Background-Motivation

Background

We at UMaryland iGEM are developing a biosensor to detect for the presence of an oyster pathogen, Perkinsus marinus. This parasite is commonly found over the eastern shore of North America, including in Maryland’s own Chesapeake Bay. P. marinus, a unicellular protist, causes an oyster infection known as Dermo (Espinosa, Winnicki, & Allam, 2013). The pathogen has been shown to infest oysters by entering their hemocytes via induced phagocytosis. When inside the hemocyte, the parasite is capable of resisting the reactive oxygen species generated by the oyster immune system and modulating the apoptosis of infected hemocyte cells to spread itself further (Espinosa et al., 2013). This leads to proliferation inside the oyster hemocyte, eventually lysing the host cell and releasing more P. marinus (Allam et al., 2013). This process leads to massive tissue damage and, ultimately, the death of the oyster.

 

It is believed that P. marinus enters utilizing a galectin, a galactose-binding protein, on the hemocyte cell surface, CvGal1, which allows it to bind preferentially to P. marinus (Schott, Pecher, Okafor, & Vasta, 2003). Our aim is to exploit this property by expressing the galectin on the surface of E. coli bacterial cells by attaching it to the C-terminus of an OmpA transmembrane protein. These bacteria will thus bind to any P. marinus present in a sample of water. Our long-term goal is to incorporate these biosensors into a lab-on-a-chip device, requiring only small samples and allowing quick response times for detecting this pathogen.

 

This year, we have focused on working with the bovine galectin-1 in place of CvGal1. At only 14 kDa in size, the bovine galectin was chosen as a simpler candidate for expression on the surface of the membrane (Liao, Kapadia, Ahmed, Vasta, & Herzberg, 1994). Furthermore, this bovine galectin-1 was one of three galectins used for the homology modeling of CvGal1 due to their similar binding domains (Tasumi & Vasta, 2007). Though bovine galectin-1 is a two-domain galectin, compared to the four domains of CvGal1 (Feng et al., 2013), it is believed that the bovine galectin has potential to bind the same ligand present on P. marinus that CvGal1 binds. Additionally, we have optimized the bovine galectin-1 gene, most notably for better protein folding and BioBrick friendliness.

 

Although laboratory methods for Dermo detection using PCR exist, they are currently not capable of detecting the disease in real time. In the time needed to conduct a laboratory test, Dermo will have easily spread to new locations. If we can pinpoint the location of Dermo on-site, then we can take action ultimately to eliminate the parasite from eastern oyster habitats.

 

References

Allam, B., Carden, W. E., Ward, J. E., Ralph, G., Winnicki, S., & Espinosa, E. P. (2013). Early host-pathogen interactions in marine bivalves: Evidence that the alveolate parasite Perkinsus marinus infects through the oyster mantle during rejection of pseudofeces. Journal of Invertebrate Pathology, 113(1), 26-34.

Espinosa, E. P., Winnicki, S., & Allam, B. (2013). Early host–pathogen interactions in a marine bivalve: Crassostrea virginica pallial mucus modulates Perkinsus marinus growth and virulence. Diseases of Aquatic Organisms, 104(3), 237-247.

Feng, C., Ghosh, A., Amin, M. N., Giomarelli, B., Shridhar, S., Banerjee, A., ... & Vasta, G. R. (2013). The galectin CvGal1 from the eastern oyster (Crassostrea virginica) binds to blood group A oligosaccharides on the hemocyte surface. Journal of Biological Chemistry, 288(34), 24394-24409.

Liao, D. I., Kapadia, G., Ahmed, H., Vasta, G. R., & Herzberg, O. (1994). Structure of S-lectin, a developmentally regulated vertebrate beta-galactoside-binding protein. Proceedings of the National Academy of Sciences, 91(4), 1428-1432.

Tasumi, S., & Vasta, G. R. (2007). A galectin of unique domain organization from hemocytes of the Eastern oyster (Crassostrea virginica) is a receptor for the protistan parasite Perkinsus marinus. The Journal of Immunology, 179(5), 3086-3098.

Schott, E. J., Pecher, W. T., Okafor, F., & Vasta, G. R. (2003). The protistan parasite Perkinsus marinus is resistant to selected reactive oxygen species. Experimental Parasitology, 105(3-4), 232-240.