A multi-adenylate cyclase regulator at the flagellar tip controls African trypanosome transmission

Abstract

Signaling from ciliary nanodomains controls developmental processes in metazoans. Trypanosome transmission requires development and migration in the tsetse vector alimentary tract. Flagellar cAMP signaling has been linked to parasite social motility (SoMo) in vitro, yet uncovering control of directed migration in fly organs is challenging. Here we show that the architecture of an adenylate cyclase (AC) complex in the flagellar tip nanodomain is essential for tsetse salivary gland (SG) colonization and SoMo. Cyclic AMP response protein 3 (CARP3) binds and regulates multiple AC isoforms. CARP3 tip localization depends on the scaffold FLAM8. Re-localization of CARP3 away from the tip nanodomain is sufficient to abolish SoMo and fly SG colonization. Since intrinsic development is normal in ∆carp3 and ∆flam8 mutant parasites, AC complex-mediated tip signaling specifically controls parasite migration and thereby transmission. Participation of several developmentally regulated receptor-type AC isoforms may indicate the complexity of the in vivo signals perceived.

Proximity-dependent biotinylation and identification of flagellar proteins inTrypanosoma cruzi

“…3E ), of which 29 have orthologs that are represented in the TrypTag database ( Supplementary Table 3 ) and 20 proteins exhibited some flagellar localization in T. brucei . Examples of confirmed flagellar proteins in other trypanosomatids that are significantly enriched in the T. cruzi flagellar proximity proteome include: flagellar membrane 8 43 , flabarin 44 , flagellar attachment zone 14 32 , casein kinase I 42 , CARP3 20 and cysteine peptidase, Clan CA, family C2 (calpain 1.3) 42 . Although a significant proportion of the flagellar candidates identified in T. cruzi epimastigotes and amastigotes fall into the ‘hypothetical’ category (i.e., no annotation), the datasets were found to be enriched in kinase domains, calpain domains, and small GTP binding protein / GTPase domains ( Fig.…”

“…Although little is known regarding the sensory capabilities of trypanosomatids in general, significant progress has been made toward a molecular understanding of pH taxis and social motility in the insect stages of T. brucei 21,23 . The sensory system involves regulation of cyclic AMP levels, modulated by flagellar receptor-type adenylate cyclases and cyclic AMP phosphodiesterases 22 and the involvement of a cyclic AMP responsive protein (CARP3), which is thought to act in a complex with adenylate cyclases 20 . Our discovery, that CARP3 is expressed in the replicative stages of T. cruzi , including intracellular amastigotes (despite the inability to localize the tagged protein in this life stage), is quite exciting given the established role of CARP3 in T. brucei 20 .…”

“…The sensory system involves regulation of cyclic AMP levels, modulated by flagellar receptor-type adenylate cyclases and cyclic AMP phosphodiesterases 22 and the involvement of a cyclic AMP responsive protein (CARP3), which is thought to act in a complex with adenylate cyclases 20 . Our discovery, that CARP3 is expressed in the replicative stages of T. cruzi , including intracellular amastigotes (despite the inability to localize the tagged protein in this life stage), is quite exciting given the established role of CARP3 in T. brucei 20 . In T. brucei CARP3 is known to co-localize with calpain 1.3 at the distal region of the flagellum, where the two proteins may physically interact 20 .…”

“…Calpain 1.3 belongs to a sub-family of cysteine peptidases that are predicted to be catalytically inactive as they lack one or more of the active site amino acid residues 48 . Proteins with these features are thought to play a role in calcium homeostasis / signaling, including the calcium-based regulation of adenylate cyclase complexes 20,49 . Despite the lack of social motility in T. cruzi, the expression of flagellar CARP3 and calpain 1. membranes (lipid rafts) 41 and forms homodimers 41 .…”

“…cilia) and non-motile cilia have emerged as important sensory organelles that are equipped with signal transduction systems and second messengers such as cyclic AMP (cAMP) 14 and calcium 15,16 that coordinate cellular responses to external stimuli. Functions beyond cell locomotion have also been ascribed to the flagellum of motile trypanosomatid life stages 11,13,[17][18][19] , where the best understood example of sensory integration in these organisms is the role of flagellar receptor-type adenylate cyclases and cAMP-depending signaling in pH taxis and social motility in the insect stage of Trypanosoma brucei [20][21][22][23] . In Leishmania, flagellar aquaporin has been implicated in osmotaxis 24 in the insect stage, and the flagellar membrane may be a critical site for glucose 25,26 and arginine 27 sensing in these parasites.…”