Novel kinetoplastid-specific cAMP binding proteins identified by RNAi screening for cAMP resistance inT. brucei

Abstract

AbstractCyclic 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 toTrypanosoma. Localization data and/or domain architecture of all CARPs predict association with theT. bruceiflagellum. 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.ImportanceTrypanosomes are major pathogens of humans and livestock. In addition they have been invaluable as a model system to investigate new biological systems, and not just of protozoa. Equally, they are known to have a lot of unique biology and biochemistry. One example of this is signal transduction by cyclic nucleotides. Some elements, including phosphodiesterases and the catalytic domains of its dozens of adenylate cyclase isoforms, are highly conserved, while the absence of G-proteins, a cAMP-responsive protein kinase A and other known effector types suggests a unique cAMP-dependent pathway, which as yet is mostly uncharacterised. Here, we identify a set of tenTrypanosoma bruceiproteins, all localised to its flagellum, that appear to be involved in the production of cAMP, or in mediating its cellular effects. These cAMP Response Proteins (CARPs) were mostly unique to trypanosomes, suggesting a completely novel pathway. Two of the CARPs were shown to bind cAMP and were found to possess structurally conserved cyclic nucleotide binding domains.