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Current Research in the Sinai laboratory focuses on two areas, which overlap at the level of the parasite cell cycle and life cycle with an emphasis on the biology of the poorly understood chronic phase of Toxoplasma infection. In our work (Watts et al. 2015 mBio) we established for the first time that parasirtes within tissue cysts, long thought to be dormant, exhibit considerbable replicative ability which could be harnessed to develop drugs against a form that is viewed as being refractory to treatment. This work pioneered the development of quantitative imaging based approaches developed in collaboration with Dr. Abhijit Patwardhan (UK College of Engineering), to quantify actual parasite (bradyzoite) burdens within purified tissue cyst. This breakthrough highlighed the fact that contrary to long held dogma (based primarily on anecdotal evidence), bradyzoites retain considerable metabolic activity, but dsiplay in patterns that remain to elucdiated. Supported by a collaborative RO1 grant that is expected to be funded in July 2019 (8th percentile score), we will further advance the imaging approches to be able etsblish not only absolute parasire burdens but also gain insights into the dynamics of replication, including the recency of replication, mitochondrial morphology/activity and the patterns of amylopectin (starch) accumulation (see below). By integrating these physiological variable along the temporal progression of the chronic infection, we expect to be able to identify the patterns associated associated with bradyzoite behavior in vivo. The dissection of these data will be facilitated by the development of computational models seeking to match the experimental data to predictive systems. This information, will be critically important to the design of drug treatment regimens, an aspect that will be addressed using these newly developed tools to determine the physiolgical consequences of drug treatments on what have been viewed as refractory organisms.
An additional area of investigation into bradyzoite biology centers on dissecting the role of amylopectin, an insoluble starch-like polymer of glucose that is the hallmark of bradyzoites. In a project with the laboratory of Dr. Matthew Gentry (Dept. of Mol. Cell. Biochem), we are currently dissecting the enzymology of AG turnvoer to establish the role of this storage polymer in the progression of the chronic infection. Given that AG patterns within bradyzoites are highly variable, we posit that the stored glucose may be a key metabolite powering the replicaiton of bradyzoites. We currently have a R21 to address the role of AG in the chronic infection pending consideration for funding.
The identification of bradyzoties as replicating entities led us to investigated the cell cycle in the chronic infection and its regulation. We focused on the contributon of cell cycle regulated Ovarian TUmor (OTU) family deubiquitinases. Our work (Dhara and Sinai mSphere 2016) establised that specific ubiqutin linkage modifications exhibited dynamic changes during the cell cycle while defining the biochemical and enzymological properties of TgOTUD3A. The targeted abalation of TgOTUD3A presented a remarkbale phenotype indicating a complex role in the regulation of the cell cycle architecture whereby mutants exhibited high fidelity replication using alternate replicaiton strategies (schizogony and endopolygeny) in addition to the normal endodyogeny (Dhara et al. mBio 2017). In addtion these mutants appear to be developmentally abberant as they express markers of the chronic infection as well as induce the expression of genes associated with the sexual cycle. The secodn major focus of the lab is to establish the molecular basis for these developmental abberations that appear to link the control of the cell cyle to the life cycle. We currently have an R21 proposal pending to address aspects of these complex phenotypes with additional grants under development.
Earlier work in the laboatory had focused on the pathogen-host interaction with the characterization of host cell pathways manipualted by the parasite to establish a productive infection. we are now exploring the role of ubiqutin/ autophagy mediated mechanisms prevalent in the Interferon resposne in human cells and potential parasite countermeasures to neutralize these defenses. The laboratory had also focused on the dissection of the role of autophagy as a regulated cell death pathway in the parasite. This is remarkable for a single cell organism and is likely to be exploitable in the development of drugs against Toxoplasma as well as Plasmodium, the agent of malaria.
7/2019- 6/2022. RO1AI145335-01A1: Quantitative approaches for drug testing in chronic toxoplasmosis: leveraging new insights into bradyzoite biology within tissue cysts in vivo. Multi-PI: Sinai, PI: Patwardhan.
This proposal aims to develop new imgaing based approches to monitor and quantify physiolgical markers to better understand the progression of the chronic infection. Integration of data assessing replication, mitochondrial function and amylopectin levels will be used to develop a computational framework to understand the evolution of the chronic infection in vivo to aid in the development of better treatment regimens.
12/2019-11/2021. R21AI150285-01: Extragenic adaptation to the loss of a deubiqutinase affecting the T. gondii cell cycle and development. PI; Sinai
The loss of TgOTUD3A, a deubiqutinase coincides with the dysregualtion of the cell cycle and ectopic expression of developmental markers, In addition, we observe the selective induciton of the Clade D1 OTU family members, the charaterization of which are the focus of this proposal.
12/2019-11/2021 R21 (NIHID: 9940207): Role of amylopectin granules in chronic toxoplasmosis, an HIV-AIDS defining infection. PI: Sinai
The accumulation of amylopectin granules is a feature of the chronic infection, the precise role of which remains unknown. With this study we propose to determine the effect of intefering with AG synthesis on the acute infection, stage conversion and the progression of the chronic phase. Finally, we will also address whether AG play a role in reactivation, the triggering event in symtomatic toxoplasmosis in HIV-AIDS.
Biology of Toxoplasma gondii bradyzoites and the chronic infection.
Watts E., Y. Zhao, A. Dhara, B. Eller , A. Patwardhan and AP Sinai (2015) Novel approaches reveal that Toxoplasma gondii bradyzoites within tissue cysts are dynamic and replicating entities in vivo MBio: 6(5): pii: e01155-15. Doi: 10.1128/mBio.01155-15
Sinai AP, EA Watts, A. Dhara, RD Murphy, MS Gentry and A Patwardhan (2016) Reexamining chronic Toxoplasma gondii infection: Surprising activity for a “dormant” parasite. Curr. Clin Micro. Rpt. Curr. Clin Micro. Rpt. 3:175 d.o.i:10.1007/s40588-016-0045-3
Watts E.A., A. Dhara and A.P. Sinai (2017) Purification of Toxoplasma gondii tissue cysts using Percoll gradients. Current Protocols in Microbiology 45:20C.2.1-20C.2.19. doi: 10.1002/cpmc.30.
Ovarian TUmor (OTU) family deubiquitinases regulating the Toxoplasma cell cycle and development
Dhara A. and AP Sinai (2016) A cell cycle-regulated Toxoplasma deubiquitinase, TgOTUD3A, targets polyubiquitins with specific lysine linkages. mSphere 1(3): e00085-16 PMID27340699
Dhara, A., R. De Paula Baptista, J.C. Kissinger, E.C. Snow and A.P. Sinai (2017) Ablation of an OTU-family deubiquitinase exposes the underlying regulation governing the plasticity of cell cycle progression in Toxoplasma gondii. mBio.8(6): e01846-1. Doi: 10.1128/mbio.01846-17.