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In addition to being an important opportunistic pathogen, the protozoan parasite Toxoplasma gondii is an excellent model for the study of Apicomplexan parasites and pathogen-host interaction. Among the early interests of the laboratory has been the dissection of the parasite and host factors involved in pathogenesis. This work focused predominantly on the dysregulation of host apoptotic pathways with an emphasis on the NFkappaB cascade and the mitochondrial contributions. Among the key findings were the confirmation of parasite mediated dysregulation of apoptosis involving a complex rewiring of signaling, metabolism and cell cycle progression in the infected cell.
The interest in host cell apoptosis (a mechanism of programmed cell death) led us to explore the possibility of regulatory cascades within Toxoplasma that may be involved in promoting parasite death. Our findings that the pathways triggered by nutrient limitation result in an unusual autophagy-related death that is associated with a selective fragmentation of the parasite mitochondrion links intermediary metabolism, to energy metabolism and the control of parasite viability. This presents an evolutionary paradox as the retention of a cell death/suicide pathway in a single cell organism does not at face value seem plausible. In the course of dissecting the molecular components regulating autophagy in Toxoplasma we now realize that while dysregulated autophagy triggered by severe acute nutrient limitation results in death, tight control of autophagy appears required for parasite replication providing a crucial insight into its evolutionary retention.We are actively exploring the role of the autophagy associated proteins TgATG8 and TgATG1 in parasite replication as mutations in both genes cause lethality due to defective replication. Studies on the intersection between the replicative and autophagic pathways are additionally focused on examining the effectiveness of PI3kinase inhibitors as potential drug targets as PI3Kinases are critical in both pathways.
Our broad interest in the regulation of both autophagy and replication led us to investigate the contribution of the ubiquitination pathway in regulating the fidelity of endodyogeny. We are focused on a cell cycle regulated member of the OTU-family of deubiquitinases the targeted deletion of which results in a myriad of defects in the replication of the parasite. Among these defects is an apparent loss in the fidelity of replication where in a subset of mutants, mother parasites generate not 2 but 3,4 and even 5 progeny. This phenotype is akin to schizogeny a replication strategy used by the malaria parasite Plasmodium spp as well sexual stages of Toxoplasma. Our studies on the autophagy pathway in Toxoplasma have led to collaboration with Dr. Paul Roepe (Georgetown University, Washington DC) where we have established a central role for autophagy associated processes in the resistance of malaria parasites to lethal doses of chloroquine. We are continuing this collaboration to develop reagents for drug screens in both Toxoplasma and Plasmodium to exploit the autophagic cascade. In addition, we are focused on further exploring the impact of autophagy on the unusual mechanisms of cytokinesis involved in the replication of Apicomplexa.
The second main area of interest deals with the establishment of tools and approaches to study the experimentally intractable tissue cyst form of the parasite associated with chronic infection. In our recently accepted work (Watts et al. 2015, MBio) we definitively demonstrate for the first time that the bradyzoite forms in tissue cysts, long considered to be dormant are actually capable of active replication in vivo contrary the prevailing dogma. In addition a collaboration with Dr. Abhijit Patwardhan, UK College of Engineering, has yielded an imaging based method to quantify bradyzoite burdens within tissue cysts that has permitted for the first time the direct quantification at the resolution of individual organisms in cysts. This technology has allowed unprecedented insights into the mechanisms and patterns of bradyzoite growth in vivo. The Patwardhan and Sinai laboratories are actively collaborating developing methodologies for the computational modelling of parasite replication in vivo to better understand the effect of drug and other treatments.
Additional studies have recently been completed to dissect the N-linked glycosylation pathway in Toxoplasma and its impact on both the chronic and acute infection. In addition, we have made significant inroads into the glycobiology of Toxoplasma cysts with evidence strongly suggesting that glycan modification in tissue cysts are hybrid entities derived from both parasite and host activities. This is particularly evident in the context of sialic acid, a sugar for which the parasite lacks any genetic capacity to synthesize or use as a modification. We are completing the studies that point to the mechanisms underlying the sialylation of parasite proteins in tissue cysts in vivo.
Finally, the laboratory is supported by a collaborative COBRE grant with Dr. Matthew Gentry (UK Department of Molecular and Cellular Biochemistry) to study the dynamics and metabolism of starch (amylopectin) granules in Toxoplasma with a focus on 2 key regulators, the starch phosphatase laforin and the kinase GWD (Glucan Water Dikinase). Our findings based on the implementation of new imaging approaches reveals that amylopectin distribution in tissue cysts is non uniform and its levels may be connected to mitochondrial activitiy as key players in overall energy metabolism.
