Biology subjects

Wilson, E. H.

Publications and source records attributed to Wilson, E. H..

8 recordsLinked to original sources

bioRxiv2025

Bradyzoite subtypes rule the crossroads of Toxoplasma development

Reactivation of toxoplasmosis is a significant health threat to chronically infected individuals, especially those who are or become immunocompromised. An estimated one-third of the world population is infected with Toxoplasma, placing millions at risk. The Toxoplasma cyst is the foundation of disease with its ingestion leading to infection and its reactivation, from slow replicating bradyzoites to fast replicating tachyzoites, leading to cell lysis in tissues such as the brain. There are no treatments that prevent or eliminate cysts in part due to our poor understanding of the mechanisms that underlie cyst formation and recrudescence. In this study, we aimed to understand the biology of bradyzoites prior to recrudescence and the developmental pathways they initiate. We have discovered ME49EW cysts from infected mice harbor multiple bradyzoite subtypes that can be identified by their expression of distinct proteins. Sorting of these subtypes revealed they initiate distinct developmental pathways in animals and in primary astrocyte cell cultures. Single bradyzoite RNA sequencing indicates 5 major bradyzoite subtypes occur within these cysts. We further show that a crucial subtype comprising the majority of bradyzoites in chronically infected mice is absent from conventional in vitro models of bradyzoite development. Altogether this work establishes new foundational principles of Toxoplasma cyst development and reactivation that operate during the intermediate life cycle of Toxoplasma.

microbiology↗

bioRxiv2024

Toxoplasma gondii infection of neurons alters the production and content of extracellular vesicles altering astrocyte phenotype and contributing to the loss of GLT-1 in the infected brain

Toxoplasma gondii (T. gondii) cyst formation in the central nervous system only occurs in neurons allowing the parasite to remain latent for the lifetime of the host. Astrocytes are fundamental to neuronal health by providing nutrients and structural support and help regulate neurotransmitters by continuous communication with neurons. It is not yet known how infection and the presence of intracellular cysts, disrupts the crucial relationship between these cells. Extracellular vesicles (EVs) function in intracellular communication and can contain proteins, lipids, DNA, miRNA, and other RNA subtypes. EVs are produced by all cells including neurons and play an important role in neuronal-astrocyte interactions including the regulation of glutamate receptors on astrocytes. Previous work has demonstrated Toxoplasma infection reduces astrocytic expression of the primary glutamate transporter, GLT-1. Here we tested if cyst infection of neurons alters the production and content of EVs. EVs were isolated from uninfected and infected primary murine cortical neurons and their size, concentration, and characterization were confirmed with nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), CD63 ELISA, liquid chromatography (LC)-mass spectrometry (MS)/MS, and microRNA Sequencing. Analysis reveals that infection of neurons reduced neuronal production of EVs and altered their protein and miRNA content. EVs from infected neurons contained secreted Toxoplasma proteins GRA1, GRA2, GRA7, MAG1 and MAG2 associated with cyst formation. Following incubation of neuronal EVs with primary astrocytes, a proportion of EVs colocalize to the nucleus. EVs from infected neurons altered gene expression of astrocytes leading to a downregulation of GLT-1 protein expression and an increase in pro-inflammatory transcriptional signatures. These results demonstrate the ability of a parasitic infection in the brain to alter EV production and the fundamental communication between neurons and astrocytes.

microbiology↗

bioRxiv2023

An Automated Scientist to Design and Optimize Microbial Strains for the Industrial Production of Small Molecules

Engineering microbes to synthesize molecules of societal value has historically been a time consuming and artisanal process, with the synthesis of each new non-native molecule typically warranting its own separate publication. Because most microbial strain engineering efforts leverage a finite number of common metabolic engineering design tactics, we reasoned that automating these design steps would help create a pipeline that can quickly, cheaply, and reliably generate so-called microbial factories. In this work we describe the design and implementation of a computational system, an Automated Scientist we call Lila, which handles all metabolic engineering design and optimization through the design-build-test-learn (DBTL) paradigm. Lila generates metabolic routes, identifies relevant genetic elements for perturbation, and specifies the design and re-design of microbial strains in a matter of seconds to minutes. Strains specified by Lila are then built and subsequently phenotyped as part of a largely automated in-house pipeline. Humans remain in-the-loop to curate choices made by the system, helping for example to refine the metabolic model or suggest custom protein modifications. Lila attempted to build strains that could produce 454 biochemically diverse molecules with precursors located broadly throughout the metabolism of two microbial hosts, Saccharomyces cerevisiae and Escherichia coli. Notably, we observed the highest published titers for the molecule naringenin, the metabolic precursor to flavonoids. In total we created hundreds of thousands of microbial strains capable of overproducing 242 molecules, of which 180 are not native to S. cerevisiae or E. coli.

