The characterization of sphingosine-1-phosphate phosphatase and genetic manipulation of sphingoid base synthesis via CRISPR/Cas9 in Leishmania major

dc.contributor.committeeChairZhang, Kai
dc.contributor.committeeMemberBrelsfoard, Corey
dc.contributor.committeeMemberKeyel, Peter
dc.creatorOkundaye, Brian
dc.creator.orcid0000-0002-3344-3444 2021
dc.description.abstractLeishmania are protozoan parasites that have dixenous life cycles that are divided into insect and mammalian host stages. These parasites are spread by sandflies, hematophagous flies that transmit the parasite to mammals, such as humans, through their mouth parts upon taking a bloodmeal. Following entry via bloodmeal into the mammalian host, Leishmania infect the immune cells of the host and propagate as intracellular parasites, giving rise to several devastating diseases known as leishmaniases (or “leishmaniasis” singularly). These leishmaniases manifest as anything ranging from relatively non-lethal ulcerations of the skin, highly lethal ulcerations of mucosal tissue, or as a chronic wasting disease with a nearly 100% lethality. In general, progress in treating these leishmaniases have largely fallen by the wayside, stalling since the early 1900’s for the most part. As such, there exists an intrinsic need to develop new treatments and/or vaccines (of which none currently exists) for leishmaniasis, which is only accomplishable through understanding the biology of the parasite. As eukaryotes, Leishmania species are known to possess an iteration of the sphingolipid biosynthesis pathway of which, while dispensable for the parasite, is required for their differentiation into mammalian infectious forms, as well as to synthesize ethanolamine-1-phosphate through the breakdown of certain sphingolipids to create bulk membrane lipids, such as phosphatidylcholine. Sphingosine-1-phosphate phosphatase (SPP) is an enzyme from the sphingolipid biosynthesis pathway that is localized to the endoplasmic reticulum and is responsible for dephosphorylating sphingosine-1-phosphate (S1P), the phosphorylated version of sphingosine, the base compound of this pathway which is phosphorylated by sphingosine kinase (SK). Previous studies of SPP in other eukaryotes, such as humans, mice, plants, and yeasts suggest the enzyme acts as a homeostatic counter to SK’s activity, with the phosphorylation of sphingosine and the dephosphorylation of sphingosine-1-phosphate signaling for cell survival and death, respectively. While this context is not thought to exist in Leishmania major (L. major), expression studies of a homolog of the gene, L. major SPP, reveal the existence of the expected biochemical activity of the gene in the parasite, while also indicating a highly unexpected localization of the enzyme to the Golgi apparatus and even more unexpected essentiality not observed of any other sphingolipid biosynthesis gene in L. major. Concurrent studies, facilitated through the adaptation of CRISPR/Cas9 gene editing in L. major, involving the localization of other uncharacterized genes from the sphingolipid biosynthesis pathway in Leishmania major, such as serine palmitoyl transferase 1 (SPT1), 3-ketodihydrosphingosine reductase (3KDSR), dihydroceramide synthase (DHCS), and dihydroceramide desaturase (DHC DES), suggest the pathway is localized to the endoplasmic reticulum in contrast to SPP’s Golgi localization. This same CRISPR/Cas9 gene editing technique would also seem to reveal an unusual, apparent essentiality associated with SPT1 in L. major in a seeming contradiction to the observation that sphingolipid biosynthesis is dispensable in the parasite. These observations, taken together with the results obtained from L. major SPP, suggest that there is more than meets the eye to the sphingolipid biosynthesis pathway in the parasite. In conclusion, SPP’s localization and essentiality would appear to carry rather profound implications as to other purposes of the pathway in not just Leishmania, but eukaryotes as a whole, in addition to SPT1’s potential essentiality in a parasite that is not thought to require to sphingolipid biosynthesis for survival.
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dc.subjectSphingosine-1-Phosphate Phosphatase
dc.subjectMolecular Biology
dc.titleThe characterization of sphingosine-1-phosphate phosphatase and genetic manipulation of sphingoid base synthesis via CRISPR/Cas9 in Leishmania major
dc.type.materialtext Sciences Tech University of Science


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