The phylum Apicomplexa is constituted exclusively by protozoan parasites, several of which pose a significant threat to public health. The apicomplexan Plasmodium falciparum is the ethiological agent of Malaria and can cause high mortality and morbidity in endemic areas. Toxoplasma, Cryptosporidium, and Cyclospora are also well known to public health authorities since they pose a threat to immunosuppressed individuals (e.g. transplanted and cancer patients), but also because sporadic outbreaks do occur (Cryptosporidium and Cyclospora) and because additional populations are at risk, i.e. pregnant women (Toxoplasma). Cryptosporidium, Cyclospora, and Toxoplasma are also listed as Category B Bioterrorist Threat Pathogens according to the Center of Disease Control, since infection occurs by oral transmission and the resistant oocysts shed by hosts can remain viable for long periods of time in soil and water. With most of the genomes of these parasites available, it becomes evident the need for heterologous expression systems that can generate recombinant proteins, a step required for characterization of protein function, structural studies, production of antigen, screening and profiling drug candidates, and understanding action mechanisms. Available heterologous systems have been used with varying degrees of success;however, there is not a single expression system universally suitable for all applications and all have drawbacks in particular applications. Hence, we propose to develop a heterologous system for production of Apicomplexa proteins using the protozoan P. marinus, a close relative of the Apicomplexa that is easily cultured in a full-defined cell-free medium. Although we have already a vector for transfection, in AIM1 we will incorporate into the vector a selectable marker and mine the P. marinus genome for targeting and secretion signals.
In AIM2, we will use selected Apicomplexa genes for proof of concept;these will include well-characterized apicomplexan genes as well as genes that have proven difficult to express in other systems. We expect to overcome some of the past difficulties and to provide a valid alternative when the available heterologous expression systems do not result in expression, optimal protein yield, or functional proteins. The success of this system would result in apicomplexan proteins suitable for protein function studies, crystal structure determination, antigen production, and drug candidates, and will ultimately provide the tools required to more effectively fight and ameliorate the effects of these diseases.

Public Health Relevance

In order to develop new drugs and vaccines for fighting parasitic diseases, a more efficient system for producing recombinant proteins is required. We propose the protozoan Perkinsus marinus as a heterologous expression system for producing Apicomplexa proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
7R21AI076797-02
Application #
7843525
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Mcgugan, Glen C
Project Start
2009-05-15
Project End
2012-04-30
Budget Start
2010-05-01
Budget End
2012-04-30
Support Year
2
Fiscal Year
2010
Total Cost
$180,903
Indirect Cost
Name
University of Maryland Baltimore
Department
Type
Schools of Medicine
DUNS #
188435911
City
Baltimore
State
MD
Country
United States
Zip Code
21201
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Wijayalath, Wathsala; Majji, Sai; Kleschenko, Yuliya et al. (2014) Humanized HLA-DR4 mice fed with the protozoan pathogen of oysters Perkinsus marinus (Dermo) do not develop noticeable pathology but elicit systemic immunity. PLoS One 9:e87435
Alemán Resto, Yesmalie; Fernández Robledo, José A (2014) Identification of MMV Malaria Box inhibitors of Perkinsus marinus using an ATP-based bioluminescence assay. PLoS One 9:e111051
Fernández Robledo, José A; Vasta, Gerardo R; Record, Nicholas R (2014) Protozoan parasites of bivalve molluscs: literature follows culture. PLoS One 9:e100872
Vasta, Gerardo R; Ahmed, Hafiz; Bianchet, Mario A et al. (2012) Diversity in recognition of glycans by F-type lectins and galectins: molecular, structural, and biophysical aspects. Ann N Y Acad Sci 1253:E14-26
Fernández Robledo, José A; Caler, Elisabet; Matsuzaki, Motomichi et al. (2011) The search for the missing link: a relic plastid in Perkinsus? Int J Parasitol 41:1217-29
Fernandez-Robledo, Jose A; Vasta, Gerardo R (2010) Production of recombinant proteins from protozoan parasites. Trends Parasitol 26:244-54