Theodore Clark, PhD
Department of Microbiology and Immunology
Professor of Parasitology and Immunology
Department of Microbiology and Immunology
Cornell University College of Veterinary Medicine
C5 163 VMC
Ithaca, NY 14853
Our laboratory focuses on the biology of host-pathogen interactions, and,
more broadly, the control of infectious disease. We have a long-standing interest in Ichthyophthirius multifiliis, a parasitic ciliate that causes “white-spot” disease, or “Ich” in freshwater fish. Ichthyophthirius is widely distributed in nature and has significant impact on commercial aquaculture worldwide. At the same time, it serves as a useful model for the study of host-pathogen interactions in lower vertebrates where adaptive immunity first evolved. In parallel with our work on Ichthyopthirius, we study the free-living non-pathogenic ciliate, Tetrahymena thermophila, as a surrogate for understanding the biology of I. multifiliis, and as an alternative platform for the production of recombinant subunit vaccinesl. Finally, we are interested in questions surrounding the evolution of adaptive immunity, and have recently identified functional homologs of mammalian dendritic cells in rainbow trout.
The Biology and Control of Ichthyophthirius. Aquaculture is the fastest growing sector of agribusiness worldwide, with farm-raised fish accounting for ~30% of the world’s annual catch. Because of its broad host range and geographic distribution, Ichthyophthirius multifiliis is generally considered among the most important protozoan pathogens of farm-raised fish. Our research is directed towards the development of vaccines and small molecule drugs for the treatment and prevention of disease. Efforts at vaccine development have focused on class of abundant GPI-anchored surface proteins known as immobilization antigens (i-antigens) that are the primary targets of the host immune response. Antibodies against these proteins immobilize the parasite in vitro, and trigger a novel evasion strategy in vivo in which parasites exit fish prematurely to evade the host immune response. Along with premature exit, recent studies show that surface antigen clustering following antibody binding can induce extrusion of large numbers of mitochondria from both Ichthyophthirius and from its free-living cousin, Tetrahymena thermophila. The biological significance of this phenomenon is now being studied. While the i-antigens remain obvious vaccine candidates, we have recently completed large-scale transcriptional profiling studies along with near complete sequencing of the I. multifiliis macronuclear genome with the hope of identifying additional vaccine and drug targets for the treatment of “white spot”. Along with its practical importance, the genome sequence provides a powerful tool for understanding the biology of this organism, including as yet unidentified stages of the parasite life cycle.
Tetrahymena as a Vaccine Production Platform. Long a model for basic research, Tetrahymena offers a number of salient features for recombinant protein production including rapid scalable growth; eukaryotic machinery for protein folding and post-translational modification; a metabolism geared towards membrane protein production; and, the ability to introduce transgenes at very high copy number leading to robust protein expression. In collaboration with an early-stage biotechnology company, Tetragenetics Inc (Cambridge, MA), we have recently shown that recombinant proteins can be directed to a post-golgi compartment comprised of dense core granules that can be induced to secrete en masse in response to various stimuli. The material released from these granules takes the form of a proteinaceous gel (termed PRiSMTM) that can be readily harvested by low speed centrifugation. By linking foreign proteins to endogenous polypeptides that normally traffic to these granules, the regulated secretory apparatus and its associated PRiSMTM matrix can now be harnessed for production and highly streamlined purification of recombinant proteins. We are currently exploring this pathway for the production of influenza and malaria vaccine candidates in particular.
Evolution of Adaptive Immunity. Dendritic cells (DCs) are specialized antigen-presenting cells that bridge innate and adaptive immunity. Although critical to the development of T-cell responses in mammals, it is not known whether DCs co-evolved with adaptive immunity, or if antigen presentation relied on a fundamentally different cell type early in evolution. We are exploring this question in rainbow trout (Oncorhynchus mykiss) as a representative of teleost fish, among the earliest extant species to have evolved acquired immune responses. Adapting protocols for generating DCs in mammals, we have developed methods to culture highly motile, non-adherent cells from trout that have irregular membrane processes and express surface MHCII. In mixed leukocyte reactions these cells have been shown to be superior to either macrophages or B-cells in inducing proliferative responses by trout T-cells. In addition they have many of the hallmarks of mammalian dendritic cells including tree-like morphology; the expression of DCs markers; the ability to phagocytose small particles; activation by toll-like receptor ligands; and, the ability to migrate in vivo. Although DCs have recently been identified in zebrafish, the ability to culture large numbers of these cells from trout hematopoietic tissues should provide a useful approach to the study of teleost DC function at the cellular and biochemical levels.
