Authors

Discussion

Little is known about Leishmania MAP kinase kinase kinases (MAPKKK). LmxMKKK19 has been annotated in TriTrypDB as a putative MAPKKK in Leishmania mexicana. To understand MAP kinase cascades in Leishmania, we targeted LmxMKKK19 for deletion and tagging using CRISPR-Cas9. Single and double allele knockout clones were generated by electroporation utilising two different resistance markers. Add-back clones were obtained implementing homologous recombination of LmxMKKK19 into the ribosomal RNA gene locus. The generated clones were screened for changes in morphology, growth and their potential to infect Balb/c mice. To unveil the localisation of LmxMKKK19 in promastigotes, the protein was tagged with mNeonGreen (mNG) using CRISPR-Cas9 at either its N- or C-terminus. Furthermore, in a CRISPR-Cas9 cell line, expressing FKBP-miniTurbo biotin ligase, a component of a 2C-BioID system, FRB, the second component, was fused to LmxMKKK19 for identification of possible interaction partners utilising biotin proximity labelling, tryptic digest of biotinylated proteins isolated using streptavidin-agarose, and mass spectrometry. Proteins encoded by LmxM.33.2090 and LmxM.07.0900 were determined as putative interaction partners of LmxMKKK19. Using CRISPR-Cas9 on a cell line already expressing mNG-tagged LmxMKKK19, monomeric red fluorescent protein (mRFP) was fused to each of these proteins and co-localisation studies were carried out employing fluorescence microscopy. Further experiments to confirm the interaction of the protein kinases are under way.