Probing druggability and biological function of essential proteins in L eishmania combining facilitated null mutant and plasmid shuffle analyses

Summary L eishmania parasites cause important human morbidity and mortality. Essential L eishmania genes escape genetic assessment by loss‐of‐function analyses due to lethal null mutant phenotypes, even though these genes and their products are biologically most significant and represent validated drug targets. Here we overcome this limitation using a facilitated null mutant approach applied for the functional genetic analysis of the MAP kinase LmaMPK 4. This system relies on the episomal expression of the target gene from vector pXNG that expresses the H erpes simplex virus thymidine kinase gene thus rendering transgenic parasites susceptible for negative selection using the antiviral drug ganciclovir. Using this system we establish the genetic proof of LmaMPK 4 as essential kinase in promastigotes. LmaMPK 4 structure/function analysis by plasmid shuffle allowed us to identify regulatory kinase sequence elements relevant for chemotherapeutic intervention. A partial null mutant, expressing an MPK 4 derivative with altered ATP ‐binding properties, showed defects in metacyclogenesis, establishing a first link of MPK 4 function to parasite differentiation. The approaches presented here are broadly applicable to any essential gene in L eishmania thus overcoming major bottlenecks for their functional genetic analysis and their exploitation for structure‐informed drug development.