Additional Studies. Our laboratory maintains an NIH-funded Tetrahymena Stock Center (http://tetrahymena.vet.cornell.edu/) responsible for the curation, housing and dissemination of >2000 native and genetically modified Tetrahymena strains to laboratories around the world. The Stock Center is dedicated to advancing Tetrahymena as a basic research tool. As part of that effort, we are creating a series of small-to-mid-sized deletions across all 5 micronuclear chromosomes to identify genes uncovered in forward mutagenic screens. The Stock Center provides primary support for the Tetrahymena Genome Database Wiki website (www.ciliate.org) maintained by Dr. Nicholas Stover at Bradley University. Additionally, we are involved in efforts to physically map the T. thermophila micronuclear genome in collaboration with the Orias laboratory at UC Santa Barbara.
PhD (SUNY at Stony Brook)
Dr. Clark joined the Department of Microbiology and Immunology in 1996 and is currently Director of the Graduate Field of Immunology and Infectious Disease. Dr. Clark received a BS from Columbia University, a PhD from the State University of New York at Stony Brook, and was a Postdoctoral Fellow in the Department of Biology at Yale University. Over the years, his research has been supported by grants from the National Institutes of Health, the National Science Foundation, the Department of Defense and the U.S. Department of Agriculture. He is a member of the Aquatic Animal Medicine Program in the College of Veterinary Medicine.
Bassity, E., Clark T.G. (2012). Functional identification of dendritic cells in the teleost model, rainbow trout (Oncorhynchus mykiss). PLoS One. 7(3):e33196.
Coyne, R.S., Hannick, L., Shanmugam, D., Hostetler, J.B., Brami, D., Joardar, V.S., Johnson, J., Radune, D., Singh, I., Badger, J., Kumar, U., Saier, M., Wang, Y., Cai, H., Gu, J., Mather, M.W., Vaidya, A.B, Wilkes, D.E., Rajagopalan, V., Asai, D.J., Pearson, C.G., Findly, R.C., Dickerson, H.W., Martens, C., Van de Peer. Y., Wu, M., Roos, D.S., Cassidy-Hanley, D.M. and T.G. Clark (2011). Comparative genomics of the pathogenic ciliate Ichthyophthirius multifiliis, its free-living relatives and a host species provide insights into adoption of a parasitic lifestyle and prospects for disease control. Genome Biology. 12:R100.
Cassidy-Hanley, D.M,; Cordonnier-Pratt, M.M., Pratt, L.H.Devine, C., Hossain, M., Dickerson, H.W. and T.G. Clark (2011) Transcriptional profiling of stage-specific gene expression in the parasitic ciliate Ichthyophthirius multifiliis. Mol. Biochem. Parastiology. 178:29-39.
Bisharyan, Y. and T. G. Clark (2011). Calcium-dependent extrusion of mitochondria from ciliated protozoa. Mitochondrion. 11:909-918.
Formigari, A., Boldrin F, Santovito G, Cassidy-Hanley D, Clark TG, Piccinni E. (2010). Functional characterization of the 5'-upstream region of MTT5 metallothionein gene from Tetrahymena thermophila. Protist. 16:71-77.
Jayaram J., Papoyan A., Bisharyan Y., Cassidy-Hanley D., Colussi P., Appleton J.A., Gagliardo L. and Clark T.G. (2010) An alternative platform for rapid production of effective subunit vaccines. BioPharm International. October 2010 Supplement: The Evolution of Vaccines. pp. 6-13.
Awards and Honors