Our interests in Science extend beyond its practice in the laboratory toward the realm of K-12 education. Our lab, together with the lab of Dr. Luke Bradley, UK- Department of Anatomy and Neurobiology have established a unique program with The Learning Center at Linlee (TLC) a Fayette County School for at risk students. At the heart of this initiative, led on the part of TLC by Dr. Scott Diamond, the science teacher and innovator in STEM education is a program to provide high school students real world research experiences. The Partnership for Authentic Student Teaching and Research (PASTR) will engage students in the application of a unique phage display technology platform developed by Dr. Bradley to the development of low cost diagnostics against parasitic diseases. A key element of this program has been the establishment of a laboratory within TLC to reinforce lessons learned when students visit our laboratories at UKThe following link is to a press release about the program: http://uknow.uky.edu/content/uk-tlcpartnership-nurtures-promising-young-....
13-15 NIH/NIAID R21AI099509-01 Host glycosyltransferases in the glycosylation of Toxoplasma proteins. PI: Sinai (direct cost $275,000) This proposal explores the potential for host glycosyltransferase involvement in the glycosylation of parasite proteins in the vacuolar space and PVM as well as the tissue cyst. The study was prompted in part by the finding of sialylated glycoproteins in the vacuole and tissue cyst wall/matrix despite the absence of any sialyltransferases in the parasite genome. Mechanisms underlying the potential delivery of such activities to the PV are explored.
15-16 COBRE: Center for Molecular Medicine, University of Kentucky College of Medicine. Carbohydrate metabolism in Toxoplasma gondii as a potential drug target Role PI: Sinai; Co-PI: Gentry (direct cost $50,000) This collaborative pilot project aims to investigating the potential of amylopectin (starch) metabolism as a potential drug target in bradyzoites. Bradyzoites are defined in part by the accumulation of amylopectin granules, the function of which has not been established. In this study we will investigate the function of Toxoplasma Laforin (TgLaforin) and Glucan Water Dikinase (TgGWD) both key enzymes in amylopectin metabolism. Dr. Gentry studies the enzymology of the mammalian, plant and algal homologs.
15-16 KSEF-3439-RDE-018 Exploiting autophagy for drug discovery in apicomplexan parasites Role: PI (direct cost $30,000) This study focuses on the development of new screenign approches to identify drugs capable of triggering the autophagy-mediated death pathway and evaluate the effectiveness of drugs targeting PI3kinases as antiparasitic agents.
15-17 NIH/NIAID 1R21 AI122894-01 The cell cycle of T. gondii bradyzoites within tissue cysts: in vivo development of an HIV-AIDS opportunistic parasite. PI: Sinai ($275,000 diret cost) Score 21/payline 30. This study focuses on the development of new strategies and tools to dissect the cell cycle of bradyzoites in vivo with an emphasis on establishing the imapact of immuen supression on the bardyzoite to tachyzoite conversion.
Recently Completed (past 3 years)
12-14 NIH/NIAID R21AI098371-01A1 Contribution of N-glycosylation to the Toxoplasma glycoproteome. Role: PI: 10% effort ($275,000 direct cost).
10-12 NIH/NIAID, RO3AI092170-02, Novel Activities and the T. gondii vacuolar membrane. Role: PI: 3% effort, ($100,000- direct cost).
