Leishmaniasis is a vector-borne disease that is caused by several species of the obligate intra-cellular protozoan parasite Leishmania. Leishmaniasis is a tropical and sub-tropical disease affecting between (12 – 15) million people worldwide. An estimated 1.5 to 2 million new cases occur and it causes 70,000 deaths per year. The flagellated protist Leishmania is one of the model organisms to study flagellar assembly. Here, we used L. mexicana as a model to investigate flagellar kinesin motor proteins. Kinesins are a large superfamily (KIFs). More than 15 kinesin families were classified by phylogenetic analysis (Wickstead et al., 2006). Kinesins are motor proteins that convert the energy from ATP hydrolysis into mechanical work to drive cargo along microtubules in a variety of cellular processes, organelle transport and cell division. Disruption of the normal function of these proteins has been shown to lead to many pathologies, including ciliopathy, neurodegenerative diseases and cancers. The current study presents a comprehensive biochemical and cell biological analysis of three kinesins thought to be associated with flagellum formation. Initially the cloning, mapping, and expression of the novel kinesin LmxKin29 were achieved. LmxKin29 is expressed in both the amastigote and promastigote life stages of L. mexicana. LmxKin29 can be assigned to the “orphan” kinesin family. Prior to the beginning of this work, the MAP kinase homologue LmxMPK3 was found to phosphorylate a peptide derived from LmxKin29 encompassing serine 551 and serine 554 (Rosenqvist, 2011; Emmerson, 2014). Here, a full-length GST-fusion protein of wild type LmxKin29 and five different mutants with substitutions of the putative serine or threonine phosphorylation sites, by alanine or aspartate, namely LmxKin29SA, LmxKin29SD, LmxKin29A2, LmxKin29A4 and LmxKin29554A were analysed. Using these mutants, it was possible to narrow down the site that is phosphorylated in activated His-LmxMPK3 as serine 554. To assess the function of LmxKin29 in L. mexicana single and double allele null mutants were generated. Morphological analysis of promastigotes displayed no obvious phenotypic differences comparing the mutants with wild type cells. Localisation studies using GFP-tagged LmxKin29 revealed that it is predominantly found in between the nucleus and the flagellar pocket, while in dividing cells LmxKin29 was found at the anterior and posterior ends of the cells. Hence, LmxKin29 might play a role in cytokinesis. Female Balb/c mice infected with ΔLmxKin29-/- null mutant promastigotes did not show a footpad lesion, whereas LmxKin29 add-back clones and single allele knockout clones caused the disease similar to wild type parasites. It was confirmed by ELISA that the serum of mice infected with L. mexicana wild type, single allele mutants and add-back mutants showed increased levels of IgG1 and IgG2a. However, the LmxKin29 null mutant scored very low similar to the level of uninfected mice serving as a negative control. The inability to cause lesions in the infected animal suggests that LmxKin29 is a potential drug target against leishmaniasis. On the other hand the absence of an immune response against the LmxKin29 null mutant clearly rules out these mutant parasites as an attenuated live vaccine. LmxOSM3.1 and LmxOSM3.2 were found to be homologous to flagellar kinesin-2 in trypanosomatids (L. major and T. brucei). They also showed sequence similarity with kinesin-2s of human kinesin KIF3A/B as well as with C. reinhardtii FLA8/FLA10 and C. elegans kinesin-like protein klp-20klp11. LmxOSM3.1GFP localisation was identified along the flagellum of L. mexicana promastigotes, while LmxOSM3.2RFP was localised at the tip of the flagellum. Hence, both kinesins might be involved in flagellum formation. LmxOSM3.1 has been identified previously as a substrate of LmxMPK3 and LmxMPK13. Kinase assays using GST-LmxOSM3.2 with activated LmxMPK3 showed that this kinase cannot phosphorylate LmxOSM3.2 in vitro. We can hypothesise that LmxOSM3.1 and LmxOSM3.2 are core IFT motors that are involved in the assembly and maintenance of the flagellum, whereas LmxOSM3.2 exhibit as an accessory motor that provides flagellum-specific functions.
|Date of Award||15 Mar 2019|
- University Of Strathclyde
|Supervisor||Martin Wiese (Supervisor) & Craig Roberts (Supervisor)|