Nucleoside Salvage and Metabolism in Trichomonas vaginalis as a target for new therapeutic approaches

Abstract

Trichomonas vaginalis is a highly prevalent human urogenital protozoan parasite and the causative agent of the most common non-viral sexually transmitted disease (STD), trichomoniasis. Despite the prevalence of trichomoniasis exceeding that of chlamydia, gonorrhoea and syphilis, T. vaginalis has not received the attention it deserves from the public health community. Numerous studies have highlighted the serious health risks associated with T. vaginalis infection, which occurs in the female lower reproductive tract and the male urethra and can result in serious health complications. These include an increased risk of secondary infections of Human Immunodeficiency Virus (HIV) and other STDs. For almost 50 years, metronidazole has been used as the primary treatment for this disease. As a result of side effects and increased resistance to the drug, the development of a substitute compound has become increasingly urgent. As in all protozoan parasites, T. vaginalis lacks de novo synthesis of purines and pyrimidine nucleotides. Instead, it realises on purine and pyrimidine salvage pathways as a source of these nutrients from the host. Indeed, in order to survive and reproduce, protozoan parasites depend completely on salvage pathways and nucleoside transporters. The salvage of nucleosides and nucleobases is believed to be dependent on the Equilibrative Nucleoside Transporter family (ENT). To date, a total of nine genes belonging to the ENT have been identified in the T. vaginalis genome. The aim of this thesis was to identify and functionally characterise these transporters in T. vaginalis. Due to cross-resistance, and the side effects associated with metronidazole (the current treatment for trichomoniasis), new drug targeting strategies and chemotherapy are urgently needed. The study of the ENTs’ ability to uptake vital nutrients could provide new therapeutic approaches as targets for inhibitors, or conduits for nucleoside antimetabolites or other drugs. To achieve this, a strategy to clone T. vaginalis ENT genes into heterologous expression systems was adopted, so they can be studied individually. To achieve this, a new expression system needed to be developed, with at most a very low level of nucleoside uptake. The strategy selected was to delete all three nucleoside transporters from Leishmania mexicana (LmexNT1.1., LmexNT1.2 and LmexNT2) promastigotes using CRISPR/cas9. The author meticulously implemented this strategy by directly knocking out NT1.1 and NT1.2, while a dedicated member from the HDK group took charge of the NT2 component. This collaborative effort ensured a comprehensive and proficient execution of the genetic modifications required for the removal of targeted nucleoside transporters within the Leishmania mexicana promastigotes. The resulting cell line displayed almost no uptake of purine or pyrimidine nucleosides and survived by salvaging purine nucleobases through its LmexNT3 transporter instead. This new cell line, named L. mexicana ‘super knock-out’ (SUPKO), constitutes an easily manipulated system for studying and understanding nucleoside transport. TvagENT genes 1 to 9 gene have previously been sub-cloned into Trypanosoma brucei brucei expression vectors and a nucleoside transport assay was performed on TvagENT3 and TvagENT6 transfected into the T. b. brucei strain TbAT1-KO. However, these cells still have a high background of uptake of almost all purine nucleosides and nucleobases, making the assessment of the unknown heterologous difficult. In this thesis, the construction of SUPKO is described, followed by the construction of nine SUPKO-based cell lines, each expressing one of the nine T. vaginalis ENT genes for separate characterisation. In addition, some of the genes were introduced in an L. mexicana NT3-KO cell line for assessment of nucleobase transporters. Among notable results from these studies were that TvagENT3, expressed in SUPKO, was found to be a high affinity transporter for adenosine and inosine and TvagENT6 was able to transport uridine, albeit with moderate affinity. High affinity uridine uptake was found to be mediated by TvagENT8, and this was inhibited effectively by cytidine and adenosine. Trichomonas foetus and Trichomonas vaginalis are two closely related parasites that cause distinct diseases in different hosts. T. foetus causes trichomoniasis in cattle, leading to infertility, abortion, and significant economic losses, while T. vaginalis is responsible for trichomoniasis in humans, potentially causing various health complications. Both parasites have a shared characteristic of being unable to produce purine and pyrimidine, making them dependent on host epithelial cells for nutrients. In this thesis, introduces the identification and characterization of TfENT transporters in T. foetus, comparing them to TvagENTs in T. vaginalis. While the genetic sequences reveal close relationships, the distinct host environments may lead to functional differences. Although progress was limited due to unforeseen challenges, this work is foundational base of TfENT further investigation and characterization. Overall, the chapter highlights the significance of these ENT genes and their functions in parasites and proposes potential therapeutic targets. The resistance mechanism of T. vaginalis against TH1012, an adenosine analogue, has been analyzed through genomics, transcriptomics, and metabolomics data. The results show that the parasite has adapted to the drug in a multifaceted and complex way over a long period. The study reveals that resistant clones have distinct and common responses to TH1012. Additionally, genomic analysis identified a specific mutation in TvagENT8, Gln264Lys, in resistant clones. However, this mutation is not the sole cause of resistance, as multiple transporters are likely involved in adenosine transport. The resistance mechanism involves a complex interplay of genetic mutations, gene expression changes, and adaptations in cellular metabolism. Moreover, metabolomic analysis points out differences in energy and purine nucleotide metabolism between WT and resistant cells. The study's findings provide comprehensive insights into the multifaceted mechanisms of drug resistance in T. vaginalis, highlighting the need for further research into its unique metabolism and biochemical pathways. Applying the knowledge gained from this these may lead to development of new anti-trichomonal compounds will open the door to developing improved anti-trichomonal control strategies.