Authors

Discussion

The protein kinase RDK2 (Repressor of differentiation 2) has been proposed to be involved in differentiation of L. mexicana.  To investigate this, we generated an RDK2 null mutant (Δrdk2)using CRISPR-Cas9 genome editing. In T. brucei, RNA inference of RDK2 promotes differentiation from bloodstream stumpy form to procyclic form. In L. mexicana in vitro, the Δrdk2 cells were able to undergo normal differentiation from promastigote to amastigote. To attempt to quantitatively measure this differentiation using flow cytometry, we generated a mNeonGreen-dynein line where the newly formed flagellum can be a measured fluorescently. We found Δrdk2 was able to transform from amastigote to promastigote in vitro, albeit at a slower rate compared to the control, during the initial phase of differentiation.

The Δrdk2 cell line was screened within a protein kinase knockout library using BarSeq to identify genes with a loss of fitness in differentiation from lesion-derived amastigotes to promastigotes. Like the in vitro assays, Δrdk2 had a delayed differentiation from amastigote to promastigote. We generated an N-terminal MYC tagged RDK2 and carried out immunoprecipitation and mass spectrometry to identify potential interacting proteins. From this we identified other protein kinases that may be involved in the same pathway as RDK2.  From these screens, we are attempting to unravel the role of RDK2 and its interactome in L. mexicana differentiation.