The contribution of amylopectin granules (AG), a branched chain storage homopolymer of glucose, to the maintenance and progression of the chronic Toxoplasma gondii infection has remained undefined. Here, we describe the role of AG in the physiology of encysted bradyzoites using a purpose-developed imaging-based application, AmyloQuant, which permitted the quantification of relative levels of AG within in vivo-derived tissue cysts during the initiation and maturation of chronic infection. Our findings establish that AG are dynamic, exhibiting considerable heterogeneity among tissue cysts at all post-infection time points examined. Quantification of relative steady-state AG levels within tissue cysts reveals a previously unrecognized temporal cycle involving both phases of AG accumulation and utilization over the first 6 weeks of the chronic infection. This AG cycle is temporally coordinated with overall bradyzoite mitochondrial activity. In addition, the staging of AG levels is defined by a period of low accumulation, leading into a phase of high accumulation, followed by apparent rapid utilization associated with a coordinated burst of intra-cyst bradyzoite replication. These findings suggest that AG may represent a key component in the licensing of bradyzoite replication, intimately linking stored metabolic potential to the course of the chronic infection, thereby extending the impact of AG beyond the previously assigned role in transmission. These findings force a fundamental reassessment of the chronic Toxoplasma infection, highlighting the critical need to address the temporal progression of this crucial stage in the parasite life cycle.IMPORTANCEAmylopectin granules (AG) represent a storage polymer of glucose within Toxoplasma gondii bradyzoites, the life cycle stage associated with the chronic infection. In this study, we report on the development of AmyloQuant, an image-based application, to investigate the levels and distribution of AG within encysted bradyzoites in the murine brain with the progression of the chronic infection. Quantification reveals that AG, although heterogeneous both within and across tissue cysts, exhibit a previously unrecognized temporal cycle that is linked to the overall mitochondrial activity and the capacity to replicate in vivo. This confirms that encysted bradyzoites, long considered dormant, retain considerable metabolic activity, with AG playing a potentially critical role in defining and perhaps licensing the progression of this life-long persistent infection.

The asexual stages of Toxoplasma gondii are defined by the rapidly growing tachyzoite during the acute infection and by the slow growing bradyzoite housed within tissue cysts during the chronic infection. These stages represent unique physiological states, each with distinct glucans reflecting differing metabolic needs. A defining feature of T. gondii bradyzoites is the presence of insoluble storage glucans known as amylopectin granules (AGs), the function of which remains largely unexplored during the chronic infection. The presence of storage glucans has more recently been established in tachyzoites, a finding corroborated by specific labeling with the anti-glycogen antibody IV58B6. The T. gondii genome encodes activities needed for glucan turnover inlcuding: a glucan phosphatase (TgLaforin; TGME49_205290) and a glucan kinase (TgGWD; TGME49_214260) that catalyze a cycle of reversible glucan phosphorylation required for glucan degradation by amylases. Disruption of TgLaforin in tachyzoites had no impact on growth under nutrient-replete conditions. Growth of TgLaforin-KO tachyzoites was however severely stunted when starved of glutamine despite being glucose replete. Loss of TgLaforin attenuated acute virulence in mice and was accompanied by a lower tissue cyst burden, without a direct impact on tissue cyst size. Quantification of relative AG levels using AmyloQuant, an imaging based application, revealed the starch-excess phenotype associated with the loss of TgLaforin is heterogeneous and linked to an emerging AG cycle in bradyzoites. Excessive AG accumulation TgLaforin-KO bradyzoites promoted intra-cyst bradyzoite death implicating reversible glucan phosphorylation as a legitimate target for the development of new drugs against chronic T. gondii infections.

