Biology subjects

White, M. W.

Publications and source records attributed to White, M. W..

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↗

bioRxiv2022

AP2XII-2 coordinates transcriptional repression for Toxoplasma gondii sexual commitment

Toxoplasma gondii is a widespread protozoan parasite that has significant impact on human and veterinary health. The parasite undergoes a complex life cycle involving multiple hosts and developmental stages. How Toxoplasma transitions between life cycle stages is poorly understood, yet central to controlling transmission. Of particular neglect are the factors that contribute to its sexual development, which takes place exclusively in feline intestines. While epigenetic repressors have been shown to play an important role in silencing spurious gene expression of sexually committed parasites, the specific factors that recruit this generalized machinery to the appropriate genes remains largely unexplored. Here, we establish that a member of the AP2 transcription factor family, AP2XII-2, is targeted to genomic loci associated with sexually committed parasites along with the epigenetic regulators of transcriptional silencing, HDAC3 and MORC. Despite widespread association with gene promoters, AP2XII-2 is required for silencing of relatively few genes. Using CUT&Tag methodology, we identify two major genes associated with sexual development downstream of AP2XII-2 control, AP2X-10 and the amino acid hydroxylase AAH1. Our findings show that AP2XII-2 is a key contributor to the gene regulatory pathways modulating Toxoplasma sexual development. IMPORTANCEToxoplasma gondii is a parasite that undergoes its sexual stage exclusively in feline intestines, making cats a major source of transmission. A better understanding of the proteins controlling the parasites life cycle stage transitions is needed for the development of new therapies aimed to treat toxoplasmosis and transmission of the infection. Genes that regulate the sexual stages need to be turned on and off at the appropriate times, activities that are mediated by specific transcription factors that recruit general machinery to silence or activate gene expression. In this study, we identify a transcription factor called AP2XII-2 as being important for repression of a subset of sexual stage genes, including a sexual stage-specific AP2 factor (AP2X-10) and a protein (AAH1) required to construct the infectious oocysts expelled by infected cats.

microbiology↗

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

Toxoplasma gondii AP2XII-2 contributes to proper progression through S-phase of the cell cycle

Toxoplasma gondii is a protozoan parasite that causes lifelong chronic infection that can reactivate in immunocompromised individuals. Upon infection, the replicative stage (tachyzoite) converts into a latent tissue cyst stage (bradyzoite). Like other apicomplexans, T. gondii possesses an extensive linage of proteins called ApiAP2s that contain plant-like DNA-binding domains. The function of most ApiAP2s is unknown. We previously found that AP2IX-4 is a cell cycle-regulated ApiAP2 expressed only in dividing parasites as a putative transcriptional repressor. In this study, we purified proteins interacting with AP2IX-4, finding it to be a component of the recently characterized microrchidia (MORC) transcriptional repressor complex. We further analyzed AP2XII-2, another cell cycle-regulated factor that associates with AP2IX-4. We monitored parallel expression of AP2IX-4 and AP2XII-2 proteins in tachyzoites, detecting peak expression during S/M phase. Unlike AP2IX-4, which is dispensable in tachyzoites, loss of AP2XII-2 resulted in a slowed tachyzoite growth due to a delay in S-phase progression. We also found that AP2XII-2 depletion increased the frequency of bradyzoite differentiation in vitro. These results suggest that multiple AP2 factors collaborate to ensure proper cell cycle progression and tissue cyst formation in T. gondii. ImportanceToxoplasma gondii is a single-celled parasite that causes opportunistic infection due to its ability to convert into a latent cyst stage. This work describes a new transcriptional factor called AP2XII-2 that plays a role in properly maintaining the growth rate of replicating parasites, which contributes to signals required for development into its dormant stage. Without AP2XII-2, Toxoplasma parasites experience a delay in their cell cycle that increases the frequency of latent cyst formation. In addition, we found that AP2XII-2 operates in a multi-subunit complex with other AP2 factors and chromatin remodeling machinery that represses gene expression. These findings add to our understanding of how Toxoplasma parasites balance replication and dormancy, revealing novel points of potential therapeutic intervention to disrupt this clinically relevant process.

microbiology↗

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

The Toxoplasma centrocone houses cell cycle regulatory factors

Our knowledge of cell cycle regulatory mechanisms in apicomplexan parasites is very limited. In this study, we describe a novel Toxoplasma gondii factor, essential for chromosome replication 1 (ECR1), that has a vital role in chromosome replication and the regulation of cytoplasmic and nuclear mitotic structures. ECR1 was discovered by complementation of a temperature sensitive (ts) mutant that suffers lethal, uncontrolled chromosome replication at 40{degrees}C similar to a ts-mutant carrying a defect in topoisomerase. ECR1 is a 52kDa protein containing divergent RING and TRAF-Sina like zinc-binding domains that is dynamically expressed in the tachyzoite cell cycle. ECR1 first appears in the centrocone compartment of the nuclear envelope in early S phase and then in the nucleus in late S phase where it reaches maximum expression. Following nuclear division, but before daughters resolve from the mother, ECR1 is down regulated and is absent in new daughter parasites. The proteomics of ECR1 identified interactions with the ubiquitin-mediated protein degradation machinery and the minichromosome maintenance complex and the loss of ECR1 led to increased stability of a key member of this complex, MCM2. ECR1 also forms a stable complex with the CDK-related kinase, TgCrk5, which shares a similar cell cycle expression and localization during tachyzoite replication. Altogether, the results of this study suggest ECR1 may be a unique E3 ligase that regulates DNA licensing and other mitotic processes. Importantly, the localization of ECR1/TgCrk5 in the centrocone indicates this Apicomplexa-specific spindle compartment houses important regulatory factors that control the parasite cell cycle.\n\nIMPORTANCEParasites of the apicomplexan family are important causes of human disease including malaria, toxoplasmosis, and cryptosporidiosis. Parasite growth is the underlying cause of pathogenesis, yet despite this importance the molecular basis for parasite replication is poorly understood. Filling this knowledge gap cannot be accomplished by mining recent whole genome sequencing because apicomplexan cell cycles differ substantially and lack many of the key regulatory factors of well-studied yeast and mammalian cell division models. We have utilized forward genetics to discover essential factors that regulate cell division in these parasites using the Toxoplasma gondii model. An example of this approach is described here with the discovery of a putative E3 ligase/protein kinase mechanism involved in regulating chromosome replication and mitotic processes of asexual stage parasites.

microbiology↗

bioRxiv2017

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 form that is invisible 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 immune compromised patients. Transcriptional changes accompany conversion of the rapidly replicating tachyzoites into the encysted bradyzoites, 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 many bradyzoite mRNAs dramatically increased beyond 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 also 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.

microbiology↗

bioRxiv2016

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 role 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 these factors have opposite functions in bradyzoite development. Conversely, conditional overexpression of FKBP-modified of 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 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 specific binds the BAG1 promoter in parasite chromatin. Altogether, these results suggest the AP2IX-9 transcriptional repressor and 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.\n\nIMPORTANCEToxoplasma infections are life-long due to 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 formation of the tissue cyst 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 mechanisms are not well defined and the operating principles of ApiAP2 mechanisms are poorly understood. Here we establish that competing ApiAP2 transcriptional mechanisms operate to regulate this clinically important developmental pathway.

microbiology↗