Discussion

Leishmania encounters a number of challenges during its life cycle following environmental changes that trigger the transition from an intracellular amastigote that replicates inside a parasitophorous vacuole in the mammalian host to the extracellular insect stages, a replicative procyclic promastigote and an infective metacyclic. The differentiation process is thought to be regulated in part by phosphorylation resulting in dramatic changes in protein expression, which allows adaptation. Here we aim to identify and characterize protein kinases involved in this process, as well as understand which mechanisms Leishmania uses to overcome stress and regulate its life cycle. We have applied the CRISPR-Cas9 genome editing system to L. mexicana in order to investigate 2 protein kinases previously characterized in Trypanosoma brucei as regulators of bloodstream to procyclic form differentiation, RDK1 and RDK2 (Repressor of Differentiation Kinase) [1]. Our data show that while RDK1 is non-essential for L. mexicana promastigotes, its absence impacts on amastigote proliferation and differentiation to promastigotes in vitro, but doesn’t affect infectivity in mice. RDK2 is located on supernumerary Chromosome 30, making it a challenging gene to study.   Progress on the application of CRISPR-Cas9 technology to generate protein kinase null mutants and investigate their involvement in Leishmania life cycle progression will be discussed.  [1] Jones et al., 2014. PMID: http://www.ncbi.nlm.nih.gov/pubmed/24453978