Search for: All records

Perkinsus marinus (Perkinsozoa), a close relative of apicomplexans, is an osmotrophic facultative intracellular marine protozoan parasite responsible for “Dermo” disease in oysters and clams. Although there is no clinical evidence of this parasite infecting humans, HLA-DR4 0 transgenic mice studies strongly suggest the parasite as a natural adjuvant in oral vaccines. P. marinus is being developed as a heterologous gene expression platform for pathogens of medical and veterinary relevance and a novel platform for delivering vaccines. We previously reported the transient expression of two rodent malaria genes Plasmodium berghei HAP2 and MSP8 . In this study, we optimized the original electroporation-based protocol to establish a stable heterologous expression method. Using 20 μg of p PmMOE[MOE1]:GFP and 25.0 × 10 6 P. marinus cells resulted in 98% GFP-positive cells. Furthermore, using the optimized protocol, we report for the first time the successful knock-in of GFP at the C-terminus of the PmMOE1 using ribonucleoprotein (RNP)-based CRISPR/Cas9 gene editing methodology. The GFP was expressed 18 h post-transfection, and expression was observed for 8 months post-transfection, making it a robust and stable knock-in system. 

Perkinsus marinus is a facultative intracellular marine protozoan parasite responsible for the Dermo disease in Crassostrea virginica oysters. Associated with mass mortalities in the Gulf Coast and Chesapeake Bay (USA), it remains one of the main hurdles for oyster reef restoration and aquaculture. Oysters take up the parasite by filter-feeding; in the pallial cavity, it can be phagocytosed by the hemocytes via CvGal1, gaining access to the internal milieu. Inside the parasitophorous vacuole, the parasite resists oxidative stress and acquires nutrients. Propagation strategies include binary fission, budding, palintomy, and schizogony. Although the effect on humans upon consumption of raw infected oysters has not been studied, humanized HLA-DR4 mice fed with P. marinus do not develop noticeable pathology but elicit systemic immunity. Parasite culture in host-free media, and the use of genetic tools, make it a tractable genetic model and a heterologous expression and vaccine-delivery system.