The mitochondrion is intimately linked to energy and overall metabolism and therefore the morphology of mitochondrion can be very informative for inferring the metabolic state of cells. In this study we report an approach for automatic classification of mitochondrial morphologies using supervised machine learning to efficiently classify them from a large number of cells at a time. Fluorescence microscopy images of the chronic encysted form of parasite Toxoplasma gondii were used for this development. Manually classifying these morphologies from the hundreds of parasites within typical tissue cysts is tedious and error prone. In addition, because of inherent biological heterogeneity in morphologies, there can be variability and lack of reproducibility in manual classification. We used image segmentation to detect mitochondrial shapes and used features extracted from them in a multivariate logistic regression model to classify the detected shapes into five morphological classes: Blobs, Tadpoles, Lasso/Donuts, Arcs, and Other. The detected shapes from a subset of images were first used to obtain consensus classification among expert users to obtain a labeled set. The model was trained using the labeled set from five cysts and its performance was tested on the mitochondrial morphologies from ten other cysts that were not used in training. Results showed that the model had an average overall accuracy of 87%. There was high degree of confidence in the classification of Blobs and Arcs (average F scores 0.91 and 0.73) which constituted the majority of morphologies (85%). Although the current development used microscopy images from tissue cysts of Toxoplasma gondii, the approach is adaptable with minor adjustments and can be used to automatically classify morphologies of organelles from a variety of cells.

Recent advances into the unique biology of Toxoplasma tissue cysts and the bradyzoites they house necessitate optimization of tissue cyst recovery from infected mouse brains. Here, we present data from 83 tissue cyst purifications of Type II ME49 tissue cysts in CBA/J mice performed over a period of 3 years.

Toxoplasma gondii is an intracellular parasite that generates amylopectin granules (AGs), a polysaccharide associated with bradyzoites that define chronic T. gondii infection. AGs are postulated to act as an essential energy storage molecule that enable bradyzoite persistence, transmission, and reactivation. Importantly, reactivation can result in the life-threatening symptoms of toxoplasmosis. T. gondii encodes glucan dikinase and glucan phosphatase enzymes that are homologous to the plant and animal enzymes involved in reversible glucan phosphorylation and which are required for efficient polysaccharide degradation and utilization. However, the structural determinants that regulate reversible glucan phosphorylation in T. gondii are unclear. Herein, we define key functional aspects of the T. gondii glucan phosphatase TgLaforin (TGME49_205290). We demonstrate that TgLaforin possesses an atypical split carbohydrate-binding-module domain. AlphaFold2 modeling combined with hydrogen-deuterium exchange mass spectrometry and differential scanning fluorimetry also demonstrate the unique structural dynamics of TgLaforin with regard to glucan binding. Moreover, we show that TgLaforin forms a dual specificity phosphatase domain-mediated dimer. Finally, the distinct properties of the glucan phosphatase catalytic domain were exploited to identify a small molecule inhibitor of TgLaforin catalytic activity. Together, these studies define a distinct mechanism of TgLaforin activity, opening up a new avenue of T. gondii bradyzoite biology as a therapeutic target.

Toxoplasma gondii is a parasite that chronically infects about a third of the world's population. During chronic infection, the parasite resides within tissue cysts in the form of poorly understood bradyzoites which can number in the thousands. Our prior work showed that these bradyzoites are metabolically active exhibiting heterogeneous replication potential. The morphological plasticity of the mitochondrion potentially informs about parasite metabolic state. We developed an image processing based program to assist manual classification of mitochondrial morphologies by trained operators to collect data and statistics from the manual classification of shapes. We sought to determine whether certain morphologies were readily classifiable and the congruence among manual classifiers, i.e. the degree to which different operators would place the same objects within the same class. Results from three operators classifying mitochondrial morphologies from 5 tissue cyst images showed that among the four classes, one (Blobs) were the easiest to classify. There was remarkable congruence between 2 of the 3 operators in classifying the objects (96%), while the agreement among all 3 operators was somewhat modest (57%). Such information would be valuable for biologists studying these parasites as well as in development of fully automated methods of morphological classification.

The addition of phosphate groups into glycogen modulates its branching pattern and solubility which all impact its accessibility to glycogen interacting enzymes. As glycogen architecture modulates its metabolism, it is essential to accurately evaluate and quantify its phosphate content. Simultaneous direct quantitation of glucose and its phosphate esters requires an assay with high sensitivity and a robust dynamic range. Herein, we describe a highly-sensitive method for the accurate detection of both glycogen-derived glucose and glucose-phosphate esters utilizing gas-chromatography coupled mass spectrometry. Using this method, we observed higher glycogen levels in the liver compared to skeletal muscle, but skeletal muscle contained many more phosphate esters. Importantly, this method can detect femtomole levels of glucose and glucose phosphate esters within an extremely robust dynamic range with excellent accuracy and reproducibility. The method can also be easily adapted for the quantification of plant starch, amylopectin or other biopolymers.

