Saudi Cultural Missions Theses & Dissertations
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Item Restricted Nucleoside Salvage and Metabolism in Trichomonas vaginalis as a target for new therapeutic approaches(University of Glasgow, 2023-10-28) Alsiari, Tahani; Koning, Harry DeTrichomonas 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.38 0Item Restricted Mechanisms of Infection in an Ecological Model of Host-Parasite Interactions(Saudi Digital Library, 2023-01-09) Alghafli, Hawra; Barribeau, SethTrypanosomatids are evolutionary successful obligate parasites that have two distinct infection life-cycles; some species complete their entire life-cycle in a single host (monoxenous) while others infect two hosts (dixenous). Monoxenous trypanosomtids mostly infect an insect host and are believed to be more primitive and widespread, though not as commonly studied as their dixenous kins. Dixenous trypanosomatids are usually vectored-parasites that encompass etiological agents of several human diseases such African Sleeping Sickness, Chagas disease and Leishmaniasis. Previous studies have examined infection prevalence for subsets of hosts and trypanosomatids, but little is known about whether monoxenous and dixenous trypanosomatids differ in infection prevalence. In chapter 2, I synthesised all published evidence of trypanosomatid infection prevalence for the last two decades using a semi-automated screening protocol. In examining the eligible and included 584 studies that describe infection prevalence, monoxenous species were found to be two-fold more prevalent than dixenous species across all hosts and among insects only. In addition, dixenous trypanosomatids have significantly lower infection prevalence in insects than their non-insect hosts. These results reveal for the first time, a fundamental difference in infection prevalence according to host specificity where vectored species might suffer from lower infection prevalence. These findings will help researchers better understand trypanosomatids in general and further tailor control strategies for various trypanosomatid diseases relevant to human and livestock health. In the Chapter 3 and Chapter 4, I focused on one monoxenous trypanosomatid, the gut parasite Crithidia bombi and its bumblebee host Bombus terrestris. C. bombi and B. terrestris have become an important model system to study host-parasite interactions and the subsequent ecological and evolutionary aspects of their interactions. In Chapter 2, I infected age-controlled workers of B. terrestris with one genotype of C. bombi (08.076; the reference strain which had been fully sequenced) and dissected their guts at 24 hours, 48 hours and 7 days post infection for full transcriptome sequencing of both the parasite and the host. Differential gene expression comparing (I) late infection versus early infection and (II) for each infection time-point versus pre-infection status (log phase in vitro cultures). I found several upregulated potential virulence genes, based on the orthology to Leishmania major, such as two surface glycoproteins and three calpain family cysteine proteases-like proteins. In addition, parasite genes under positive selection, are more dynamically expressed during all the infection time-points than genes that are not under positive selection. Furthermore, I correlated host immune genes with parasite genes and found that co-regulated host and parasites genes were largely antagonistic (negatively correlated) with distinctive clusters of potential gene-gene interactions. Moreover, I also compared the expression patterns of C. bombi with other dixenous and monoxenous trypanosomatids to examine if differentiation responses are governed by conserved gene expression profiles. Overall, I found a slight but significant predictive power across trypanosomatids that is more pronounced among closer related species. My results help shape a better overall understanding of the co-evolved interactions in this host-parasite system and the breadth of similarity and differences in differentiation responses across trypanosomatids that have distinctive life-cycle strategies. Co-infection by multiple strains, or species, of parasites can have important implications for both host immune responses and the manifestation of virulence (a feature of infection that is determined by both parasite and host traits). The process by which co-infection arises may also be important in determining these effects. To understand the role of temporal spacing between infectious exposures and parasite genotype in infection, in Chapter 4, I performed a series of within-host competition assays using transmission load (i.e. the number of parasite cells in the infected host’s faeces) as a fitness proxy. My assays show dramatic increase in faecal parasite abundance when hosts are re-infected by the same genotype. Notably, this advantage of repeated exposure only held when host bees were exposed to two doses of the same genotype but not when they were administered together, or when two different genotypes were used in any combination or order of delivery. This suggests a form of immunosuppression leading to genotype specific precedence based facilitation. Taken together, my work forms a clearer understanding of the previously poorly characterized C. bombi strategies of infection during single and mixed infections. Additionally, the work also highlights a potential fundamental difference between trypanosomatids in infection prevalence based on their life cycle complexity.6 0Item Restricted IL36 and IL37Cytokines, Mediators or Potential Modulators of Airway Infection and Inflammation?(Saudi Digital Library, 2022-10-01) Alshammari, Jamilah; Flannagan, BrianRespiratory tract infections caused by viruses or bacteria are among the most common human diseases worldwide. More specifically, respiratory syncytial virus (RSV) is a major cause of acute lower respiratory infection (ALRI), and this frequently results in hospitalisation in young children, particularly among those under the age of 5, and the elderly. Infection with RSV is also correlated with various airway respiratory diseases, such as bronchiolitis and asthma. In vivo testing suggests that levels of IL-36 alpha, IL-36 gamma, and IL-37 protein can be very high in nasopharyngeal aspirate (NPA) during RSV and rhinovirus (RV) infections. This work thus hypothesises that, during RSV and RV infections, the epithelial cells in the airways may express IL-36α, IL-36γ, and their respective receptors in a manner that plays a crucial pro-inflammatory role in RSV and RV infections. In addition, this work hypothesises the possibility of the further expression of IL-36Rα and IL-37 cytokines by airway epithelial cells (AECs) in response to RSV and RV infections. The main aim of this work, as described in this thesis, is thus to investigate whether RSV infection can trigger AECs to express IL-36 and IL-37 cytokines and their receptors.2 0