Cyclic AMP signalling in trypanosomes differs from most eukaryotes due to absence of known cAMP effectors and cAMP independence of PKA. We have previously identified four genes from a genome-wide RNAi screen for resistance to the cAMP phosphodiesterase (PDE) inhibitor NPD-001. The genes were named cAMP Response Protein (CARP) 1 through 4. Here, we report an additional six CARP candidate genes from the original sample, after deep sequencing of the RNA interference target pool retrieved after NPD-001 selection (RIT-seq). The resistance phenotypes were confirmed by individual RNAi knockdown. Highest level of resistance to NPD-001, approximately 17-fold, was seen for knockdown of CARP7 (Tb927.7.4510). CARP1 and CARP11 contain predicted cyclic AMP binding domains and bind cAMP as evidenced by capture and competition on immobilised cAMP. CARP orthologues are strongly enriched in kinetoplastid species, and CARP3 and CARP11 are unique to Trypanosoma. Localization data and/or domain architecture of all CARPs predict association with the T. brucei flagellum. This suggests a crucial role of cAMP in flagellar function, in line with the cell division phenotype caused by high cAMP and the known role of the flagellum for cytokinesis. The CARP collection is a resource for discovery of unusual cAMP pathways and flagellar biology.

Parasitic 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 they colonize as 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 (PKA) in this differentiation process. Here, we describe a new, divergent, regulatory subunit (PKAR3) found only in a limited member of the family Kinetoplastidae. In L. donovani, phosphorylation of PKAR3 was regulated by the differentiation signal and coincided with parasite morphogenesis during stage development. LdPKAR3 was bound to the subpellicular microtubules cell cortex via a formin homology (FH2)-like domain at the tip of a large and divergent N-terminal domain. Immunoprecipitation, fluorescence resonance energy transfer (FRET), proteomics analyses, and structural modeling showed that PKAR3 selectively binds the C3 isoform of the PKA catalytic subunit in a holoenzyme complex. In promastigotes, PKAR3 recruited PKAC3 to the subpellicular microtubules at the central cortex. After exposure to a differentiation signal, PKAR3 homogenously distributed across the entire cortex, in concert with cell rounding. Deleting the genes encoding either the R3 or C3 subunit resulted in premature rounding of the promastigote population, indicating that PKA determines the normal elongated shape. Regulation of Leishmania developmental morphogenesis by interaction with the subpellicular microtubule corset is a novel function for a unique PKA complex not present in the host cell.

Cyclic AMP signalling in trypanosomes differs from most eukaryotes due to absence of known cAMP effectors and cAMP independence of PKA. We have previously identified four genes from a genome-wide RNAi screen for resistance to the cAMP phosphodiesterase (PDE) inhibitor NPD-001. The genes were named cAMP Response Protein (CARP) 1 through 4. Here, we report an additional six CARP candidate genes from the original sample, after deep sequencing of the RNA interference target pool retrieved after NPD-001 selection (RIT-seq). The resistance phenotypes were confirmed by targeted RNAi knockdown and highest level of resistance to NPD-001, approximately 17-fold, was seen for knockdown of CARP7 (Tb927.7.4510). CARP1 and CARP11 contain predicted cyclic AMP binding domains and bind cAMP as evidenced by capture and competition on immobilised cAMP. CARP orthologues are strongly enriched in kinetoplastid species, and CARP3 and CARP11 are unique to Trypanosoma. Localization data and/or domain architecture of all CARPs predict association with the T. brucei flagellum. This suggests a crucial role of cAMP in flagellar function, in line with the cell division phenotype caused by high cAMP and the known role of the flagellum for cytokinesis. The CARP collection is a resource for discovery of unusual cAMP pathways and flagellar biology.