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Using the Plasmodium BEM46-like Protein to Characterize the Parasitic Plasma Membrane Do You Want to Build a Parasite? Darrell Lockhart, Saddie Burkentine, and Anna Groat-Carmona Biomedical Sciences Division, University of Washington Tacoma, Tacoma WA Methodology Figure 4. Gel-electrophoresis confirming restriction digests of target vector (A) pSL0401 C-BirA Tag, (B) pUC57-0028 fragements cut with ApaLI, and (C) pUC57-0028 with PBLP insert selected for gel-isolation. To the left of the gels are band sizes the fragments were found. Results Malaria is a vector-borne illness caused by the Plasmodium parasite, which cycles between its mammalian host and mosquito vector. The female Anopheles mosquito transmits the Plasmodium parasite to its host upon taking a blood meal. Malaria is prevalent in tropical and subtropical regions, such as the majority of Africa as well as parts of southeast Asia and South America, causing roughly 430,000 deaths annually (Moxon 2020). Even though there are treatments for malaria, including antimalarial drugs such as chloroquine phosphate, artemisinin-based combination therapies, primaquin phosphate, and mefloquine, there is a growing number of drug-resistant strains circulating worldwide. The malaria life cycle consists of three stages, the liver-, blood- and mosquito-stages (Fig. 1). Figure 1. A diagram showing the malaria life cycle. When the Anopheles mosquito vector takes a blood meal, it transmits sporozoites to the mammalian host. Once the parasite enters the blood stream, the sporozoite travels to the liver where the parasite infects suitable hepatocytes (Moxon 2020). After replication (schizogeny), the schizont bursts and releases tens of thousands of merozoites into the blood stream where they infect red blood cells (Moxon 2020). After a few days, gametes are produced and may be transmitted to another mosquito vector when taking a blood meal (Moxon 2020). Image made with BioRender. Throughout liver-stage development, a member of the α/β -hydrolase super family, the Plasmodium BEM46-like protein (PBLP), is expressed on the parasitic plasma membrane (PPM) (Groat-Carmona 2015). Not much is known about the specific mechanism, but PBLB is associated with the production of invasive forms throughout the parasite life cycle (Groat-Carmona 2015). Regardless of PBLP’s functional role in parasite development, we are taking advantage of the protein’s ubiquitous nature to identify the parasitic proteins expressed on the PPM during liver-stage development (Groat- Carmona 2015). The PPM changes its protein composition throughout liver-stage development but not all the proteins on the PPM have been identified (Groat-Carmona 2015). This research will ultimately allow us to characterize the network of proteins on the PPM at any given period throughout liver-stage development. To engineer this transgenic parasite, we have ligated the PBLP coding sequence as well as the endogenous PBLP promoter into an existing parasite cloning vector of that adds a C-terminal Bir-A tag (Fig. 2). Introduction Acknowledgements Conclusions Figure 2. (A) A schematic of PBLP tagged with the biotin ligase BirA and the protein network present on the PPM. (B) The BirA tag biotinylates the proximal proteins on the PPM. The proteins tagged with biotin molecules will be purified using Streptavidin and then identified through mass spectrometry. Image made with BioRender. At this time, cloning of our final parasite cloning plasmid is still in progress, but we will begin wtih screening our transformed E. coli colonies. • The gel-electrophoresis from our target vector pSL0401 BirA+4xMyc sequential digest confirms the vector has been properly cut preceding ligation (Fig. 4A). • The gel-electrophoresis from the pUC57-0028 restriction digest with ApaLI shows 3 fragments (roughly 498, 1247, and 3981 base pairs), confirming that our custom plasmid is viable (Fig. 4B). • The PBLP fragment is 3013 base pairs, and the gel- electrophoresis on the pUC57-0028 plasmid, digested with SacII and Not I, allowed us to isolate the PBLP fragment. This insert was used for ligation with our target vector. (Fig. 4C). • Isolated colonies on our transformation plates have formed, indicating possible success of our ligated pAG0028 (Fig. 5B). Once we confirm that our ligation is successful, and after our pAG0028 plasmid is sequenced, we will start parasite cloning in Swiss Webster mice. Days after the mice are infected with Plasmodium blood-stage parasites, late-stage schizonts will be extracted, transfected with our linearized PBLP-BirA plasmid, and introduced back into the mice. Blood from parasite-positive mice will be extracted and used for additional rounds of cloning. Figure 3. Initial parasite cloning plasmid had a C-terminal BirA coding sequence attached to UIS4 so restriction enzymes SacII and Not I were used to cut out the UIS4 sequence (pSL0401 BirA+4xMyc) (A). CIP (phosphatase) was added to remove the 5’ phosphates on the target vector and prevent re-ligation (B). A custom plasmid containing a PBLB coding sequence (pUC57-0028) was ordered and digested with Sac II and Not I to obtain the PLBP insert (C,D). Note that the double arrowhead implies ligation to generate our final vector (pAG0028 PBLP C- BirATag), which should have the BirA sequence following the PBLP coding sequence (E). Images with BioRender and SnapGene. Figure 5. A schematic of the transformation of our final plasmid pAG0028 PBLP C-BirA Tag into E. coli. (A) Shows the ligation of pAG0028, which was quickly transformed into E. coli. (B) Transformation plates containing a putative transgenic E. coli with isolated colonies selected for screening (indicated by red circles). I’d like to thank my mentor and research supervisor, Dr. Groat- Carmona, for allowing me to participate in this research and keeping me on as the research continues. I also want to thank my partner, Saddie Burkentine, for orienting me onto the project as well as working diligently with me. References Groat‐Carmona AM, Kain H, Brownell J, Douglass AN, Aly ASI, Kappe SHI. 2015. A plasmodium α/β‐hydrolase modulates the development of invasive stages. Cellular Microbiology. 17(12):1848-67. Moxon CA, Gibbins MP, McGuinness D, Milner DA, Marti M. 2020. New insights into malaria pathogenesis. Annual Review of Pathology: Mechanisms of Disease. 15(1). Kain HS, Glennon EKK, Vijayan K, Arang N, Douglass AN, Fortin CL, Zuck M, Lewis AJ, Whiteside SL, Dudgeon DR, et al. 2020. Liver stage malaria infection is controlled by host regulators of lipid peroxidation. Cell Death and Differentiation. 27(1):44-54. Bir-A is a promiscuous biotinylating ligase, which when expressed in the liver- stage, will tag the proximal on the PPM with biotin (Fig. 2). Once we have collected these proteins using Streptavidin, we will then be able to identify them through mass specphotometry. A B C
