Description:
<jats:title>Abstract</jats:title><jats:p><jats:italic>Leishmania</jats:italic> parasites are thought to control protein activity at the post-translational level, e.g. by protein phosphorylation. In the pathogenic amastigote, the mammalian stage of <jats:italic>Leishmania</jats:italic> parasites, heat shock proteins show increased phosphorylation, indicating a role in stage-specific signal transduction. Here we investigate the impact of phosphosites in the <jats:italic>L</jats:italic>. <jats:italic>donovani</jats:italic> heat shock protein 90. Using a chemical knock-down/genetic complementation approach, we mutated 11 confirmed or presumed phosphorylation sites and assessed the impact on overall fitness, morphology and <jats:italic>in vitro</jats:italic> infectivity. Most phosphosite mutations affected the growth and morphology of promastigotes <jats:italic>in vitro</jats:italic>, but with one exception, none of the phosphorylation site mutants had a selective impact on the <jats:italic>in vitro</jats:italic> infection of macrophages. Surprisingly, aspartate replacements mimicking the negative charge of phosphorylated serines or threonines had mostly negative impacts on viability and infectivity. HSP90 is a substrate for casein kinase 1.2-catalysed phosphorylation <jats:italic>in vitro</jats:italic>. While several putative phosphosite mutations abrogated casein kinase 1.2 activity on HSP90, only Ser<jats:sub>289</jats:sub> could be identified as casein kinase target by mass spectrometry. In summary, our data show HSP90 as a downstream client of phosphorylation-mediated signalling in an organism that depends on post-transcriptional gene regulation.</jats:p>