synthetic biology↗

bioRxiv2022

Neutrophils in the brain are sources of neuroprotective molecules and demonstrate functional heterogeneity during chronic Toxoplasma gondii infection

Infection with the protozoan parasite Toxoplasma gondii leads to the formation of lifelong cysts in neurons of the brain that can have devastating consequences in the immunocompromised. However, despite the establishment of a chronic inflammatory state and infection-induced neurological changes, there are limited signs of clinical neuropathology resulting in an asymptomatic infection in the immunocompetent. This suggests the work of neuroprotective mechanisms to prevent clinical manifestations of disease. However, such sources of neuroprotection during infection remain largely unknown. This study identifies a population of neutrophils chronically present in the brain during Toxoplasma infection that express the neuroprotective molecules NRG-1, ErbB4, and MSR1. Further phenotyping of this population via flow cytometry and singe-cell RNA sequencing reveals two distinct subsets of neutrophils based on age that display functional heterogeneity. This includes cells transcriptionally prepared to function both as anti-parasitic effector cells and in a more alternative protective manner. Chronic depletion of neutrophils results in increased parasite burden and infection-induced vascular pathology. Lack of neutrophils during chronic infection also deleteriously affects neuronal regeneration and repair mechanisms. In conclusion, this work identifies and demonstrates a functionally diverse chronic neutrophil population that plays a dynamic role in controlling infection outcome in the CNS by balancing classical responses with neuroprotective functions. Author SummaryThe predominantly asymptomatic nature of chronic Toxoplasma gondii infection despite the life-long infection of neurons suggests that there are neuroprotective mechanisms at work in the brain to maintain homeostasis and integrity. This study identifies neutrophils, normally considered a first-responding innate immune cell, as a prominent source of neuroprotective molecules during Toxoplasma infection. Aged neutrophils in the brain exhibit an ability to be functionally flexible expressing signatures of classical proinflammatory responses; and neuroprotective, pro-angiogenic indicators. Lack of neutrophils during chronic infection leads to increased parasite burden, increased vascular damage, and decreased neuronal regeneration. We conclude that chronic brain neutrophils are a functionally dynamic population and a source of neuroprotection during infection and suggest that this is a potentially novel target to promote brain tissue repair without compromising anti-microbial activity.

immunology↗

bioRxiv2022

Engineering Toxoplasma transgenic tissue cysts.

Current approaches to find therapeutic solutions to treat and prevent reactivation of toxoplasmosis have suffered from limited accessibility to the relevant Toxoplasma stages and a lack of accurate in vitro developmental models. The loss of developmental competency in vitro that is exacerbated during the generation of transgenic tachyzoites is also a major impediment to understanding the molecular basis of bradyzoite recrudescence, which is the central parasite stage of reactivation. We have successfully modified Toxoplasma genes in the bradyzoite tissue cyst stage using ex vivo bradyzoite-based methods. Our new protocols validated the clonality of individual cysts and confirmed that single tissue cysts can robustly infect mice. We demonstrated these protocols by generating in vivo bradyzoites carrying a knockout of the Toxoplasma HXGPRT gene or the gene encoding the ApiAP2 transcription factor, AP2IX-9. Unexpectedly, the knockout of the AP2IX-9 gene in the Type II ME49EW strain eliminated one of the three developmental pathways initiated by the bradyzoite: host-dependent bradyzoite-to-bradyzoite replication. Our genetic protocols were further validated by producing in vivo bradyzoites lacking the bradyzoite-specific cyclin, TgCYC5. Interestingly, genetic ablation of TgCYC5 led to a large number of small cysts that formed from single mother cysts in mouse brain. Further study revealed the cause of the small-cyst phenotype in TgCYC5 knockout parasites was a disruption in the normal balance of bradyzoite subtypes that enhanced the bradyzoite-to-bradyzoite developmental pathway. These new data demonstrate the feasibility of generating transgenic parasites in a developmentally competent strain using ex vivo bradyzoite-based methods. Furthermore, by targeting the AP2IX-9 and TgCYC5 genes, whose transcripts are upregulated specifically in bradyzoites, we have clarified how these factors influence the pathways of tissue cyst recrudescence.

