Discussion

L. donovani is the causative agent of visceral leishmaniasis, a major public health problem worldwide. We are interested in understanding how Leishmania interacts with its host, and particularly the impact of secreted parasitic proteins on intracellular survival. Casein Kinase 1 isoform 2 (CK1.2), a protein kinase involved in signaling, has been identified in exosomes by proteome analysis and is important for intracellular parasite survival. However very little is known about the functions it may carry outside of the parasite. One possibility would be to target host proteins to modulate host cell biology and evade the innate anti-leishmanial immune response. We addressed this hypothesis by:
(i) Investigating whether CK1.2 is essential for intracellular parasite using a null mutant approach.
(ii) Determining the localization of CK1.2 in extra- and intracellular parasites and establishing its presence inside the host cell cytoplasm using cell imaging. Our preliminary results suggest that the localization of V5-tagged CK1.2 is vesicular in promastigotes and intracellular amastigotes, supporting the hypothesis of its presence in exosomes.
(iii) Identifying CK1.2 binding partners allowing it to carry out its functions, in particular to be secreted into the host cell by exosomes. Applying immunoprecipitation and mass spectrometry analyses on promastigote and axenic amastigote extracts allowed us to establish a list of potential binding partners. In promastigotes, we identified proteins involved mostly in vesicular processes, folding and transport, whereas in axenic amastigotes CK1.2 partners were involved in metabolism translation and transport. We are currently performing similar experiments to identify host partners