12-13 KSEF-2624-RDE-015 (Kentucky Science and Engineering Foundation). Autophagy in the protozoan parasite Toxoplasma gondii. Role: PI: ($50,000 direct cost)
Article in Press
Watts E, Y. Zhao, A. Dhara, B. Eller, A. Patwardhan and A.P. Sinai (2015) Novel approaches reveal that Toxoplasma gondii bradyzoites within tissue cysts are dynamic replicating entities in vivo. MBio in press
- Watts, E.A.;Dhara, A.;Sinai, A.P. "Purification Toxoplasma gondii Tissue Cysts Using Percoll Gradients." Current protocols in microbiology 45, (2017): 20C.2.1-20C.2.19. [PubMed Link] | [ Full text ]
- Dhara, A.;Sinai, A.P. "A Cell Cycle-Regulated Toxoplasma Deubiquitinase, TgOTUD3A, Targets Polyubiquitins with Specific Lysine Linkages." mSphere 1, 3 (2016): [PubMed Link] | [ Full text ]
- Sinai, A.P.;Watts, E.A.;Dhara, A.;Murphy, R.D.;Gentry, M.S.;Patwardhan, A. "Reexamining Chronic <i>Toxoplasma gondii</i> Infection: Surprising Activity for a "Dormant" Parasite." Current clinical microbiology reports 3, 4 (2016): 175-185. [PubMed Link] |
- Watts, E.;Zhao, Y.;Dhara, A.;Eller, B.;Patwardhan, A.;Sinai, A.P. "Novel Approaches Reveal that Toxoplasma gondii Bradyzoites within Tissue Cysts Are Dynamic and Replicating Entities In Vivo." mBio 6, 5 (2015): e01155-15. [PubMed Link] | [ Full text ]
- Weiss, L.M.;Cushion, M.T.;Didier, E.;Xiao, L.;Marciano-Cabral, F.;Sinai, A.P.;Matos, O.;Calderon, E.J.;Kaneshiro, E.S. "The 12th International Workshops on Opportunistic Protists (IWOP-12)." The Journal of eukaryotic microbiology 60, 3 (2013): 298-308. [PubMed Link] | [ Full text ]
- Weilhammer, D.R.;Iavarone, A.T.;Villegas, E.N.;Brooks, G.A.;Sinai, A.P.;Sha, W.C. "Host metabolism regulates growth and differentiation of Toxoplasma gondii." International journal for parasitology 42, 10 (2012): 947-59. [PubMed Link] | [ Full text ]
- Sinai, A.P.;Roepe, P.D. "Autophagy in Apicomplexa: a life sustaining death mechanism?" Trends in parasitology 28, 9 (2012): 358-64. [PubMed Link] | [ Full text ]
- Ghosh, D.;Walton, J.L.;Roepe, P.D.;Sinai, A.P. "Autophagy is a cell death mechanism in Toxoplasma gondii." Cellular microbiology 14, 4 (2012): 589-607. [PubMed Link] | [ Full text ]
- Carmen, J.C.;Sinai, A.P. "The Differential Effect of Toxoplasma Gondii Infection on the Stability of BCL2-Family Members Involves Multiple Activities." Frontiers in microbiology 2, (2011): 1. [PubMed Link] | [ Full text ]
- Liu, T.;Martin, A.M.;Sinai, A.P.;Lynn, B.C. "Three-layer sandwich gel electrophoresis: a method of salt removal and protein concentration in proteome analysis." Journal of proteome research 7, 10 (2008): 4256-65. [PubMed Link] | [ Full text ]
- Molestina, R.E.;El-Guendy, N.;Sinai, A.P. "Infection with Toxoplasma gondii results in dysregulation of the host cell cycle." Cellular microbiology 10, 5 (2008): 1153-65. [PubMed Link] | [ Full text ]
- Nelson, M.M.;Jones, A.R.;Carmen, J.C.;Sinai, A.P.;Burchmore, R.;Wastling, J.M. "Modulation of the host cell proteome by the intracellular apicomplexan parasite Toxoplasma gondii." Infection and immunity 76, 2 (2008): 828-44. [PubMed Link] | [ Full text ]
- Carmen, J.C.;Southard, R.C.;Sinai, A.P. "The complexity of signaling in host-pathogen interactions revealed by the Toxoplasma gondii-dependent modulation of JNK phosphorylation." Experimental cell research 314, 20 (2008): 3724-36. [PubMed Link] | [ Full text ]
- Sinai, A.P. "Biogenesis of and activities at the Toxoplasma gondii parasitophorous vacuole membrane." Sub-cellular biochemistry 47, (2008): 155-64. [PubMed Link] |
- Herman, R.K.;Molestina, R.E.;Sinai, A.P.;Howe, D.K. "The apicomplexan pathogen Neospora caninum inhibits host cell apoptosis in the absence of discernible NF-kappa B activation." Infection and immunity 75, 9 (2007): 4255-62. [PubMed Link] | [ Full text ]
- Carmen, J.C.;Sinai, A.P. "Suicide prevention: disruption of apoptotic pathways by protozoan parasites." Molecular microbiology 64, 4 (2007): 904-16. [PubMed Link] | [ Full text ]
