Thesis

Investigating the importance of LmxOSM3.1 phosphorylation in flagella development and determining LmxOSM3.1 and LmxMPK13 interaction partners

Creator
Rights statement
Awarding institution
  • University of Strathclyde
Date of award
  • 2024
Thesis identifier
  • T17035
Person Identifier (Local)
  • 202082328
Qualification Level
Qualification Name
Department, School or Faculty
Abstract
  • Parasitic infection induced by the protozoan Leishmania from the Trypanosomatidae family causes Leishmaniasis with the female sand fly being the primary vector of transmission. Leishmaniasis is endemic across many tropical regions. However, climate change, urbanisation and deforestation are propagating its global distribution to unaffected regions. Hence, the estimated global population of 12 million people with Leishmaniasis is anticipated to increase. Current anti-leishmaniasis drugs including pentavalent antimonials, amphotericin B, miltefosine, paromomycin, and pentamidine are limited in their drug potency, and demonstrate extensive drug resistance. Novel drug treatments are needed to combat these limitations. Kinases and kinesins are suggested to be effective drug targets that could target flagella growth in Leishmania. LmxOSM3.1 is proposed to be a plus end-directed 170kDa, homodimeric N-kinesin involved in mediating anterograde transport in the flagellum. CeOSM3 p802 null mutants induce a loss in the sensory ciliary distal domain of Caenorhabditis elegans with a 37-50% decrease in cilium length. LmxOSM3.1 is therefore proposed to be important in the regulation of flagella length in L. mexicana. The kinase domain of LmxMPK13 displays homology to the Homo sapiens MOK and Chlamydomonas reinhardtii LF4 with MOK/LF4 downregulation promoting ciliary elongation. As LmxOSM3.1 and LmxMPK13 are involved in flagella regulation, it is hypothesised that LmxOSM3.1 is a substrate of LmxMPK13 and together they mediate flagellum length. To determine if LmxOSM3.1 and LmxMPK13 interact with each other, the 2C-miniTurbo assay was conducted. Using CRISPR-Cas9, the fusion protein FRB which binds to FKBP was tagged to the 5’ end of LmxMPK13 and 3’ end of LmxOSM3.1 for proximity labelling. To achieve this donor DNA (FRBLmxMPK13 and LmxOSM3.1-FRB) was generated by PCR and purified. Then electroporated into the LmxA1B5 cell line containing the CRISPR-Cas9 machinery with the miniTurbo biotin ligase fused to FKBP. Following electroporation, 6 transfectants were selected and cultured. Lmx_A2 and Lmx_E5 carrying FRB-LmxMPK13 and LmxOSM3.1-FRB, respectively, were treated with rapamycin and biotin to permit FRB-FKBP oligomerisation and biotinylation of potential proteins in close vicinity to LmxMPK13 and LmxOSM3.1, and then analysed by mass spectrometry. For technical reasons mass spectrometry analysis failed for clone Lmx_E5 and could not be repeated due to time constraints. Mass spectrometry for clone Lmx_A2 resulted in a considerable list of putative interaction partners but it failed to yield LmxOSM3.1 as an interaction partner of LmxMPK13. Further research is necessary to examine the potential interaction between LmOSM3.1 and LmxMPK13. The phosphorylation of kinesins via kinases has shown to be important in governing the primary motor activity of kinesins. Thus, the effects of phosphorylating LmxOSM3.1 towards flagella length was explored in vivo by electroporating into Leishmania plasmids to replace LmxOSM3.1 with LmxOSM3.1GFP encoding C-terminally GFP tagged wild-type, S477A, and S477D LmxOSM3.1 mutants. However, Leishmania cells transfected with LmxOSM3.1GFP, LmxOSM3.1SAGFP and LmxOSM3.1SDGFP failed to show green fluorescent signals when analysed by microscopy. The Cterminal fusion of GFP to LmxOSM3.1 and the N-terminal 6xHis tag may have impacted LmxOSM3.1 activity causing no green fluorescent signals to be observed in the clones. Therefore, the hypothesis could not be supported. Further research is required to support LmxOSM3.1 as a substrate of LmxMPK13 and to examine the role of phosphorylating Ser477 in LmxOSM3.1 to mediate flagella length.
Advisor / supervisor
  • Wiese, Martin (Researcher on Leishmania)
Resource Type
DOI
Date Created
  • 2023
Embargo Note
  • This thesis is restricted to Strathclyde users only.

Relações

Itens

Miniatura Título Data de carga Acesso Ações
file details File 2024-08-12 University of Strathclyde