Autophagic-related cell death of Trypanosoma brucei induced by bacteriocin AS-48

Abstract

The parasitic protozoan Trypanosoma brucei is the causative agent of human African trypanosomiasis (sleeping sickness) and nagana. Current drug therapies have limited efficacy, high toxicity and/or are continually hampered by the appearance of resistance. Antimicrobial peptides have recently attracted attention as potential parasiticidal compounds. Here, we explore circular bacteriocin AS-48's ability to kill clinically relevant bloodstream forms of T. brucei gambiense, T. brucei rhodesiense and T. brucei brucei. AS-48 exhibited excellent anti-trypanosomal activity in vitro (EC₅₀ = 1-3 nM) against the three T. brucei subspecies, but it was innocuous to human cells at 10⁴-fold higher concentrations. In contrast to its antibacterial action, AS-48 does not kill the parasite through plasma membrane permeabilization but by targeting intracellular compartments. This was evidenced by the fact that vital dye internalization-prohibiting concentrations of AS-48 could kill the parasite at 37 °C but not at 4 °C. Furthermore, AS-48 interacted with the surface of the parasite, at least in part via VSG, its uptake was temperature-dependent and clathrin-depleted cells were less permissive to the action of AS-48. The bacteriocin also caused the appearance of myelin-like structures and double-membrane autophagic vacuoles. These changes in the parasite's ultrastructure were confirmed by fluorescence microscopy as AS-48 induced the production of EGFP-ATG8.2-labeled autophagosomes. Collectively, these results indicate AS-48 kills the parasite through a mechanism involving clathrin-mediated endocytosis of VSG-bound AS-48 and the induction of autophagic-like cell death. As AS-48 has greater in vitro activity than the drugs currently used to treat T. brucei infection and does not present any signs of toxicity in mammalian cells, it could be an attractive lead compound for the treatment of sleeping sickness and nagana.

Keywords: AS-48; Antimicrobial peptides; Autophagy; Sleeping sickness; Trypanocidal drugs; Trypanosoma brucei.

Graphical abstract

Fig. 1

AS-48 kills BSF of T. brucei (a) AS-48 inhibits the proliferation of T. brucei. PCF (open circles) or BSF (filled circles) of T. brucei and control MRC-5 human cells (filled triangles) were incubated with increasing concentrations of AS-48 at 37 °C and for 72 h. Cell viability was determined using an alamarBlue^®-based assay and expressed as the percentage of untreated control samples. The results show the mean ± SD for one of the three independent experiments performed in triplicate. (b) Cytocidal effect of AS-48 on BSF of T. brucei. BSF of T. brucei were incubated with 100 nM AS-48 for 60 min at 37 °C and the cells were fixed and stained with DAPI (left panel). The middle panel corresponds to Nomarsky (DIC) images and the right panel shows the images once merged. Scale bar: 2 μm.

Fig. 2

The trypanocidal effect of AS-48 is temperature-dependent. BSF of T. brucei were incubated with 100 nM AS-48 for 30 and 60 min at 4 °C and 37 °C, respectively. Parasites were then fixed and stained with DAPI (upper panel). The middle panel shows the DIC images and the lower panel shows the merged images. Scale bar: 2 μm. These images are representative of four independent experiments performed in duplicate.

Fig. 3

The uptake of fluorescent AS-48 by BSF of T. brucei is temperature-dependent. BSF were incubated at 4 °C or 37 °C with 0.2 μM of fluorescent FITC-AS-48 for 10, 30, 45 and 60 min. AS-48 accumulation was analyzed by flow cytometry, as described in Material and Methods. The results are the mean ± SD of three experiments. Significant differences were determined using Student's t-test (_*, p < 0.05 versus 4 °C).

Fig. 4

AS-48 is internalized by BSF of T. brucei through clathrin-dependent endocytosis. (a) Clathrin-depleted cells were incubated with 0 (control), 0.05, 0.1 and 0.5 μM AS-48 for 10, 30 and 60 min and the number of live cells counted. The data are represented as cell survival percentages with respect to the control (cells incubated for 10 min in the absence of AS-48). Experiments were performed in triplicate upon 4 h of TbCLH RNAi induction. The results are shown as the mean ± SD of three experiments (_*, p < 0.05 versus non depleted controls). (b) Representative DIC pictures extracted from videos displayed in Supplementary data (See Supp. Data). T. b. brucei WT (upper panel) vs. TbCLT KD (lower panel), with (right) or without (left) AS-48.

Fig. 5

AS-48 binds VSG at the surface of BSF of T. brucei (a) FITC-AS48 binds to the surface of the parasites. 1 μM FITC-AS-48 was added to BSF parasites and cells were immediately fixed before being observed by fluorescence microscopy. The representative image shows how FITC-AS-48 label the plasma membrane in most cells. Scale bar: 5 μm (b) AS-48 interact with isolated VSG. 96-well ELISA plate coated with purified VSG were incubated with AS-48 or PBS (control), and the peptide bound to VSG was detected with anti-AS-48 (rabbit), HRP-conjugated anti-rabbit IgG antibodies and the HRP substrate ABTS. The results shows ELISA well absorbance at 450 nm and are the mean ± SD of two independent experiments performed in triplicate. Significant differences were determined using Student's t-test (* p < 10⁻⁵ versus control).

Fig. 6

Ultrastructural alterations on BSF cells caused by AS-48. Representative transmission micrographs of ultrathin resin sections of BSF parasites before (a) or after (b–i) treatment with 100 nM AS-48 for 60 min; note the morphological features of autophagic cell death. Autophagic-like vacuoles (av), myelin-like structures (mls), multivesicular structures (mvs) and dilated nuclear membrane (dnm) are shown with black arrows. f, flagellum; fp, flagellar pocket; m, mitochondrion; n, nucleus; Ac, acidocalcisome. Scale bars are indicated on the images.

Fig. 7

AS-48 increases the number of autophagosomes on BSF of T. brucei. (a) BSF parasites transfected with EGFP-ATG8.2 were incubated in the presence of 100 nM AS-48 for 0, 30 and 60 min at 37 °C. The parasites were then fixed and autophagy was monitored by counting the number of EGFP-ATG8.2-labeled autophagosomes per cell and represented as the percentage of cells containing more or less than one autophagosome for each set of conditions. The results are the mean ± SD of two independent experiments performed in duplicate. Significant differences were determined using Student's t-test (_*, p < 0.05 versus control) (b). A representative image showing the fluorescent autophagosomes at the same time as those described above. Scale bar: 5 μm.