A divergent protein kinase A in the human pathogen Leishmania is associated with developmental morphogenesis

Abstract

SummaryParasitic protozoa of the genus Leishmania cause human leishmaniasis. They cycle between the phagolysosome of mammalian macrophages, where they reside as round intracellular amastigotes, and the mid-gut of female sand flies, which are colonized by elongated extracellular promastigotes. Shifting promastigotes to a lysosome-like environment (pH 5.5 and 37°C, 5% CO2) initiates their development into amastigotes. Previous studies suggested a role for protein kinase A in this differentiation process. Here we describe a new, divergent, regulatory subunit of protein kinase A (PKAR3) present only in a limited group of kinetoplastids. In L. donovani, phosphorylation of PKAR3 is regulated by the differentiation signal and coincides with parasite morphogenesis during stage development. LdPKAR3 is bound to the subpellicular microtubules cell cortex via a formin FH2-like domain at the tip of a large and divergent N-terminal domain. Immunoprecipitation, fluorescence resonance energy transfer (FRET) and proteomics analyses show that PKAR3 selectively binds the C3 isoform of the PKA catalytic subunit in a holoenzyme complex, as supported by structural modeling. In promastigotes, PKAR3 recruits PKAC3 to the subpellicular microtubules at the cell’s central cortex. After exposure to the differentiation signal, PKAR3 relocates evenly to the entire cortex in concert with cell rounding. Deleting either the R3 or C3 subunit resulted in premature rounding of the promastigote population, indicating that PKA determines their normal elongated shape. Regulation of Leishmania developmental morphogenesis by interaction with the subpellicular microtubule corset is a novel function for an unusual PKA complex not present in the host cell.