Lab: (859) 323-1341
The Sinai lab is currently open to accept new graduate students through the UK Integrated Biomedical Sciences Program. We also welcome undergraduate students for research experiences.
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 parasites within tissue cysts, long thought to be dormant, exhibit considerable 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 highlighted the fact that, contrary to long held dogma (based primarily on anecdotal evidence), bradyzoites retain considerable metabolic activity, but display in patterns that remain to be elucidated. Supported by a recently funded collaborative RO1 grant, we will further advance the imaging approches to be able etsblish not only absolute parasite 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 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 physiological 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 turnover 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 replication of bradyzoites. We currently have an R21 to address the role of AG in the chronic infection pending consideration for funding.
Research Focus 2
The identification of bradyzoites as replicating entities led us to investigate the cell cycle in the chronic infection and its regulation. We focused on the contribution of cell cycle regulated Ovarian TUmor (OTU) family deubiquitinases. Our work (Dhara and Sinai mSphere 2016) established that specific ubiquitin linkage modifications exhibited dynamic changes during the cell cycle while defining the biochemical and enzymological properties of TgOTUD3A. The targeted ablation of TgOTUD3A presented a remarkable phenotype indicating a complex role in the regulation of the cell cycle architecture whereby mutants exhibited high fidelity replication using alternate replication strategies (schizogony and endopolygeny) in addition to the normal endodyogeny (Dhara et al. mBio 2017). In addition these mutants appear to be developmentally aberant as they express markers of the chronic infection as well as induce the expression of genes associated with the sexual cycle. The second major focus of the lab is to establish the molecular basis for these developmental aberations that appear to link the control of the cell cycle to the life cycle. We currently have an R21 proposal pending, which has secured what is likely to be a fundable score to aspects of these complex phenotypes. Should the proposal be funded at NIH council (in October 2019), we expect funding to commence in December 2019. Additional grants proposals for this area of investigation are under development.
Earlier work in the laboratory 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 ubiquitin / autophagy mediated mechanisms prevalent in the Interferon response 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.
Active Funded Projects
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.
04/2020-04/2022 R21 AI150631: 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.
- Sinai, AP ; Suvorova, ES "The RESTRICTION checkpoint: a window of opportunity governing developmental transitions in Toxoplasma gondii." Current opinion in microbiology 58, (2020): 99-105. [PubMed Link] | [ Full text ]
- McPhillie, MJ ; Zhou, Y.; Hickman, MR ; Gordon, JA ; Weber, CR ; Li, Q.; Lee, PJ ; Amporndanai, K ; Johnson, RM ; Darby, H ; Woods, S.; Li, ZH ; Priestley, RS ; Ristroph, KD ; Biering, SB ; Bissati, El ; Hwang, S ; Hakim, FE ; Dovgin, SM ; Lykins, JD ; Roberts, L.; Hargrave, K ; Cong, H ; Sinai, AP ; Muench, SP ; Dubey, JP ; Prud'homme, RK ; Lorenzi, HA ; Biagini, GA ; Moreno, SN ; Roberts, CW ; Antonyuk, SV ; Fishwick, CWG ; McLeod, R "Potent Tetrahydroquinolone Eliminates Apicomplexan Parasites." Frontiers in cellular and infection microbiology 10, (2020): 203. [PubMed Link] |
- Haydar, D.; Cory, TJ ; Birket, SE ; Murphy, BS ; Pennypacker, KR ; Sinai, AP ; Feola, DJ "Azithromycin Polarizes Macrophages to an M2 Phenotype via Inhibition of the STAT1 and NF-κB Signaling Pathways." Journal of immunology (Baltimore, Md. : 1950) 203, 4 (2019): 1021-1030. [PubMed Link] | [ Full text ]
