

Inhibition of Calcium Dependent Protein Kinase 1 (CDPK1) by Pyrazolopyrimidine Analogs Decreases Establishment and Reoccurrence of Central Nervous System Disease by Toxoplasma gondii.

Calcium dependent protein kinase 1 (CDPK1) is an essential enzyme in the opportunistic pathogen Toxoplasma gondii. CDPK1 controls multiple processes that are critical to the intracellular replicative cycle of T. gondii including secretion of adhesins, motility, invasion, and egress. Remarkably, CDPK1 contains a small glycine gatekeeper residue in the ATP binding pocket making it sensitive to ATP-competitive inhibitors with bulky substituents that complement this expanded binding pocket. Here we explored structure-activity relationships of a series of pyrazolopyrimidine inhibitors of CDPK1 with the goal of increasing selectivity over host enzymes, improving antiparasite potency, and improving metabolic stability. The resulting lead compound 24 exhibited excellent enzyme inhibition and selectivity for CDPK1 and potently inhibited parasite growth in vitro. Compound 24 was also effective at treating acute toxoplasmosis in the mouse, reducing dissemination to the central nervous system, and decreasing reactivation of chronic infection in severely immunocompromised mice. These findings provide proof of concept for the development of small molecule inhibitors of CDPK1 for treatment of CNS toxoplasmosis.

Massive diltiazem and metoprolol overdose rescued with extracorporeal life support.

The management of overdoses of cardioactive medications in the emergency department can be challenging. The reversal of severe toxicity from one or more types of cardioactive medication may fail maximal medical therapies and require extreme invasive measures such as transvenous cardiac pacing and extracorporeal life support. We present a case of massive diltiazem and metoprolol overdose refractory to maximal medical therapy, including intravenous calcium, glucagon, vasopressors, high dose insulin, and lipid emulsion. The patient experienced refractory bradydysrhythmia that responded only to transvenous pacing. Extracorporeal life support was initiated and resulted in successful organ perfusion and complete recovery of the patient. This case highlights the potential utility of extracorporeal life support in cases of severe toxicity due to multiple cardioactive medications.

Opposing Transcriptional Mechanisms Regulate Toxoplasma Development.

The Toxoplasma biology that underlies human chronic infection is developmental conversion of the acute tachyzoite stage into the latent bradyzoite stage. We investigated the roles of two alkaline-stress-induced ApiAP2 transcription factors, AP2IV-3 and AP2IX-9, in bradyzoite development. These factors were expressed in two overlapping waves during bradyzoite development, with AP2IX-9 increasing expression earlier than AP2IV-3, which peaked as AP2IX-9 expression was declining. Disruption of the AP2IX-9 gene enhanced, while deletion of AP2IV-3 gene decreased, tissue cyst formation, demonstrating that these factors have opposite functions in bradyzoite development. Conversely, conditional overexpression of FKBP-modified AP2IX-9 or AP2IV-3 with the small molecule Shield 1 had a reciprocal effect on tissue cyst formation, confirming the conclusions of the knockout experiments. The AP2IX-9 repressor and AP2IV-3 activator tissue cyst phenotypes were borne out in gene expression studies that determined that many of the same bradyzoite genes were regulated in an opposite manner by these transcription factors. A common gene target was the canonical bradyzoite marker BAG1, and mechanistic experiments determined that, like AP2IX-9, AP2IV-3 regulates a BAG1 promoter-luciferase reporter and specifically binds the BAG1 promoter in parasite chromatin. Altogether, these results suggest that the AP2IX-9 transcriptional repressor and the AP2IV-3 transcriptional activator likely compete to control bradyzoite gene expression, which may permit Toxoplasma to better adapt to different tissue environments and select a suitable host cell for long-term survival of the dormant tissue cyst. IMPORTANCEToxoplasma infections are lifelong because of the development of the bradyzoite tissue cyst, which is effectively invisible to the immune system. Despite the important clinical consequences of this developmental pathway, the molecular basis of the switch mechanisms that control tissue cyst formation is still poorly understood. Significant changes in gene expression are associated with tissue cyst development, and ApiAP2 transcription factors are an important mechanism regulating this developmental transcriptome. However, the molecular composition of these ApiAP2 complexes and the operating principles of ApiAP2 mechanisms are not well defined. Here we establish that competing ApiAP2 transcriptional mechanisms operate to regulate this clinically important developmental pathway.

Toxoplasma gondii AP2IX-4 Regulates Gene Expression during Bradyzoite Development.

Toxoplasma gondii is a protozoan parasite of great importance to human and animal health. In the host, this obligate intracellular parasite persists as a tissue cyst that is imperceptible to the immune response and unaffected by current therapies. The tissue cysts facilitate transmission through predation and give rise to chronic cycles of toxoplasmosis in immunocompromised patients. Transcriptional changes accompany conversion of the rapidly replicating tachyzoites into the encysted bradyzoites, and yet the mechanisms underlying these alterations in gene expression are not well defined. Here we show that AP2IX-4 is a nuclear protein exclusively expressed in tachyzoites and bradyzoites undergoing division. Knockout of AP2IX-4 had no discernible effect on tachyzoite replication but resulted in a reduced frequency of tissue cyst formation following alkaline stress induction-a defect that is reversible by complementation. AP2IX-4 has a complex role in regulating bradyzoite gene expression, as the levels of many bradyzoite mRNAs dramatically increased beyond those seen under conditions of normal stress induction in AP2IX-4 knockout parasites exposed to alkaline media. The loss of AP2IX-4 also resulted in a modest virulence defect and reduced cyst burden in chronically infected mice, which was reversed by complementation. These findings illustrate that the transcriptional mechanisms responsible for tissue cyst development operate across the intermediate life cycle from the dividing tachyzoite to the dormant bradyzoite. IMPORTANCEToxoplasma gondii is a single-celled parasite that persists in its host as a transmissible tissue cyst. How the parasite converts from its replicative form to the bradyzoites housed in tissue cysts is not well understood, but the process clearly involves changes in gene expression. Here we report that parasites lacking a cell cycle-regulated transcription factor called AP2IX-4 display reduced frequencies of tissue cyst formation in culture and in a mouse model of infection. Parasites missing AP2IX-4 lose the ability to regulate bradyzoite genes during tissue cyst development. Expressed in developing bradyzoites still undergoing division, AP2IX-4 may serve as a useful marker in the study of transitional forms of the parasite.

Toxoplasma development - turn the switch on or off?

Toxoplasma gondii exhibits a complex, multi-stage life cycle in which the need for parasite expansion is balanced with the production of transmissible forms. For human disease the key developmental switch is from the tachyzoite to the mature bradyzoite, which is not well understood at the molecular level. This review highlights the role of the tachyzoite in regulating the initiation of bradyzoite differentiation through newly discovered transcription factors of the ApiAP2 family that must be turned off for development to unfold. Exit from the tachyzoite cell cycle is also tightly co-ordinated with the induction of bradyzoite gene expression, which is strongly influenced by the host cell environment. New evidence suggests a parasite casein kinase II and host anti-growth factor CDA1 can influence specific pathways that are responsible for sensing the host cell environment and informing the parasites decision to continue replication or to develop into bradyzoites. These results indicate tachyzoite gene expression mechanisms and signal transduction pathways likely hold the keys to tissue cyst formation in Toxoplasma.