An Investigation of TbLpn Methylation in Trypanosoma brucei

Abstract

Human African trypanosomiasis (HAT), more commonly referred to as African Sleeping Sickness, is a disease transmitted through the bite of an infected tsetse fly harboring the human pathogenic parasite, Trypanosoma brucei, a hemoflagellate of the taxonomic class Kinetoplastea. The disease impacts primarily populations of sub-Saharan Africa, and exists in two primary stages: the bloodstream and the central nervous system (CNS) stage. In the first stage, T. brucei will migrate through the lymph and blood, continually proliferating and causing unspecific signs and symptoms. Over time, the parasite will penetrate the blood-brain barrier and migrate to the central nervous system, where various neurological disturbances can occur, greatly increasing the severity of the disease. It is at this stage that the disease becomes difficult to treat, as antibodies that circulate the body can no longer reach the parasite, and the brain immune response is less efficient than other immune responses within the body. However, early intervention is also difficult, due to the lack of specific signs or symptoms present during the bloodstream stage of the disease. In the event of early diagnosis of HAT, the drugs that are currently available for treatment will likely cause negative side effects. Vaccination is also not possible due to the trypanosome's antigenic variation, which allows the T. brucei to successfully elude the host’s immune responses through their expression of the variant surface glycoproteins (VSGs). VSGs cover the Trypanosoma cell with a frequently-changing “protective coat,” allowing the parasite to avoid detection by host antibodies. Five Protein Arginine Methyltransferase enzymes (PRMTs) are also found in T. brucei, which suggests that arginine methylation plays a predominant role in the parasite’s life cycle. PRMTs have been shown to interact with a trypanosome homologue of lipin, termed TbLpn, which acts as phosphatidate phosphatase (PAP) enzyme, and actively converts phosphatidate to diacylglycerol (DAG) during the synthesis of triacylglycerol (TAG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). PE is particularly important in T. brucei’s synthesis of glycosylphosphatidylinositol (GPI), which is utilized to anchor VSGs and give the parasite its ability to evade the immune system through antigenic variation. TbLpn is the only phosphatidic acid phosphatase known to date to exhibit arginine methylation. One of the main objectives of this project was to confirm the importance of TbLpn, and to verify that it is the major enzyme responsible for the dephosphorylation of phosphatidic acid in T. brucei. To determine this, a phosphatidic acid phosphatase (PAP) assay was performed, and the results showed that that the wild type TbLpn produced 48.05 (± 26.09) nmol of phosphate per minute per mg of protein, whereas the TbLpn-depleted cells produced only 21.96 (± 14.59) nmol Pi/min/mg. These results confirmed the importance of TbLpn for the dephosphorylation of phosphatidic acid, and its effect on enzymatic activity in T. brucei, as the cells with trace amounts of TbLpn displayed significantly less activity. The other main objective of this experiment was to determine the effect of TbLpn methylation by TbPRMT7 on its enzymatic activity. Following an additional PAP assay, the phosphatase activity of wild type TbPRMT7 T. brucei cells was compared to that of TbPRMT7- depleted cells, and the effect of methylation on TbPRMT7 PAP activity was deemed insignificant, with minimal difference in measured enzymatic activity. It can therefore be concluded that the methylation of TbPRMT7 is not important for the enzymatic activity of TbLpn. However, it cannot be confirmed from these results that TbPRMT7 methylation plays no role in Trypanosoma brucei. Further experiments must be conducted to determine what role arginine methylation has on TbLpn enzymatic activity.