title: Transient Expression of Plasmodium berghei MSP8 and HAP2 in the Marine Protozoan Parasite Perkinsus marinus.
abstract: Perkinsus marinus is a protozoan parasite of molluscs that can be propagated in vitro in a defined culture medium, in the absence of host cells. We previously reported that P. marinus trophozoites can be transfected with high efficiency by electroporation using a plasmid based on MOE, a highly expressed gene, and proposed its potential use as a "pseudoparasite." This is a novel gene expression platform for parasites of medical relevance for which the choice of the surrogate organism is based on phylogenetic affinity to the parasite of interest, while taking advantage of the whole engineered surrogate organism as a vaccination adjuvant. Here we improved the original transfection plasmid by incorporating a multicloning site, an enterokinase recognition sequence upstream of GFP, and a His-tag and demonstrate its potential suitability for the heterologous expression of Plasmodium sp. genes relevant to the development of anti-malarial vaccines. Plasmodium berghei HAP2 and MSP8, currently considered candidate genes for a malaria vaccine, were cloned into p[MOE]:GFP, and the constructs were used to transfect P. marinus trophozoites. Within 48 hr of transfection we observed fluorescent cells indicating that the P. berghei genes fused to GFP were expressed. The expression appeared to be transient for both P. berghei genes, as florescence of the transfectants diminished gradually over time. Although this heterologous expression system will require optimization for integration and constitutive expression of Plasmodium genes, our results represent attainment of proof for the "pseudoparasite" concept we previously proposed, as we show that the engineered P. marinus system has the potential to become a surrogate system suitable for expression of Plasmodium spp. genes of interest, which could eventually be used as a malaria vaccine delivery platform. The aim of the present study was to test the ability of marine protozoan parasite P. marinus to express genes of P. berghei .
authors.affiliation: Bigelow Laboratory for Ocean Sciences, East Boothbay, Maine 04544.
authors.forename: Emma R
authors.initials: ER
authors.lastname: Cold
authors.affiliation: Bigelow Laboratory for Ocean Sciences, East Boothbay, Maine 04544.
authors.forename: Gerardo R
authors.initials: GR
authors.lastname: Vasta
authors.affiliation: Bigelow Laboratory for Ocean Sciences, East Boothbay, Maine 04544.
authors.forename: José A Fernández
authors.initials: JA
authors.lastname: Robledo
mesh_terms: D056893:Alveolata / Q000235:genetics / Q000276:immunology*; D000818:Animals; D000953:Antigens, Protozoan / Q000276:immunology / Q000378:metabolism*; D005260:Female; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D008288:Malaria / Q000517:prevention & control; D017780:Malaria Vaccines / Q000235:genetics / Q000276:immunology; D008297:Male; D010957:Plasmids; D010962:Plasmodium berghei / Q000235:genetics / Q000276:immunology*; D015800:Protozoan Proteins / Q000276:immunology / Q000378:metabolism*; D014162:Transfection
chemical_list: D000953:Antigens, Protozoan; D017780:Malaria Vaccines; D015800:Protozoan Proteins; C431669:merozoite surface protein 8, Plasmodium