developmental biology↗

bioRxiv2020

An ex vivo model of Toxoplasma recrudescence

Toxoplasma has been a useful parasite model for decades because it is relatively easy to genetically modify and culture, however, attempts to generate and study the recrudescence of tissue cysts have come up short with lab-adapted strains generating low numbers of tissue cysts in vivo. Here we have established a new model of Toxoplasma recrudescence using bradyzoites from an unadapted Type II ME49 strain (ME49EW) isolated from murine brain tissue. Ex vivo bradyzoite infection of fibroblasts and astrocytes produced sequential tachyzoite growth stages; a fast-growing stage was followed by formation of a slower-growing stage. In astrocytes, but not in fibroblasts, bradyzoites also initiated a second recrudescent pathway involving bradyzoite to bradyzoite replication. Intraperitoneal infections of mice with either bradyzoites or the fast-growing tachyzoite stage efficiently disseminated to brain tissue leading to high numbers of tissue cysts, while infections with the slow-growing tachyzoite stage were largely retained in the peritoneum. Poor infection and cyst formation of slow-growing tachyzoites was reversible by serial tissue cyst passage, while the poor tissue cyst formation of lab-adapted tachyzoites was not reversible by these approaches. To distinguish strain developmental competency, we identified Toxoplasma genes highly expressed in ME49EW in vivo tissue cysts and developed a qPCR approach that differentiates immature from mature bradyzoites. In summary, the results presented describe a new ex vivo bradyzoite recrudescence model that fully captures the growth and developmental processes during toxoplasmosis reactivation in vivo opening the door to the further study of these important features of the Toxoplasma intermediate life cycle.

microbiology↗

bioRxiv2017

Large-scale interpretation of chemical-genetic interaction profiles using a genetic interaction network

Chemical-genetic interactions - observed when the treatment of mutant cells with chemical compounds reveals unexpected phenotypes - contain rich functional information linking compounds to their cellular modes of action. To systematically identify these interactions, an array of mutants is challenged with a compound and monitored for fitness defects, generating a chemical-genetic interaction profile that provides a quantitative, unbiased description of the cellular function(s) perturbed by the compound. Genetic interactions, obtained from genome-wide double-mutant screens, provide a key for interpreting the functional information contained in chemical-genetic interaction profiles. Despite the utility of this approach, integrative analyses of genetic and chemical-genetic interaction networks have not been systematically evaluated. We developed a method, called CG-TARGET (Chemical Genetic Translation via A Reference Genetic nETwork), that integrates large-scale chemical-genetic interaction screening data with a genetic interaction network to predict the biological processes perturbed by compounds. CG-TARGET compared favorably to a baseline enrichment approach across a variety of benchmarks, achieving similar accuracy while substantially improving the ability to control the false discovery rate of biological process predictions. We applied CG-TARGET to a recent screen of nearly 14,000 chemical compounds in Saccharomyces cerevisiae, integrating this dataset with the global S. cerevisiae genetic interaction network to prioritize over 1500 compounds with high-confidence biological process predictions for further study. Upon investigation of the compatibility of chemical-genetic and genetic interaction profiles, we observed that one-third of observed chemical-genetic interactions contributed to the highest-confidence biological process predictions and that negative chemical-genetic interactions overwhelmingly formed the basis of these predictions. We present here a detailed characterization of the CG-TARGET method along with experimental validation of predicted biological process targets, focusing on inhibitors of tubulin polymerization and cell cycle progression. Our approach successfully demonstrates the use of genetic interaction networks in the functional annotation of compounds to biological processes.

systems biology↗

bioRxiv2017

Transcriptional repression by ApiAP2 factors is central to chronic toxoplasmosis

Bradyzoite 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 important Toxoplasma transcriptional 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-{gamma} 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 for Toxoplasma to establish a chronic infection in the immune-competent host.\n\nAuthor SummaryThe Toxoplasma biology 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 chronic Toxoplasma infections.

microbiology↗