- Martin, A.M.;Liu, T.;Lynn, B.C.;Sinai, A.P. "The Toxoplasma gondii parasitophorous vacuole membrane: transactions across the border." The Journal of eukaryotic microbiology 54, 1 (2007): 25-8. [PubMed Link] | [ Full text ]
- Sinai, A.P. "The toxoplasma kinase ROP18: an active member of a degenerate family." PLoS pathogens 3, 2 (2007): e16. [PubMed Link] | [ Full text ]
- Martin, A.M.;Liu, T.;Lynn, B.C.;Sinai, A.P. "Elimination of affinity reagent interference for the mass spectrometric detection of low-abundance proteins following immunoprecipitation." Journal of proteome research 6, 12 (2007): 4758-62. [PubMed Link] | [ Full text ]
- Carmen, J.C.;Hardi, L.;Sinai, A.P. "Toxoplasma gondii inhibits ultraviolet light-induced apoptosis through multiple interactions with the mitochondrion-dependent programmed cell death pathway." Cellular microbiology 8, 2 (2006): 301-15. [PubMed Link] | [ Full text ]
- Molestina, R.E.;Sinai, A.P. "Detection of a novel parasite kinase activity at the Toxoplasma gondii parasitophorous vacuole membrane capable of phosphorylating host IkappaBalpha." Cellular microbiology 7, 3 (2005): 351-62. [PubMed Link] | [ Full text ]
- Molestina, R.E.;Sinai, A.P. "Host and parasite-derived IKK activities direct distinct temporal phases of NF-kappaB activation and target gene expression following Toxoplasma gondii infection." Journal of cell science 118, Pt 24 (2005): 5785-96. [PubMed Link] | [ Full text ]
- Sinai, A.P.;Payne, T.M.;Carmen, J.C.;Hardi, L.;Watson, S.J.;Molestina, R.E. "Mechanisms underlying the manipulation of host apoptotic pathways by Toxoplasma gondii." International journal for parasitology 34, 3 (2004): 381-91. [PubMed Link] | [ Full text ]
- Karsten, V.;Hegde, R.S.;Sinai, A.P.;Yang, M.;Joiner, K.A. "Transmembrane domain modulates sorting of membrane proteins in Toxoplasma gondii." The Journal of biological chemistry 279, 25 (2004): 26052-7. [PubMed Link] | [ Full text ]
- Payne, T.M.;Molestina, R.E.;Sinai, A.P. "Inhibition of caspase activation and a requirement for NF-kappaB function in the Toxoplasma gondii-mediated blockade of host apoptosis." Journal of cell science 116, Pt 21 (2003): 4345-58. [PubMed Link] | [ Full text ]
- Molestina, R.E.;Payne, T.M.;Coppens, I.;Sinai, A.P. "Activation of NF-kappaB by Toxoplasma gondii correlates with increased expression of antiapoptotic genes and localization of phosphorylated IkappaB to the parasitophorous vacuole membrane." Journal of cell science 116, Pt 21 (2003): 4359-71. [PubMed Link] | [ Full text ]
- Nath, A.;Sinai, A.P. "Cerebral Toxoplasmosis." Current treatment options in neurology 5, 1 (2003): 3-12. [PubMed Link] |
- Sinai, A.P.;Joiner, K.A. "The Toxoplasma gondii protein ROP2 mediates host organelle association with the parasitophorous vacuole membrane." The Journal of cell biology 154, 1 (2001): 95-108. [PubMed Link] | [ Full text ]
- Sinai, A.P.;Paul, S.;Rabinovitch, M.;Kaplan, G.;Joiner, K.A. "Coinfection of fibroblasts with Coxiella burnetti and Toxoplasma gondii: to each their own." Microbes and infection 2, 7 (2000): 727-36. [PubMed Link] | [ Full text ]
- Coppens, I.;Sinai, A.P.;Joiner, K.A. "Toxoplasma gondii exploits host low-density lipoprotein receptor-mediated endocytosis for cholesterol acquisition." The Journal of cell biology 149, 1 (2000): 167-80. [PubMed Link] | [ Full text ]
- Sinai, A.P.;Webster, P.;Joiner, K.A. "Association of host cell endoplasmic reticulum and mitochondria with the Toxoplasma gondii parasitophorous vacuole membrane: a high affinity interaction." Journal of cell science 110 ( Pt 17), (1997): 2117-28. [PubMed Link] | [ Full text ]
- Sinai, A.P.;Joiner, K.A. "Safe haven: the cell biology of nonfusogenic pathogen vacuoles." Annual review of microbiology 51, (1997): 415-62. [PubMed Link] | [ Full text ]