Conservation and divergence within the clathrin interactome of Trypanosoma cruzi

Abstract

Trypanosomatids are parasitic protozoa with a significant burden on human health. African and American trypanosomes are causative agents of Nagana and Chagas disease respectively, and speciated about 300 million years ago. These parasites have highly distinct life cycles, pathologies, transmission strategies and surface proteomes, being dominated by the variant surface glycoprotein (African) or mucins (American) respectively. In African trypanosomes clathrin-mediated trafficking is responsible for endocytosis and post-Golgi transport, with several mechanistic aspects distinct from higher organisms. Using clathrin light chain (TcCLC) and EpsinR (TcEpsinR) as affinity handles, we identified candidate clathrin-associated proteins (CAPs) in Trypanosoma cruzi; the cohort includes orthologs of many proteins known to mediate vesicle trafficking, but significantly not the AP-2 adaptor complex. Several trypanosome-specific proteins common with African trypanosomes, were also identified. Fluorescence microscopy revealed localisations for TcEpsinR, TcCLC and TcCHC at the posterior region of trypomastigote cells, coincident with the flagellar pocket and Golgi apparatus. These data provide the first systematic analysis of clathrin-mediated trafficking in T. cruzi, allowing comparison between protein cohorts and other trypanosomes and also suggest that clathrin trafficking in at least some life stages of T. cruzi may be AP-2-independent.

The Plasmodium falciparum Artemisinin Susceptibility-Associated AP-2 Adaptin μ Subunit is Clathrin Independent and Essential for Schizont Maturation

“…5). A similar result was obtained for AP-2 in T. cruzi using comparable techniques, suggesting clathrin and AP-2 may not be universally associated, as previously thought (45). These data imply that despite the shared ␤ subunit, which canonically links the AP complex to clathrin, other unknown P. falciparum subunits or factors may be involved in the selection of coat proteins.…”

“…The AP-2 complex is clathrin independent and associates with Kelch10. To identify AP-2 interacting partners, early schizont (32 to 36 h postinvasion) cell lysates of P. falciparum AP-2-3xHA were prepared using cryomilling and detergent lysis, a cell disruption technique that has generated high-resolution interactomes in other organisms, although not previously deployed in Plasmodium (45). Using both Triton X-100 and CHAPS-containing lysis buffers, originally derived for the extraction of clathrininteracting proteins from Trypanosoma species, we lysed the frozen parasites, immunoprecipitated AP-2 using anti-HA-conjugated beads and performed mass spectrometry (MS).…”

“…A 300-mg portion of the milled powder per replicate was lysed in buffer A (20 mM HEPES, 100 mM NaCl, 0.1% [vol/vol] Triton X-100, 0.1 mM TLCK [N␣-p-tosyl-L-lysine chloromethyl ketone], and protease inhibitors [Complete protease inhibitor cocktail tablet, EDTA-free, Roche]; pH 7.4) or buffer B (20 mM HEPES, 250 mM sodium citrate, 0.1% [wt/vol] CHAPS, 1 mM MgCl 2 , 10 mM CaCl 2 , 0.1 mM TLCK, and protease inhibitors; pH 7.4). Buffer B was previously optimized to immunoprecipitate clathrin heavy chain from trypanosomes (45). The lysate was sonicated on ice with four cycles of 3 s on at 30% amplitude, followed by 10 s off and clarified by centrifugation.…”

Shotgun Label-Free Proteomic Analyses of the Oyster Parasite Perkinsusmarinus

“…Clathrin-mediated trafficking is known to be responsible for endocytosis and postGolgi transport in trypanosomes. It also represents an important interface with the host, in addition to play multiple roles in immune evasion and host cell invasion that are vital for effective infection and persistence [31].…”

Unlocking the biological potential ofEuglena gracilis: evolution, cell biology and significance to parasitism

“…Both the cell surface and flagellum are of significant importance to life cycle progression, interactions with the environment and, in the case of parasitic trypanosomes, pathogenesis and immune evasion (Langousis andHill, 2014, Perdomo, et al, 2016). The surface macromolecules of trypanosomatids are highly lineage-specific with roles in life cycle progression (Jackson, et al, 2013, 2016, Kalb, et al, 2016, Zoltner, et al, 2016, Hovel-Miner, et al, 2016, Devault and Banuls, 2008, Chamakh-Ayari, et al, 2014, but it remains to be determined to what extent E. gracilis shares ! 4 surface protein families or other aspects of their cell biology with parasitic kinetoplastids.…”