Knockout of the gamma subunit of the AP-1 adaptor complex in the human parasite Trypanosoma cruzi impairs infectivity and differentiation and prevents the maturation and targeting of the major protease cruzipain

Abstract

The AP-1 Adaptor Complex assists clathrin-coated vesicle assembly in the trans-Golgi network (TGN) of eukaryotic cells. However, the role of AP-1 in the protozoan Trypanosoma cruzi—the Chagas disease parasite—has not been addressed. Here, we studied the function and localization of AP-1 in different T. cruzi life cycle forms, by generating a gene knockout of the large AP-1 subunit gamma adaptin (TcAP1-γ), and raising a monoclonal antibody against TcAP1-γ. Co-localization with a Golgi marker and with the clathrin light chain showed that TcAP1-γ is located in the Golgi, and it may interact with clathrin in vivo, at the TGN. Epimastigote (insect form) parasites lacking TcAP1-γ (TcγKO) have reduced proliferation and differentiation into infective metacyclic trypomastigotes (compared with wild-type parasites). TcγKO parasites have also displayed significantly reduced infectivity towards mammalian cells. Importantly, TcAP1-γ knockout impaired maturation and transport to lysosome-related organelles (reservosomes) of a key cargo—the major cysteine protease cruzipain, which is important for parasite nutrition, differentiation and infection. In conclusion, the defective processing and transport of cruzipain upon AP-1 ablation may underlie the phenotype of TcγKO parasites.

Deletion of a Golgi protein in Trypanosoma cruzi reveals a critical role for Mn2+ in protein glycosylation needed for host cell invasion and intracellular replication

“…After selection with G418, resistant cells were analyzed by immunofluorescence (IFA) (Fig 1B -1D) and western blot (Fig 1E) using monoclonal anti-HA antibodies in all three life cycle stages of the parasite. Fluorescence microscopy images showed that TcGDT1 localizes to the Golgi complex, as detected by partial co-localization with the TcAP1γ (Fig 1B -1D), a recently identified marker for Golgi complex in T. cruzi [28]. This co-localization could be observed in all three life cycle stages of the parasite.…”

“…Fluorescence microscopy images showed that TcGDT1 localizes to the Golgi complex, as detected by partial co-localization with the TcAP1γ ( Fig 1B–1D ), a recently identified marker for Golgi complex in T . cruzi [ 28 ]. This co-localization could be observed in all three life cycle stages of the parasite.…”

Transcriptional remodeling during metacyclogenesis in Trypanosoma cruzi I

“…Previous studies evaluated AP-2 expression in EPs and CDTs of T. cruzi ; thus, we verified the ID TcSYL_0201950 that codes for this protein in our dataset (Table S2). We observed that although EPs express this protein, its expression is greater in MTs [ 38 , 39 ]. To what extent the increased expression of this protein can confer certain properties to MTs remains unknown, but these results suggest that MT expression levels are regulated to modify an entire metabolic pathway.…”

A novel receptor for platelet‐activating factor and lysophosphatidylcholine in Trypanosoma cruzi

“…To obtain enough DNA by mass for transfection (25‐ to 50‐μg cassettes), 20 cycles of PCR, each in a volume of 50 μL, were performed to amplify each cassette. T. cruzi transfection was performed as described (Moreira et al., 2017). The epimastigote form of the T. cruzi Dm28c clone was grown in LIT medium (Camargo, 1964) to a density of 2 × 10 7 parasites/ml.…”