Analysis of LmxMPK4 and LmxMPK6, two mitogen-activated protein kinases of Leishmania mexicana

Abstract

The focus of this thesis was the functional analysis of two MAP kinase homologues, LmxMPK4 and LmxMPK6, of Leishmania mexicana. LmxMPK4 is essential in both Leishmania life stages and therefore a potential drug target. The STE7-like protein kinase LmxMKK5 was successfully identified as the in vitro activator of LmxMPK4, resulting in active, recombinant protein, which is now available for drug screenings. An inhibitor-sensitised mutant was employed to investigate the function of LmxMPK4 in vivo. The inhibition of LmxMPK4 led to a reversible growth arrest of insect-stage promastigotes, yet it was demonstrated that LmxMPK4 was not involved in regulating progression through specific cell cycle checkpoints. The growth arrest seemed rather to be due to a metabolic arrest. It was furthermore demonstrated that fully functional LmxMPK4 is required for the establishment of infection in the mammalian host. LmxMPK6 could not be deleted from the Leishmania genome and is therefore supposed to be essential. Recombinant LmxMPK6 was successfully expressed and purified from E. coli as an active protein. The kinase activity of LmxMPK6 was demonstrated to be dependent on the presence of 78 amino acids after the end of the kinase domain, but not on the presence of the remaining C-terminus.