The Intracellular Amastigote of Trypanosoma cruzi Maintains an Actively Beating Flagellum

Abstract

Understanding the interplay between intracellular pathogens and their hosts is vital to the development of new treatments and preventive strategies. The intracellular “amastigote” stage of the Chagas disease parasite, Trypanosoma cruzi , is a critical but understudied parasitic life stage.

Proximity-dependent biotinylation and identification of flagellar proteins inTrypanosoma cruzi

“…T. cruzi strains expressing TcSMP1-1GFP were previously generated 6 . A plasmid containing the TurboID sequence was a kind gift of Jeffrey Dvorin (Harvard Medical School).…”

“…Four of the 6 proteins were successfully tagged and three of which exhibited flagellar localization in T. cruzi The functional capabilities of the T. cruzi flagellum are broadly uncharacterized, beyond its role in propelling motile life stages and anchoring epimastigotes to the rectal mucosa in the insect vector 4,45,46 . However, the recent recognition that the flagellum of cytosolically-localized intracellular amastigotes is capable of beating and establishes physical contact with host mitochondria 3,6 , points to a potential role for the amastigote flagellum in host environmental sensing. 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 .…”

“…It has long been assumed that the minimal amastigote flagellum serves no function other than to provide a structural platform for flagellar outgrowth during differentiation to motile life stages 11 . However, recent observations that the flagellum of intracellular T. cruzi amastigotes undergoes low frequency aperiodic 'beating' inside mammalian host cells 6 and makes physical contact with the host mitochondria 3,6 , indicate that the amastigote flagellum has a functional role within the host cell. The interaction between the T. cruzi amastigote flagellum and host mitochondria is comparable to the intimate contact observed between the flagellum of intracellular Leishmania mexicana amastigotes and the host parasitophorous vacuole membrane 12 .…”

“…FCaBP is expressed in all T. cruzi life stages and is conserved across other trypanosomatid species, but its precise role in the biology of these organisms is unknown beyond its role as a calcium binding protein 36,38,39 . Additionally, we have recently localized small myristoylated protein 1-1 (TcSMP1-1) to the flagellum in amastigotes 6 , but the overall proteomic landscape of the T. cruzi flagellum remains largely uncharacterized.…”

“…in the parasite flagellum, as an in-frame fusion with C-terminal FLAG-tagged T. cruzi small myristoylated protein 1-1 (TcSMP1-1) (Fig.1A,B; SMP1-1-FLAG-TurboID; 'F-Turbo'). TcSMP1-1 was chosen as the endogenous 'bait' protein for flagellar localization of TurboID given its near exclusive localization in the flagellum in both replicative stages of T. cruzi, epimastigotes and amastigotes6 (Fig.1A), and because TcSMP1-1 contains the N-myristoylation sequence motif (MGXXXS/T) required for localization and tethering to the inner flagellar membrane, as demonstrated in Leishmania41 . The strategy of targeting TurboID to the flagellum using TcSMP1-1 is expected to increase the likelihood of identifying flagellar membrane and associated proteins while minimizing capture of axonemal proteins.…”