Investigation towards the role of LmxOSM3.1 phosphorylation and the identification of its interaction partners using an improved method of proximity labelling

Leishmaniasis is a parasitic infection provoked by vector-borne protozoan Leishmania, of the family Trypanosomatidae, transmitted by female hematophagous sand flies. It is endemic to tropical/subtropical regions. However, its geographical distribution is anticipated to diversify, due to climate change, urbanisation, and deforestation. Therefore, the global figure of those affected (12 million) is likely to increase. Examples of current leishmanias is therapeutics include pentavalent antimonials, amphotericin B, miltefosine, paromomycin, and pentamidine. Unfortunately, they do not come without limitations, hence, more effective treatments are needed. Kinases and kinesins have been shown to be promising drug targets.;LmxOSM3.1 is expected to be a plus end-directed 170kDa, homodimeric N-kinesin, whose function is to mediate anterograde transport (IFT) in the flagellum. CeOSM3 p802 null mutants provoke a complete loss in the (sensory) ciliary distal domain of C. elegans and a 37.5% to 50% decrease in cilium length. Thus, LmxOSM3.1 is anticipated to be fundamental for flagellar length regulation in L. mexicana. The kinase domain of LmxMPK13 exhibits a high degree of homologyto the H. sapiens MOK and C. reinhardtii LF4, whereby down regulation of either protein promotesciliary elongation. As both LmxOSM3.1 and LmxMPK13 are implicated in flagellar regulation, it is hypothesised that LmxOSM3.1 functions as a substrate of LmxMPK13 and together they coordinate flagellum length.;In 2C-mTID, a proximity-based assay to define potential protein-protein interactions (PPIs), miniTurbo (mTID) and LmxOSM3.1 (PoI) are fused to FKBP and FRB (of the FRB-FKBPmammalian oligomerisation system), respectively. A cell line expressing the CRISPR-Cas9 machinery and mTID fused to FKPB was generated but not yet assessed on a molecular level. Likewise, LmxOSM3.1 and LmxMPK13 tagged with FRB still require to be integrated into the genome of this cell line using CRISPR to prove LmxOSM3.1 as a substrate of LmxMPK13. To investigate the importance of phosphorylation of LmxOSM3.1 in vivo, LmxOSM3.1 is replaced with LmxOSM3.1GFP encoding C-terminally GFP-tagged wild type, S477A, and S477DLmxOSM3.1 mutants using CRISPR. Three A1OSM3SDGFP promastigote transfectants were generated (A5, C6, and E7) appearing immobile and aflagellated, which may be due to the Cterminal GFP disrupting IFT cargo binding, LmxOSM3.1 inactivation by phosphorylation mimicked by D477, or the mutation generally interfering with LmxOSM3.1 activity, e.g. by restricting flagellar access or promoting kinesin degradation.;However, some A1OSM3SDGFP promastigote transfectants (A6, B2, B4, C3, and C10) appeared normal. Therefore, the latter Leishmania are presumed to have a single allele replacement of LmxOSM3.1 maintaining a fully functional wild type allele. So far, no A1OSM3SAGFP promastigote transfectants could be generated. To conclude, further research is required to corroborate LmxOSM3.1 as a substrate of LmxMPK13 with phosphorylation on Ser477, LmxOSM3.2 as a PPI of LmxOSM3.1, and GFP or Ser477Asp as the causative factor for a flagellated LmxOSM3.1SD.

Name dc:type.qualificationname

mres

Level dc:type.qualificationlevel

masters-pg

Grantor dc:publisher.institution

University of Strathclyde

Year dc:date.issued

2020

Author dc:creator

• Clyne, Bethany

Advisor dc:contributor.advisor

• Wiese, Martin (Researcher on Leishmania)

DOI

10.48730/dmwc-ga49

Identifier

T15734

Author Identifier

201984124

OAI identifier oai:identifier

oai:strathclyde:hq37vn56b