- Dhara, A.; Paula Baptista, R.; Kissinger, JC ; Snow, EC ; Sinai, AP "Ablation of an Ovarian Tumor Family Deubiquitinase Exposes the Underlying Regulation Governing the Plasticity of Cell Cycle Progression in <i>Toxoplasma gondii</i>." mBio 8, 6 (2017): [PubMed Link] | [ Full text ]
- Watts, EA ; Dhara, A.; Sinai, AP "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, AP "A Cell Cycle-Regulated Toxoplasma Deubiquitinase, TgOTUD3A, Targets Polyubiquitins with Specific Lysine Linkages." mSphere 1, 3 (2016): [PubMed Link] | [ Full text ]
- Sinai, AP ; Watts, EA ; Dhara, A.; Murphy, RD ; Gentry, MS ; 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, AP "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 ]
- Gaviria, D ; Paguio, MF ; Turnbull, LB ; Tan, A.; Siriwardana, A ; Ghosh, D.; Ferdig, MT ; Sinai, AP ; Roepe, PD "A process similar to autophagy is associated with cytocidal chloroquine resistance in Plasmodium falciparum." PloS one 8, 11 (2013): e79059. [PubMed Link] | [ Full text ]
- Weilhammer, DR ; Iavarone, AT ; Villegas, EN ; Brooks, GA ; Sinai, AP ; Sha, WC "Host metabolism regulates growth and differentiation of Toxoplasma gondii." International journal for parasitology 42, 10 (2012): 947-59. [PubMed Link] | [ Full text ]
- Sinai, AP ; Roepe, PD "Autophagy in Apicomplexa: a life sustaining death mechanism?" Trends in parasitology 28, 9 (2012): 358-64. [PubMed Link] | [ Full text ]
- Ghosh, D.; Walton, JL ; Roepe, PD ; Sinai, AP "Autophagy is a cell death mechanism in Toxoplasma gondii." Cellular microbiology 14, 4 (2012): 589-607. [PubMed Link] | [ Full text ]
- Carmen, JC ; Sinai, AP "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, AM ; Sinai, AP ; Lynn, BC "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 ]
- El-Guendy, N.; Sinai, AP "Potential problems inherent in cell-based stable NF-kappaB-GFP reporter systems." Molecular and cellular biochemistry 312, 1-2 (2008): 147-55. [PubMed Link] | [ Full text ]
- Molestina, RE ; El-Guendy, N.; Sinai, AP "Infection with Toxoplasma gondii results in dysregulation of the host cell cycle." Cellular microbiology 10, 5 (2008): 1153-65. [PubMed Link] | [ Full text ]
- Nelson, MM ; Jones, AR ; Carmen, JC ; Sinai, AP ; Burchmore, R.; Wastling, JM "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, JC ; Southard, RC ; Sinai, AP "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, AP "Biogenesis of and activities at the Toxoplasma gondii parasitophorous vacuole membrane." Sub-cellular biochemistry 47, (2008): 155-64. [PubMed Link] |
- Burleigh, BA ; Sinai, AP "Molecular mechanisms of parasite invasion. Preface." Sub-cellular biochemistry 47, (2008): xix-xx. [PubMed Link] |
- Herman, RK ; Molestina, RE ; Sinai, AP ; Howe, DK "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, JC ; Sinai, AP "Suicide prevention: disruption of apoptotic pathways by protozoan parasites." Molecular microbiology 64, 4 (2007): 904-16. [PubMed Link] | [ Full text ]
- Martin, AM ; Liu, T.; Lynn, BC ; Sinai, AP "The Toxoplasma gondii parasitophorous vacuole membrane: transactions across the border." The Journal of eukaryotic microbiology 54, 1 (2007): 25-8. [PubMed Link] | [ Full text ]
- Martin, AM ; Liu, T.; Lynn, BC ; Sinai, AP "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 ]
- Petersen, CA ; Krumholz, KA ; Carmen, J ; Sinai, AP ; Burleigh, BA "Trypanosoma cruzi infection and nuclear factor kappa B activation prevent apoptosis in cardiac cells." Infection and immunity 74, 3 (2006): 1580-7. [PubMed Link] | [ Full text ]
- Carmen, JC ; Hardi, L.; Sinai, AP "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, RE ; Sinai, AP "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, RE ; Sinai, AP "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, AP ; Payne, TM ; Carmen, JC ; Hardi, L.; Watson, SJ ; Molestina, RE "Mechanisms underlying the manipulation of host apoptotic pathways by Toxoplasma gondii." International journal for parasitology 34, 3 (2004): 381-91. [PubMed Link] | [ Full text ]
- Payne, TM ; Molestina, RE ; Sinai, AP "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, RE ; Payne, TM ; Coppens, I.; Sinai, AP "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, AP "Cerebral Toxoplasmosis." Current treatment options in neurology 5, 1 (2003): 3-12. [PubMed Link] |