Systematic functional analysis of Leishmania protein kinases identifies regulators of differentiation or survival

Abstract

Differentiation between distinct stages is fundamental for the life cycle of intracellular protozoan parasites and for transmission between hosts, requiring stringent spatial and temporal regulation. Here, we apply kinome-wide gene deletion and gene tagging in Leishmania mexicana promastigotes to define protein kinases with life cycle transition roles. Whilst 162 are dispensable, 44 protein kinase genes are refractory to deletion in promastigotes and are likely core genes required for parasite replication. Phenotyping of pooled gene deletion mutants using bar-seq and projection pursuit clustering reveal functional phenotypic groups of protein kinases involved in differentiation from metacyclic promastigote to amastigote, growth and survival in macrophages and mice, colonisation of the sand fly and motility. This unbiased interrogation of protein kinase function in Leishmania allows targeted investigation of organelle-associated signalling pathways required for successful intracellular parasitism.

Unpicking the Roles of DNA Damage Protein Kinases in Trypanosomatids

“…However, as for ATM, VE-821-treated cells were significantly more sensitive to H 2 O 2 , suggesting that ATR may act during the response to oxidative stress, similar to ATM, though work is needed to validate ATR as the target of VE-821 and to assess whether inhibiting ATR also sensitizes cells to other genotoxins. Unlike in T. brucei, and perhaps consistent with the VE-821 inhibition data, ATR has been reported to be dispensable for L. mexicana survival in vitro, though effects of ATR loss were not investigated further (Baker et al, 2021).…”

“…T. brucei, T. cruzi, and Leishmania encode for 190 (Jones et al, 2014), 190 (Parsons et al, 2005, and 206 (Baker et al, 2021) PKs, respectively, with several aPKs identified and members of all ePK groups represented, except for tyrosine-like and tyrosine kinases (Figure 2B). Over the last decade, with the implementation of genome-wide and kinome-focused screens in T. brucei, the roles of PKs have been investigated during drug resistance (Alsford et al, 2012), cell cycle control (Jones et al, 2014), and in vivo survival (Fernandez-Cortes et al, 2017).…”

“…Whether KU-55933 treatment induces a more generalized sensitivity to genotoxins requires further investigation, as we lack information about how selective this inhibitor is for ATM in trypanosomatids. In a recent study, an unexpected role for the ATM gene in L. mexicana was uncovered (Baker et al, 2021). Deletion of ATM in promastigotes prevented the establishment of infections in the sandfly vector, implicating ATM (and perhaps the wider DDR directed by the PK) in a previously unappreciated role in parasite transmission, though the basis for this defect is unexplained.…”

“…Dysregulation of aurora kinase family members is associated with the formation of cancer, and the PKs play prominent roles during mitosis (Tang et al, 2017). The function of AUK2 is unknown in Leishmania, but null mutants could not be recovered in promastigote cells, suggesting that it is essential (Baker et al, 2021). In T. cruzi, only AUK1 (homologous to AURKB) function has been assessed, with evidence suggesting it acts canonically during mitosis and nuclear division, alongside being required during kinetoplast duplication (Fassolari and Alonso, 2018).…”

“…Other peculiarities have recently emerged, with components of the 9-1-1 complex playing non-canonical roles in Leishmania genome replication, as facilitators of genomic plasticity (Damasceno et al, 2016(Damasceno et al, , 2018. Moreover, DDR PK activity has been implicated in developmental transitions between host and vector (Baker et al, 2021) and as a driver of host immune evasion (Black et al, 2020). Thus, the trypanosomatid DDR and its associated PKs have potential for the discovery of novel biology and the prospect of parasite-specific drug targets.…”