Toxoplasma gondii is an opportunistic, protozoan parasite that infects roughly one‐third of humans worldwide. Shortly after infection, T. gondii establishes cysts, occupied by the slow‐growing bradyzoite stage in the CNS or muscle tissue where it will persist for the lifetime of the host. Bradyzoite biology is poorly understood, especially in regards to energetics. Accumulation of insoluble carbohydrate structures known as amylopectin granules (AGs) in the cytoplasm is a distinguishing morphological feature of bradyzoites, and thus suggests a potential source of energetic and biosynthetic potential. We have identified multiple enzymes involved in maintaining energetic homeostasis in the T. gondii. We have characterized these enzymes using bioinformatics and biochemical approaches. These studies seek to characterize this set of enzymes as gatekeepers to accessing the metabolic potential stored in AGs.Support or Funding InformationThis project is funded by the IDeA award from NIH/NIGMS 5P30GM110787 (COBRE for the Center for Molecular Medicine. PI Louis B Hersh, University of Kentucky) through the University of Kentucky Center for Molecular Medicine.

PURPOSE OF REVIEW:Despite over a third of the world's population being chronically infected with Toxoplasma gondii, little is known about this largely asymptomatic phase of infection. This stage is mediated in vivo by bradyzoites within tissue cysts. The absence of overt symptoms has been attributed to the dormancy of bradyzoites. In this review, we reexamine the conventional view of chronic toxoplasmosis in light of emerging evidence challenging both the nature of dormancy and the consequences of infection in the CNS.RECENT FINDINGS:New and emerging data reveal a previously unrecognized level of physiological and replicative capacity of bradyzoites within tissue cysts. These findings have emerged in the context of a reexamination of the chronic infection in the brain that correlates with changes in neuronal architecture, neurochemistry, and behavior that suggest that the chronic infection is not without consequence.SUMMARY:The emerging data driven by the development of new approaches to study the progression of chronic toxoplasma infection reveals significant physiological and replicative capacity for what has been viewed as a dormant state. The emergence of bradyzoite and tissue cyst biology from what was viewed as a physiological "black box" offers exciting new areas for investigation with direct implications on the approaches to drug development targeting this drug-refractory state. In addition, new insights from studies on the neurobiology on chronic infection reveal a complex and dynamic interplay between the parasite, brain microenvironment, and the immune response that results in the detente that promotes the life-long persistence of the parasite in the host.

Epidemiological evidence indicates that cadmium and arsenic exposure increase lung cancer risk. Cadmium and arsenic are environmental contaminants that act as endocrine disruptors (EDs) by activating estrogen receptors (ERs) in breast and other cancer cell lines but their activity as EDs in lung cancer is untested. Here, we examined the effect of cadmium chloride (CdCl2) and sodium arsenite (NaAsO2) on the proliferation of human lung adenocarcinoma cell lines. Results demonstrated that both CdCl2 and NaAsO2 stimulated cell proliferation at environmentally relevant nM concentrations in a similar manner to 17β-estradiol (E2) in H1793, H2073, and H1944 cells but not in H1792 or H1299 cells. Further studies in H1793 cells showed that 100 nM CdCl2 and NaAsO2 rapidly stimulated mitogen-activated protein kinase (MAPK, extracellular-signal-regulated kinases) phosphorylation with a peak detected at 15 min. Inhibitor studies suggest that rapid MAPK phosphorylation by NaAsO2, CdCl2, and E2 involves ER, Src, epidermal growth factor receptor, and G-protein coupled ER (GPER) in a pertussis toxin-sensitive pathway. CdCl2 and E2 activation of MAPK may also involve ERβ. This study supports the involvement of membrane ER and GPER signaling in mediating cellular responses to environmentally relevant nM concentrations of CdCl2 and NaAsO2 in lung adenocarcinoma cells.