Biology subjects

White, M. W.

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

3 recordsLinked to original sources

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↗